Giovanni Salzano

Formation of biogenic amines as criteria for the selection of wine yeasts

This work deals with biogenic amine production by yeast strains isolated from grapes and wines. A total of 50 strains were tested for their capacity to produce biogenic amines in wine. In general, all the species produced very low or non-detectable amounts of histamine, whereas methylamine and agmatine were formed by all the species considered. The highest concentration of total biogenic amines was formed by Brettanomyces bruxellensis, with an average value of 15 mg/l, followed by Saccharomyces cerevisiae with an average of 12.14 mg/l. The other species formed less than 10 mg of total biogenic amines per litre. Wines fermented with the most fermentative strains of S. cerevisiae species had the highest contents of ethanolamine, from 2.3 to 16 mg/l, and of agmatine, from 3.1 to 7.5 mg/l. The strains of the other species, which exhibited a low fermentative ability, Kloeckera apiculata, B. bruxellensis and Metschnikowia pulcherrima, varied in the production of agmatine and phenylethylamine. A significant variability in the production of cadaverine was characteristic of Candida stellata strains, which varied also in ethanolamine production. Our results emphasize the importance of using selected strains of S. cerevisiae, not only for the expression of desirable technological traits, but also to avoid potentially negative effects on human health. Therefore, the characterization of strains of S. cerevisiae for the production of biogenic amines becomes of applicative interest.

Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy)

Lactic acid bacteria (LAB) from traditional fermented sausages of the Basilicata region were investigated by ARDRA-PCR and RAPD-PCR for taxonomic identification at species and strain level and characterized on the basis of the growth and acidification at different temperatures, incubation times, levels of NaCl and KNO(2), hydrolysis of sarcoplasmatic and myofibrillar proteins and antimicrobial, peptide/amino acid release and nitrate reductase activities. Lactobacillus sakei was the predominant species (67%) followed by Pediococcus pentosaceus (16%), Leuconostoc carnosum (8%), Lactobacillus plantarum (4%), Lactobacillus brevis (2%) and Leuconostoc pseudomesenteroides (2%). The technological characterization revealed that most of the isolates had good acidifying and proteolytic properties. Moreover, Lb. sakei strains showed antimicrobial ability, while Leuconostoc strains the highest reduction of nitrates. This work was a preliminary study in the formulation of autochthonous starter cultures in order to standardize the production process of sausages, to preserve their typical organoleptic and sensory characteristics and to improve the quality of final product.

Technological and safety characterization of coagulase-negative staphylococci from traditionally fermented sausages of Basilicata region (Southern Italy).

Thirty-seven strains of coagulase-negative staphylococci, isolated from traditional fermented sausages of Basilicata region, were screened on the basis of their technological properties (nitrate reductase, proteolytic, lipolytic, amino acid-decarboxylase and antimicrobial activities) and for their ability to grow in the presence of different salt concentrations, temperatures and pH values, to elucidate their possible role during meat fermentation process. All strains grew at 20°C and 30°C in all conditions, while at 10°C, only few isolates showed a good growth at pH values ranging from 6 to 5.2. Staphylococci strains had different enzymatic profiles. A significant level of proteolytic activity was observed in 75.6% of the strains and 23 staphylococci hydrolysed sarcoplasmatic proteins. Nitrate was reduced by 62% of the strains, while most of isolates (80.9%) were able to decarboxylate at least one of the tested amino acids. This is a preliminary study focused on the selection of autochthonous starter cultures to standardize the production of fermented sausages, to preserve their organoleptic and sensory properties and to improve the quality of final product.

Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper content in wine

Two wine strains of Saccharomyces cerevisiae, characterized by a different degree of copper resistance, were tested in grape must fermentation in the presence of different copper concentrations. The sensitive strain SN9 was strongly affected by copper concentration (32 ppm, (32 mg/l)), whereas the resistant strain SN41 exhibited a good growth activity in presence of 32 ppm of copper and only a reduced activity in presence of 320 ppm. The different strain fermentation performance in response to the copper addition corresponded to a different capability to accumulate copper inside the cells. Both strains exhibited the capacity to reduce the copper content in the final product, eventhough a significantly greater reducing activity was exerted by the resistant strain SN41, which was able to reduce by 90% the copper concentration in the final product and to accumulate the metal in great concentrations in the cells. As high concentrations of copper can be responsible for wine alterations, the selection of S. cerevisiae strains possessing high copper resistance and the ability to reduce the copper content of wine has a great technological interest, in particular for the fermentation of biological products. From the results obtained, the technique proposed is not only suitable for the assay of copper residues in must, wine and yeast cells, but it also offers the advantage of easy sample preparation and low detection limit in the ppb (μg/l) range.

Adaptive changes in geranylgeranyl pyrophosphate synthase gene expression level under ethanol stress conditions in Oenococcus oeni

The aim of this study was to investigate the effect of ethanol exposure on the expression level of geranylgeranyl pyrophosphate synthase gene involved in the metabolism of Oenococcus oeni to probe the mechanisms of ethanol tolerance correlated with adaptive changes.

Automated multiple development method for determination of glycerol produced by wine yeasts

A rapid and efficient analytical method for the determination of glycerol in wines is described. This method utilizes high-performance thin layer chromatography (HPTLC) plates coupled with an automated multiple development system with an elution gradient based on acetonitrile–acetone–hexane on silica gel layers. The absence of clean-up procedures, sometimes only centrifugation, makes this method suitable also for the large-scale control of alcoholic beverages. In particular the capacity of different wine yeast species (Saccharomyces cerevisiae, Zygosaccharomyces bailii, Kloeckera apiculata and Saccharomycodes ludwigii) to produce glycerol was determined. Generally, the strains of S. cerevisiae produced elevated amounts of glycerol together with Z. bailii, whereas K. apiculata strains formed the lowest amounts of glycerol, exhibiting also a great strain variability.

Fingerprinting analysis of Oenococcus oeni strains under stress conditions

Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12-13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 degrees C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.

Genotypic and technological diversity of Leuconostoc mesenteroides and Lactobacillus paracasei subsp. paracasei strains for use as adjunct starter cultures in Pecorino di Filiano cheese

Seventeen Leuconostoc mesenteroides and 33 Lactobacillus paracasei subsp. paracasei from traditional Pecorino di Filiano cheese were tested for their potential use as adjunct starters, and a study of their genetic variability was carried out. Forty one per cent (41%) of Leu.xa0mesenteroides and 46% of Lb.xa0paracasei subsp. paracasei showed medium–high proteolytic activity, while high lipolytic activity was detected in 18% of Lb.xa0paracasei subsp. paracasei. The aminopeptidase activity of Lb.xa0paracasei subsp. paracasei was higher than that of Leu.xa0mesenteroides. Strain diversity by RAPD analysis showed a high degree of heterogeneity. This study identified strains with unusual properties that could be good candidates as adjuncts in a starter to manufacture PF cheese.

Assessment of the genetic polymorphism and physiological characterization of indigenous Oenococcus oeni strains isolated from Aglianico del Vulture red wine

The aim of this study was a reliable intra-species discrimination and strain biodiversity in Oenococcus oeni populations of two different Aglianico wineries by molecular, biochemical, and physiological characterization. Pulsed field gel electrophoresis (PFGE) analysis revealed a high polymorphism related to the origin (winery) of strains, while differential display PCR (DD-PCR) allowed a further discrimination of strains from the same winery. Moreover, the heterogeneity of these natural populations was investigated by capillary electrophoresis and enzymatic assays. A variability related to a different surface charge distribution was observed among strains, linked to their origin. Malolactic activity study evidenced strain-specific differences in malic acid degradation, and then, only the presence of L(-)-malic acid in the medium induced the mle gene. This study provided evidences on the importance of intra-species biodiversity of malolactic bacterial populations in wine ecosystems, as each wine possess peculiar winemaking conditions and physical–chemical properties which make specific the bacterial survival and growth. This study highlighted a great biodiversity among O. oeni strains that can be also winery specific. Such biodiversity within a certain winery and winemaking area is important for selecting malolactic starters, and strain-specific trait identification is especially important to match individual strains to specific industrial process.

Stress response assessment of Lactobacillus sakei strains selected as potential autochthonous starter cultures by flow cytometry and nucleic acid double-staining analyses.

The aim of this study was to apply the flow cytometry to Lactobacillus sakei strains, selected as potential autochthonous starters, to investigate dynamics and physiological heterogeneity of microbial behaviour under different stress conditions.