Simona Guerrini

Biogenic amine production by Oenococcus oeni.

The biogenic amine-producing capability of several Oenococcus oeni strains, originally isolated from different Italian wines, was determined. The amine-producing capability was quali-quantitatively variable among the strains: out of the 44 strains investigated under optimal growth conditions, more than 60% were able to produce histamine, at concentrations ranging from 1.0 to 33 mg/L, and about 16% showed the additional capability to form both putrescine and cadaverine, to different extents and variable relative proportions. The amine-producing behavior of the strains was confirmed under stress culture conditions, while performing malolactic fermentation. In wine, one randomly chosen strain was very effective in forming putrescine from ornithine. The formation of putrescine from arginine by some strains has been also demonstrated. Consequently, O. oeni can really and significantly contribute to the overall biogenic amine content of wines. Practical consequences of these findings are discussed.

Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines

A phenotypic and genotypic characterization of 84 Oenococcus oeni isolates from Italian wines of different oenological areas was carried out. Numerical analysis of fatty acid profiles grouped the isolates into two clusters at low level of similarity (63%), the minor cluster containing seven isolates besides the type and the reference strains. Forthy-eight O. oeni isolates, representative of the two clusters, showed no differences in their metabolic properties (heterolactic fermentation pattern, citrate degradation capability and formation of some secondary metabolites). Moreover, the analysis of species-specific randomly amplified polymorphic DNA and 16S-23S rDNA intergenic spacer region polymorphism as well as the sequence-specific separation of V3 region from 16S rDNA by denaturing gradient gel electrophoresis demonstrated a substantial homogeneity among the isolates. On the basis of ApaI Pulsed Field Gel Electrophoresis (PFGE) restriction patterns, the 84 isolates were grouped into five different clusters at 70% similarity, but no correlation with the phenotypic groups could be demonstrated. However, by combining phenotypic and genotypic data, the 84 O. oeni isolates grouped into eight phenotypic-genotypic combined profiles and a relationship between the origin of the isolates and their combined profile became evident, so that a sort of strain specificity can be envisaged for each wine-producing area.

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.

Stable and non-competitive association of Saccharomyces cerevisiae, Candida milleri and Lactobacillus sanfranciscensis during manufacture of two traditional sourdough baked goods

The microbiota occurring in all the manufacturing phases of two Italian sourdough sweet-leavened baked goods (a typical Genoese dry biscuit, Lagaccio, and a soft stuffed North Italian typical cake, Panettone) were investigated over a period of three years. The two sourdough mother sponges were characterized by the stable presence of three dominant microbial species in potential competition for carbohydrates: Lactobacillus sanfranciscensis, Candida milleri, and Saccharomyces cerevisiae. Genotypic and phenotypic characterizations of microbial isolates pointed out that each mother sponge harbored its own strains, well distinguishable by molecular methods of analysis but not differing in their main metabolic properties from those known for the corresponding species. The microbial and biochemical evolution during the whole production protocol of both manufactures demonstrated that the three microbial species grew at almost the same growth rates, without exhausting any of the main carbon substrates (maltose, glucose and fructose). The quite similar growth dynamics under practical conditions and the constant presence of all fermentable carbohydrates were recognized as responsible for the stable non competitive association of maltose-positive and maltose-negative species in both sourdoughs. However, the two sourdoughs were characterized by quite different LAB to yeast ratio, with values significantly higher in Panettone than in Lagaccio. The cause of this difference could mainly be ascribed to the temperature of the mother sponge regeneration phase, that, in the case of Panettone manufacture, occurred under conditions of moderate refrigeration.

Effect of Oleic Acid on Oenococcus oeni Strains and Malolactic Fermentation in Wine

A different capability to assimilate oleic acid from the culture medium has been demonstrated among malolactic Oenococcus oeni strains. Strains possessing higher percentages of oleic acid and its methylated derivative, dihydrosterculic acid, in their fatty acid profile showed higher cell viability and carried out a complete malolactic fermentation after their transfer into a wine lacking oleic acid. Wine supplementation with Tween 80 (polyoxyethylene-sorbitan-mono-oleate) enhanced cell survival of strains with lower capability to assimilate oleic acid and caused cell growth of strains with higher assimilative capacity, suggesting that oleic acid may act in wine as a survival factor for the former strains and as a growth factor for the latter strains. Practical consequences of these findings are also discussed.

Typing of Lactobacillus sanfranciscensis isolates from traditional sourdoughs by combining conventional and multiplex RAPD-PCR profiles.

In the present work, a rapid and reproducible molecular method, based on the combination of conventional and multiplex RAPD-PCR reactions, was developed for typing Lactobacillus sanfranciscensis isolates from traditional sourdoughs. At first, four random primers, two used singly and two combined with the primer RD1, were chosen on the basis of their differentiating capability and reproducibility. The four resulting profiles for each isolate were integrated into a unique profile to be statistically treated by cluster analysis. The method was validated on 58 L. sanfranciscensis isolates coming from three traditional Italian sourdoughs. This new RAPD method was useful for determining the genomic diversity within the L. sanfranciscensis species. In particular, the intraspecific diversity of this species seemed to be related to the sourdough origin.

Amino Acid Metabolisms and Production of Biogenic Amines and Ethyl Carbamate

In winemaking process, a wide range of volatile and non-volatile compounds originate from microbial catabolism of amino acids. Among these catabolites, biogenic amines, low molecular weight organic bases produced by decarboxylation of their respective free precursor amino acids, are receiving much attention in wine science because of their potential implication for human health. This chapter, after a brief overview on the occurrence of biogenic amines in wines, deals with the role played by yeasts and lactic acid bacteria in the formation and accumulation of these molecules during winemaking, giving emphasis to the most frequently found amines (histamine, tyramine and putrescine) and their physiological significance in bacterial cells. Moreover, the most suitable methods to detect biogenic amine-producing lactic acid bacteria or to quantify biogenic amine in wine as well as strategies to reduce biogenic amine content in wine are reported. Finally, a note on the formation of ethyl carbamate, a carcinogen compound originating in wine through a non-enzymatic reaction between ethanol and microbial catabolites containing a carbamyl group, is furnished.

Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions

Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area.

Biogenic amine producing capability of bacterial populations isolated during processing of different types of dry fermented sausages

Abstract In order to assess the distribution of the biogenic amine (BA) producing capability within the bacterial populations occurring during production of dry fermented sausages, four different types of sausage processing, three with the use of starter cultures and one without, were investigated. All the main bacterial populations involved in the sausage processing showed a diffuse and strain dependent capability to produce BAs. However, quantitative determination of individual BAs produced by the bacterial isolates suggests an important role of enterococci in the accumulation of tyramine, the most abundant biogenic amine found in all investigated sausages.

Effect of selected strains of lactobacilli on the antioxidant and anti-inflammatory properties of sourdough

Sourdough fermentation of cereal foods is an excellent source of obtaining peptides due to the ability of lactic acid bacteria to activate cereal proteases and produce strain-specific peptidases. With the aim of identifying the lactic acid bacterial strains potentially most effective in producing bioactive peptides, 131 lactobacilli isolates from Italian sourdoughs, used in baking technology, have been screened for proteolytic and peptidase activity. Of these, 23 strains were selected and singly inoculated in liquid sourdoughs from which a Low Molecular Weight fraction containing peptides was obtained. Evaluation of the antioxidant and anti-inflammatory activities of the extracts was performed on cultured cells (RAW 264.7 murine macrophage, murine H-end endothelium cells and Human intestinal Caco-2 cells) by assaying Reactive Oxygen Species (ROS) content, NFkB/IkB expression level and Interleukin-1β production. As a result, three lactobacilli strains showed a high antioxidant and anti-inflammatory ability enabling the development of model sourdoughs that will potentially increase the nutritional benefits of bread.