S. Massa

Environmental Stress Response in Wine Lactic Acid Bacteria: Beyond Bacillus subtilis

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are traditionally used to produce fermented foods. The industrialization of food transformations has increased the economical importance of LAB, as they play a crucial role in the development of the organoleptic and hygienic quality of fermented products. However, the strains selected for industrial purposes, should tolerate adverse conditions encountered in industrial processes, either during starter handling and storage (freeze-drying, freezing, or spray-drying) or during food processing in which abiotic stresses such as heat, cold, acidity, and high concentration of NaCl or ethanol are common. Wine LAB have to deal with several stresses including an acidic pH, a high alcoholic content, non optimal growth temperatures, and growth-inhibitory compounds such as fatty acids and tannins, originated from yeast and bacteria metabolism. Wine LAB have developed several mechanisms to escape or to tolerate wine conditions. They carry out a malolactic fermentation in this stressful environment. In addition to the regulation of the expression of specific genes, bacteria have evolved adaptive networks to face the challenges of a changing environment and to survive under conditions of stress. The so called Global Regulatory Systems control the simultaneous expression of a large number of genes in response to a variety of environmental stress factors. CIRCE sequences able to bind the HrcA repressor, σB dependent promoters and CtsR regulatory elements have been observed in several genes identified from wine LAB. Improved knowledge of regulators and a better understanding of LAB stress responses could constitute a basis of comparison with the well known model microorganisms, Escherichia coli and Bacillus subtilis. Moreover, it can provide an important insight into improving current industrial starter strains.

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.

A microbiological investigation on probiotic pharmaceutical products used for human health

Many and different probiotic pharmaceutical products are presently commercialised in the world. On this regard, a microbiological investigation was carried out to screen the microorganisms incorporated into these products, commonly used for human health. After determination of the cell number and viability of bacteria, several experiments were performed in vitro in order to characterise the microorganisms and to evaluate their probiotic value. Among all the strains identified, best results were obtained with Lactobacillus rhamnosus, Enterococcus faecium and Saccharomyces cerevisiae as far as regards growth rates, pH and bile salts tolerance. Moreover, the identification profiles of microorganisms showed a better reliability for the products containing a single species whereas the ones composed of different strains were usually not satisfactory. In some cases, the presence of Lactobacillus and Saccharomyces species was in disagreement with the claimed composition of the product and some species of lactobacilli, bifidobacteria and streptococci were found not viable. In defined mixed cultures experiments, the antagonism of Lactobacillus acidophilus and Enterococcus faecium versus Yersinia enterocolitica was demonstrated and explained as acid and/or antimicrobials production.

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.

Fate of Escherichia coli O157 : H7 in unpasteurized milk stored at 8 °C

Escherichia coli O157 : H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157 : H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 °C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from <2 log to 3 log cfu ml−1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157 : H7 in raw milk when stored at 8 °C and reaffirm the need for pasteurization and holding the milk at ≤5 °C.

Cloning, molecular characterization and expression analysis of two small heat shock genes isolated from wine Lactobacillus plantarum.

Aim:  Understanding the molecular response to stress tolerance of wine Lactobacillus plantarum.

In Vivo PCR-DGGE Analysis of Lactobacillus plantarum and Oenococcus oeni Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.

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.

Drug resistant bacteria in non carbonated mineral waters

The presence of antibiotic resistant bacteria was revealed among bacteria isolated from non carbonated mineral waters bottled in plastic (PVC) and in glass containers. Heterotrophic plate count values ranged between < 10 and 4.3 x 10(3) and between < 10 and 1.2 x 10(4) colony forming units/ml for the waters bottled in PVC and glass, respectively. The greatest resistance to a single antibiotic, 39.1% of 320 isolates from mineral waters, was found for nalidixic acid. Resistance to the other antibiotics was as follows: ampicillin (26.2%), bacitracin (19.7%), cotrimoxazole (18.7%), streptomycin (15.0%), tetracycline (14.4%), gentamycin (11.6%), chloramphenicol and rifampin (9.7%). The strains resistant to two or more antibiotics (multiple antibiotic resistant, MAR) provided 51% of the total isolates. Identification of 127 MAR strains showed that in the mineral waters gram-positive cocci dominated. The second, third and fourth group of identified MAR phenotypes were, in order to importance, gram-negative non-fermentative rods, gram-positive rods and gram-negative fermentative rods. The importance of the antibiotic resistant bacteria in mineral water is discussed.

Fresh-Cut Pineapple as a New Carrier of Probiotic Lactic Acid Bacteria

Due to the increasing interest for healthy foods, the feasibility of using fresh-cut fruits to vehicle probiotic microorganisms is arising scientific interest. With this aim, the survival of probiotic lactic acid bacteria, belonging to Lactobacillus plantarum and Lactobacillus fermentum species, was monitored on artificially inoculated pineapple pieces throughout storage. The main nutritional, physicochemical, and sensorial parameters of minimally processed pineapples were monitored. Finally, probiotic Lactobacillus were further investigated for their antagonistic effect against Listeria monocytogenes and Escherichia coli O157:H7 on pineapple plugs. Our results show that at eight days of storage, the concentration of L. plantarum and L. fermentum on pineapples pieces ranged between 7.3 and 6.3 log cfu g−1, respectively, without affecting the final quality of the fresh-cut pineapple. The antagonistic assays indicated that L. plantarum was able to inhibit the growth of both pathogens, while L. fermentum was effective only against L. monocytogenes. This study suggests that both L. plantarum and L. fermentum could be successfully applied during processing of fresh-cut pineapples, contributing at the same time to inducing a protective effect against relevant foodborne pathogens.