Influence of sequential inoculum of Starmerella bacillaris and Saccharomyces cerevisiae on flavonoid composition of monovarietal Sangiovese wines.

The selection of Starmerella bacillaris strains to be used with Saccharomyces cerevisiae as mixed cultures has been recently suggested in order to produce wines containing lower ethanol and higher glycerol concentrations and to promote fructose degradation due to their fructophilic character. However, studies about effects of such mixed starter cultures on phenolic compounds, which are responsible for the colour and health-enhancing properties in red wines, are currently lacking. Therefore, in this work, the influence of sequential inoculated fermentation (SIF) with Starm. bacillaris and S. cerevisiae on phenolic content of monovarietal Sangiovese wine was evaluated by fermentations at laboratory scale. Axenic fermentations (AXFs) with S. cerevisiae were performed as control. S. cerevisiae attained higher cell densities in AXF compared with SIF. The experimental wines obtained by SIF showed significant lower ethanol and higher glycerol concentrations, whereas no significant difference was detected in colour intensity. The total phenol index reached significantly lower values in SIF. Furthermore, the wines produced by SIF contained higher concentrations of vitisin A that has a greater colour stability than the anthocyanin monomer. Finally, a lower content of both free anthocyanins and flavan-3-ols, key compounds for wine quality possessing also health-enhancing properties, was found in wines obtained by SIF. On the contrary, no significant difference was detected on flavonol concentration between SIF and AXF. This study highlighted that the use of sequential inoculum of Starm. bacillaris and S. cerevisiae can contribute to increasing the colour stability of red wines, even if at the expense of compounds with health properties.

Enumeration and rapid identification of yeasts during extraction processes of extra virgin olive oil in Tuscany.

The aim of this study was to evaluate the occurrence of yeast populations during different olive oil extraction processes, carried out in three consecutive years in Tuscany (Italy), by analysing crushed pastes, kneaded pastes, oil from decanter and pomaces. The results showed yeast concentrations ranging between 10(3) and 10(5) CFU/g or per mL. Seventeen dominant yeast species were identified by random amplified polymorphic DNA with primer M13 and their identification was confirmed by restriction fragments length polymorphism of ribosomal internal transcribed spacer and sequencing rRNA genes. The isolation frequencies of each species in the collected samples pointed out that the occurrence of the various yeast species in olive oil extraction process was dependent not only on the yeasts contaminating the olives but also on the yeasts colonizing the plant for oil extraction. In fact, eleven dominant yeast species were detected from the washed olives, but only three of them were also found in oil samples at significant isolation frequency. On the contrary, the most abundant species in oil samples, Yamadazyma terventina, did not occur in washed olive samples. These findings suggest a phenomenon of contamination of the plant for oil extraction that selects some yeast species that could affect the quality of olive oil.

Management of in-Amphora "Trebbiano Toscano" Wine Production: Selection of Indigenous Saccharomyces cerevisiae Strains and Influence on the Phenolic and Sensory Profile.

The use of earthenware amphorae in winemaking can give wines unique attributes enhancing their typicity. Therefore, in this study, spontaneous and inoculated in-amphora fermentations of Trebbiano Toscano grape must were monitored to assess the Saccharomyces cerevisiae strains occurring in each fermentation as well as the chemical characteristics of the wines. Strain typing via Interdelta analyses pointed out that the commercial starters did not dominate, showing 24% and 13% implantation percentages, and that 20 indigenous strains were present at different percentages, ranging from 2 to 20%, in inoculated and spontaneous fermentations. The assessment of the technical characteristics of the indigenous strains via fermentations at lab and pilot scale (20 L amphorae) and the sensory analysis of the experimental wines allowed for the selection of two indigenous strains to be used as starter cultures in comparison to a commercial strain in 300-L-amphorae vinifications in the cellar. The observed fermentative performances and sensory analysis of the experimental wines highlighted that one indigenous S. cerevisiae strain dominated the process and conferred distinctive sensory characteristics to the Trebbiano Toscano wine, demonstrating its effectiveness in managing the in-amphora fermentations. In addition, the results demonstrated the ability of amphorae to protect the polyphenolic compounds from oxidation during wine ageing. Indeed, the concentration of both hydroxycinnamic acids and flavonols decreased, with an average reduction of 30% and 14%, respectively, while hydroxybenzoic acids remained unchanged.

Exploitation of Selected Sourdough Saccharomyces cerevisiae Strains for the Production of a Craft Raspberry Fruit Beer.

Recent interest in the special beer category has encouraged the search for novel brewing materials, including new ingredients and novel yeast strains, in order to differentiate the finished products. The aim of this work was to select non-brewing S. cerevisiae strains for the production of a fruit beer with raspberry. The in vitro tests and the wort fermentations allowed the selection of two sourdough S. cerevisiae strains, showing high maltose and maltotriose consumption, high ethanol production, and high viability. Fruit beers (FB) and control beers (CB) without raspberries were prepared. Fruit addition accelerated sugar consumption (7 days compared to 13 days) and increased ethanol and glycerol production by yeasts. Raspberry addition and the inoculated yeast strongly affected the aroma profile of beers. FB samples showed a higher amount of volatile organic compounds (VOCs); the most represented classes were alcohols, followed by esters and acids. FB inoculated by the selected S. cerevisiae SD12 showed the highest VOCs concentration (507.33 mg/L). Results highlighted the possible application of sourdough yeast strains for the brewing process, which, combined with raspberry addition, can be exploited for the production of beers with enhanced aromatic features and suitable chemical properties.

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.

Antioxidant Properties of Sourdoughs Made with Whole Grain Flours of Hull-Less Barley or Conventional and Pigmented Wheat and by Selected Lactobacilli Strains.

The use of sourdough fermentation and whole grain flours in baked goods manufacturing are known to enhance their functional and nutritional features. In this context, it is necessary to select the most suitable lactic acid bacteria strains and flour combination to achieve this goal. A characterization of 70 lactobacilli strains based on pro-technological and nutritional properties was carried out. The screening allowed the selection of 10 strains that were used to ferment sourdoughs made with two varieties of common wheat, the conventional red-grained cv Aubusson, a blue-grained variety rich in anthocyanins cv Skorpion, and a hull-less barley variety, cv Rondo. From each fermented sourdough, a water soluble extract was obtained and evaluated for its antioxidant activity performed on cultured cells (RAW 264.7 murine macrophage) by assaying Reactive Oxygen Species (ROS) content. Sourdoughs made with pigmented wheat and barley, had an antioxidant activity greater than that recovered in those made with conventional wheat flour, in spite they have been inoculated with the same LAB strains. Results highlighted the interdependence between flour and the inoculated lactic acid bacteria that has to be taken into account for the development of healthy breads exploiting high functional value cereals through biotechnological processes.

Antioxidant and anti-inflammatory properties of sourdoughs containing selected Lactobacilli strains are retained in breads.

Sourdough fermentation influences several properties of leavened baked goods also because Lactic acid bacteria (LAB) and yeasts produce bioactive peptides with a positive effect on human health. In an early study, three Lactobacilli strains (L. farciminis H3 and A11 and L. sanfranciscensis I4) possessing different proteolytic activities were used to produce sourdoughs containing peptides equipped with anti-inflammatory and/or antioxidant properties. This work was aimed to assess whether these properties could be retained after cooking. The selected LABs were used to produce breads from which low molecular weight (LMW-) peptides were extracted. The results provide solid proofs of keeping both antioxidant and anti-inflammatory activities of peptides from cooked products. Sequences of LMW-peptides either from doughs and breads were determined by mass spectrometry: differences have been noticed in amino acidic composition and in sequences, however, all the strains produce peptides equipped with antioxidant and anti-inflammatory activities.

Extra Virgin Olive Oil Quality as Affected by Yeast Species Occurring in the Extraction Process.

In extra virgin olive oil (EVOO) extraction process, the occurrence of yeasts that could affect the quality of olive oil was demonstrated. Therefore, in this work, at first, the yeasts occurring during different extractive processes carried out in a Tuscany oil mill, at the beginning, in the middle, and the end of the harvesting in the same crop season, were quantified. Then, possible effects on quality of EVOO caused by the predominant yeast species, possessing specific enzymatic activities, were evaluated. Yeast concentrations were higher in extraction processes at the end of the harvesting. Twelve yeast species showing different isolation frequencies during olive oil extractive process and according to the harvesting date were identified by molecular methods. The yeast species dominating olive oil samples from decanter displayed enzymatic activities, potentially affecting EVOO quality according to zymogram analysis. HS-SPME-GC-MS analysis of the volatile compounds in commercial EVOO, inoculated with three yeast species (Nakazawaea molendini-olei, Nakazawaea wickerhamii, Yamadazyma terventina), pointed out significant differences depending on the strain inoculated. In conclusion, during the olive oil extractive processes, some yeast species colonize the extraction plant and may influence the chemical and sensory characteristics of EVOO depending on the cell concentrations and their enzymatic capabilities.

Use of Selected Lactobacilli to Increase γ-Aminobutyric Acid (GABA) Content in Sourdough Bread Enriched with Amaranth Flour.

γ-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter of the central nervous system and possesses various physiological functions. GABA production can be obtained thanks to lactic acid bacteria activity in different foods such as sourdoughs. Recently, breads made from blends of pseudocereals and wheat flours have attracted much attention. Amaranth is especially interesting because of its high nutritional value, having a high protein content and containing different antioxidant compounds. Therefore, this study aimed to obtain sourdough breads enriched with GABA thanks to bacterial activity and to investigate the effect of amaranth flour addition on the antioxidant and sensorial properties of bread. Eighteen lactobacilli strains were assayed for GABA production in amaranth and wheat flour liquid sourdoughs. Two strains, Lactobacillus brevis A7 and Lactobacillus farciminis A11, demonstrated high GABA producing capability; hence, they were used to prepare breads containing 20% amaranth flour. The results confirmed the capability of the two strains to increase GABA concentrations (up to 39 mg/kg) in breads. Samples with amaranth addition showed a significantly higher total phenolic content compared to the control bread (more than 15 mg GAE 100 g-1 dwb); sensory analysis showed that breads with amaranth were moderately acceptable. Nevertheless, their general liking evaluation was significantly lower compared to the control bread. The addition of pseudocereal to traditional wheat sourdough and selection of lactobacilli allowed the production of baked goods with enhanced GABA content and antioxidant capacity, but recipes have to be developed to increase the organoleptic acceptability of the final products.

Quantifying the Effects of Ethanol and Temperature on the Fitness Advantage of Predominant Saccharomyces cerevisiae Strains Occurring in Spontaneous Wine Fermentations.

Different Saccharomyces cerevisiae strains are simultaneously or in succession involved in spontaneous wine fermentations. In general, few strains occur at percentages higher than 50% of the total yeast isolates (predominant strains), while a variable number of other strains are present at percentages much lower (secondary strains). Since S. cerevisiae strains participating in alcoholic fermentations may differently affect the chemical and sensory qualities of resulting wines, it is of great importance to assess whether the predominant strains possess a "dominant character." Therefore, the aim of this study was to investigate whether the predominance of some S. cerevisiae strains results from a better adaptation capability (fitness advantage) to the main stress factors of oenological interest: ethanol and temperature. Predominant and secondary S. cerevisiae strains from different wineries were used to evaluate the individual effect of increasing ethanol concentrations (0-3-5 and 7% v/v) as well as the combined effects of different ethanol concentrations (0-3-5 and 7% v/v) at different temperature (25-30 and 35°C) on yeast growth. For all the assays, the lag phase period, the maximum specific growth rate (µmax) and the maximum cell densities were estimated. In addition, the fitness advantage between the predominant and secondary strains was calculated. The findings pointed out that all the predominant strains showed significantly higher µmax and/or lower lag phase values at all tested conditions. Hence, S. cerevisiae strains that occur at higher percentages in spontaneous alcoholic fermentations are more competitive, possibly because of their higher capability to fit the progressively changing environmental conditions in terms of ethanol concentrations and temperature.

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-1ß 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.

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.

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.

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.

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.

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.

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.