Luciano Beneduce

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines.

Aims:  To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach.

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

Aims: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese.

Biogenic amine in wines

Biogenic amines (BA) are a group of organic nitrogenous compounds formed and degraded by the metabolism of living organisms (microorganisms, plants and animals). The main BA associated with wine are putrescine, histamine, tyramine and cadaverine, followed by phenylethylamine, spermidine, spermine, agmatine and tryptamine. The variability in the BA content of wine could be explained on the basis of differences in the winemaking process, time and storage conditions, raw material quality, and possible microbial contamination during winery operations. BA are formed by decarboxylation of the corresponding amino acids by microorganisms through substrate-specific decarboxylase enzymes. This property is usually strain dependent. Decarboxylase enzymes are generally induced at acidic pH and therefore they have a possible role in maintaining pH homeostasis or extending the microbial growth period by detoxification of the extracellular medium. The presence of these compounds is considered by some authors a fundamental parameter for the detriment of wine.

Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine

Aims:  The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway.

Determination of biogenic amines in wine by thin-layer chromatography/densitometry

Biogenic amines are small-molecular-weight organic bases that can be encountered in all fermented foods, including wine. Ingestion of wine containing biogenic amines, and especially histamine, can result in health nuisances. HPLC is the analytical technique most often employed in the determination of biogenic amines in wine but HPLC-based methods are expensive and time-consuming. A new method, based upon amine dansylation and TLC/densitometry, was developed and validated. This allowed for the determination of histamine, tyramine, putrescine and cadaverine in wine at concentrations between 1 and 20mg/L. Analytical performances adequately complied with the needs of routine wine analysis, moreover the method was high-throughput and inexpensive. A simpler, semi-quantitative version of the method, based on visual evaluation of spot intensity, was also developed.

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine.

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.

Selection of yeasts as starter cultures for table olives.

Ninety-nine yeasts were isolated from Bella di Cerignola table olives; first, the strains were studied in relation to their ability to produce biogenic amines in a laboratory medium and 49 strains were positive to this assay and cut off from the research. The remaining 50 strains were characterized for their enzymatic traits (β-glucosidase, catalase, pectolytic, xylanolytic, and lipolytic activities) and for their ability to grow at different temperatures, pHs, with salt or lactic/acetic acids added. Data were used for the evaluation of growth index and submitted to cluster and principal component analyses to choose the most promising 4 strains. In the final step of the research, the strains were inoculated as a cocktail in a model brine, containing different amounts of salt (4% to 12%) and glucose (0% to 3%), and adjusted to different pHs (4.0 to 9.0). Data analysis through a multiple regression procedure highlighted that salt, glucose, and pH acted in a different way within the storage and NaCl affected yeast growth only for few days, and then glucose and pH played a major role.

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR.

Aims: Physiological and molecular analysis such as PCR species‐specific and randomly amplified polymorphic PCR (RAPD‐PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. 
 Methods and Results: Phenotypic tests such as API 50CH and evaluation of D‐L‐lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species‐specific oligonucleotides based on short sequences of the recA gene. 
 Conclusions: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD‐PCR. The RAPD‐PCR profiles were similar in all strains that had positive results for species‐specific PCR, suggesting that the four L. plantarum strains were closely related. 
 Significance and Impact of the study: Using PCR species‐specific as a preliminary screening test and then RAPD‐PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.

A Fast, Reliable, and Sensitive Method for Detection and Quantification of Listeria monocytogenes and Escherichia coli O157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR

In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

Aims:  Developing and evaluating a rapid real‐time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre‐enrichment step before DNA extraction.