1,721,052 research outputs found
Applicazione di tecniche PCR per l’identificazione della microflora tipica nella produzione di Panettone
No full textLa possibilità di estrarre DNA amplificabile di origine microbica direttamente da matrici alimentari sottoposte a trattamento termico potrebbe consentire l’identificazione delle specie presenti all’origine nel prodotto. Tale evenienza può comportare un impatto notevole sulla tracciabilità nella filiera dei prodotti cotti in cui hanno luogo fermentazioni microbiche.
A tal scopo si è voluto verificare se, e fino a quale livello di concentrazione, sia possibile determinare la presenza e il tipo di microrganismi attraverso l’utilizzo di PCR direttamente da campioni di impasto per Panettone prima e dopo la cottura in forno. Per l’estrazione del DNA sono stati selezionati tre differenti protocolli; il grado di purezza ed integrità dell’acido nucleico ottenuto da campioni di madre, impasto finale e prodotto cotto sono stati valutati attraverso elettroforesi. La quantità di DNA estratta è correlata alla composizione delle matrici, in quanto alcune sostanze presenti sono in grado di interferire con la fase di estrazione riducendo il limite di rilevazione del segnale. Sugli estratti sono state poi sperimentate alcune tecniche PCR con differenti primers quali l’amplificazione 16S rDNA specie-specifica per la rilevazione di Lb. sanfranciscensis e l’amplificazione della regione spaziatrice ribosomiale ITS1-5.8S-ITS2 per la rilevazione di C. humilis e S. cerevisiae. Per i campioni non sottoposti a cottura (madri e impasti finali) è stato possibile ottenere segnali positivi inferiori, in media, di circa due cicli logaritmici rispetto a quelli determinati attraverso conte in piastra. Per i prodotti cotti i risultati ottenuti hanno evidenziato una frammentazione del DNA dovuta al drastico trattamento termico e all’effetto dell’acidificazione legata alla fermentazione batterica. Il disegno di primers che generano un prodotto di amplificazione di dimensioni ridotte (<200 bp) aumenta la probabilità di riuscita dell’amplificazione
Comparative study of nine Lactobacillus fermentum bacteriophages
No full textAims: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization.
Methods and Results: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations.
Conclusions: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage.
Significance and Impact of the Study: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation
What does it happen when a bacteriophage infects a sourdough microbial population?
No full textA phage infection is a real risk in food plants since viruses are able to quickly destroy bacterial population, stopping the fermentative process. Actually the manufacture of sourdough products based on traditional batch technology is less involved in this phenomenon than dairy or wine productions. By now the main belief is that the presence of several different strains and the selection of spontaneous phage-resistant mutants can explain the ability of natural mixed cultures to overcome viral infections.
In this work the propagation of Lactobacillus sanfranciscensis phage EV3 in solid sourdough, liquid sourdough and cultural medium was investigated. Lactobacillus sanfranciscensis H2A strain and the Candida humilis 21R strain were used respectively as phage host culture and yeast culture; different experiments were carried out at 25°C for 24 hours. In solid state (Dough Yield = 160) the averages of increase of dough volume and pH variation between the infected and the uninfected doughs found out to be identical. Either for yeasts and for lactobacilli counts no significant difference were observed between concentration mean values in infected and control samples, at all different times (o “at all the tested times” o “at different times”). During incubation, yeasts augmented of 1.2 log cycles while lactobacilli of 2.0 log cycles; a scarce phage multiplication (1.5 log cycles) was observed without causing any variation in fermentation parameters.
In liquid sourdough (Dough Yield = 320) the changes of pH between the infected and the uninfected samples were very similar; the growth of yeast population (1.2 log cycles) was not affected by phage infection. Also for lactobacilli counts no significant differences were detected between mean values of infected and control tests, but there was an increasing of only 1.0 log cycle. The phage concentration rises of 3,0 log cycle
Influence of cheese making process on STEC bacteriophage release
Full text linkShiga toxin-producing Escherichia coli (STEC) are foodborne pathogens implicated in diseases including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). The main virulence factor are Shiga toxins; their production and secretion are by-products of the expression of late genes of prophages upon sub-lethal environmental stimuli exposure. Hence, the lysogenic prophage after a stress switch to lytic cycle spreading the Stx phages. In the present study, 35 STEC were screened for the presence and the ability to release Shiga toxin-encoding bacteriophages. Three bacterial strains showed signals of prophage presence both in plate and in PCR. Subsequently, these bacterial strains were subjected to stressors that simulate cheese manufacturing conditions: NaCl (1, 1.5 and 2% w/v), lactic acid (0.5, 1.5 and 3% v/v), anaerobic growth, pasteurization (72°C for 15 s), UV irradiation. The ability to release prophage was evaluated by Real Time qPCR. Induction of the prophages showed that the addition of NaCl at 1.5 and 2% significantly increased viral release compared to control. Conversely, the addition of lactic acid had a significant repressive effect. The other applied stressors had no significant effect in phage release according to the experimental conditions adopted
PCR detection of Lactobacillus sanfranciscensis in sourdough and Panettone baked product
No full textSourdoughs, in which Lactobacillus sanfranciscensis is the predominant bacterial species, are distinctive of some traditional Italian sweet baked products like Panettone. The direct extraction of amplifiable bacterial DNA from products subjected to heat treatment represents a valid tool to identify and trace microbial species originally present in the food matrices. Three types of protocols for the isolation and clean-up of DNA (CTAB, Wizard (R) DNA Clean-Up System, NucleoSpin (R) Food) were applied on mother, final dough and end-product samples and compared through the determination of the maximum amplifiable dilution by a PCR reaction targeting two fragments (1460 and 153 bp long) of 16S rDNA region of Lb. sanfranciscensis. CTAB extracting protocol was revealed to be the best for isolating DNA. In dough samples the amplification with the 153 bp fragment showed signals at concentration levels that are comparable with the values obtained from the plate counts, and two log cycles higher than those found with the amplification targeting the 1460 bp fragment. In the cooked samples only the 153 bp amplicon was detected, indicating that oven cooking degrades DNA into small fragments
Exploiting a MPN-PCR technique to quantify Escherichia coli in minced meat
No full textMPN-culture and MPN-PCR methods were used to determine Escherichia coli concentration in 54 retail minced meat samples. The MPN-PCR is based on the combination between the conventional Most Probable Number and the Polymerase Chain Reaction technique targeting the uidA gene. The conventional MPN-culture technique revealed about 94% of the samples to be positive for E. coli, while MPN-PCR showed only 87%; this difference in detection can be attributed to different factors. MPN-PCR showed values of relative accuracy, sensitivity and specificity of, respectively, 85,8%, 81,6% and 93,5%, and the analysis took only one day, compared with the 4-6 days needed using MPN-culture. The alternative method is reliable when microrganisms are present at low concentration levels
Isolation and characterization of a virulent Lactobacillus sanfranciscensis bacteriophage and its impact on microbial population in sourdough
No full textThirty-five sourdough samples used for sweet and salted Italian baked products were checked for the presence of a virus active on Lactobacillus sanfranciscensis species. One phage, named EV3, was isolated and its phenotypic and genotypic features were investigated. It belonged to the Siphoviridae family (morphotype B1); its life cycle at 25°C lasted 3 hours with a burst size of about 30 viral particles per infected cell. SDS-PAGE showed one major structural protein of 35 kDa and four minor proteins. The genome, approximately 32 kb long, was a double stranded linear DNA molecule with a pac-type system. Phage spreading into sourdough did not adversely affect acidification and volume increase of the dough neither lactobacilli counts; the propagation of viral particles showed to be hindered. This is the first report of the isolation of a L. sanfranciscensis phage
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