<i>Tetrapisispora phaffii</i> killer toxin is a highly specific β-glucanase that disrupts the integrity of the yeast cell wall

Background. Killer yeasts have been used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and in the medical field, to develop novel antimycotics for the treatment of human and animal fungal infections. Among these killer yeasts, Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) secretes a glycoprotein known as Kpkt that is lethal to spoilage yeasts under winemaking conditions. In the present study, the mode of action of Kpkt, and the specific damage produced by this toxin on sensitive yeasts is investigated. Results. The use of castanospermine, a β-glucanase inhibitor, demonstrated that β-glucanase activity is essential for the Kpkt killer activity in vivo. Accordingly, Kpkt has no killer activity on either sensitive yeast spheroplasts or whole sensitive cells in the presence of isosmothic medium (0.8 molar sorbitol). Kpkt induces ultrastructural modifications in the cell wall of sensitive strains, as shown by confocal microscopy, laser-scanning electron microscopy, and atomic force microscopy. The Kpkt killer action is mediated by the glucidic portion of the toxin. This, in turn, appears to be involved both in the stronger cytocidal activity and in the selectivity for the sensitive strain shown by Kpkt compared to laminarinase. Conclusion. Collectively, these data indicate that the mode of action of Kpkt is directed towards the disruption of cell-wall integrity, and that this is mediated by a highly specific β-glucanase activity. In this, Kpkt differs from other microbial β-glucanases that do not show killer activities

Pilot Scale Fermentations of Sangiovese: An Overview on the Impact of Saccharomyces and Non-Saccharomyces Wine Yeasts

none6openRomani, Cristina; Lencioni, Livio; Biondi Bartolini, Alessandra; Ciani, Maurizio; Mannazzu, Ilaria; Domizio, PaolaRomani, Cristina; Lencioni, Livio; Biondi Bartolini, Alessandra; Ciani, Maurizio; Mannazzu, Ilaria; Domizio, Paol

Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies.

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts

Potential spoilage non- Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae

With the aim of exploring the possibility to improve wine quality through the utilization of wine-related yeasts generally considered as spoilage, mixed cultures of Saccharomyces cerevisiae with Hanseniaspora osmophila, Pichia fermentans, Saccharomycodes ludwigii and Zygosaccharomyces bailii were inoculated in grape juice. All the fermentations got to completion and most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces yeasts, and considered detrimental for wine quality, did not reach the threshold taste level in mixed fermentations with S. cerevisiae. Interestingly, the association of S. cerevisiae with P. fermentans, S. ludwigii and Z. bailii produced significant increases in the production of polysaccharides as compared to pure cultures of S. cerevisiae. Since polysaccharides improve wine taste and body, and exert positive effects on aroma persistence and protein and tartrate stability, a possible use for these yeasts can be envisaged in mixed starter cultures with S. cerevisiae for the enhancement of the final quality of wine

Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria.

Abstract Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l−1 h−1 of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure l-lactic acid can be obtained

Occurrence of wine yeasts on grapes subjected to different pesticide treatments

As a contribution to the study and preservation of indigenous wine yeast populations, we initiated a programme of isolation and characterization of yeast strains from grapes of the Italian region of Marche. During the 1996 vintage, grape samples were collected from three groups of vineyards differing in the pesticide treatments given. Of the 279 yeasts isolated, thirty-nine were assigned to the species Saccharomyces cerevisiae. The majority of the isolates and all thirty-nine S. cerevisiae came from vineyards which had not been treated with systemic pesticides, indicating that these pesticides may exert a negative effect on the biodiversity of the grape microflora, and in particular on the occurrence of S. cerevisiae on grapes. The thirty-nine S. cerevisiae isolated were subjected to microfermentation mals, in which ten of them showed promising fermentative capabilities

Lactic acid bacteria and yeasts from wheat sourdoughs of the Marche region

The need for a greater diversification of baked products has given rise to the on-going search for yeast and lactic acid bacteria (LAB) strains with optimal baking potential. Thirty-six yeasts and 118 LAB, isolated from nine type I sourdoughs that were sampled in bakeries located in the Marche region (central Italy), were molecularly and phenotypically characterized. The polyphasic approach used revealed the biodiversity of the microbial communities investigated and two yeasts and ten LAB cultures with the potential to be used in sourdough bread-making processes were identified

Propionibacteria as promising tools for the production of pro-bioactive scotta: a proof-of-concept study

Dairy propionibacteria are Gram positive Actinomycetota, routinely utilized as starters in Swiss type cheese making and highly appreciated for their probiotic properties and health promoting effects. In this work, within the frame of a circular economy approach, 47 Propionibacterium and Acidipropionibacterium spp. were isolated from goat cheese and milk, and ewe rumen liquor, and characterized in view of their possible utilization for the production of novel pro-bioactive food and feed on scotta, a lactose rich substrate and one of the main by-products of the dairy industry. The evaluation of the Minimum Inhibitory Concentration (MIC) of 13 among the most common antibiotics in clinical practice revealed a general susceptibility to ampicillin, gentamycin, streptomycin, vancomycin, chloramphenicol, and clindamycin while confirming a lower susceptibility to aminoglycosides and ciprofloxacin. Twenty-five isolates, that proved capable of lactose utilization as the sole carbon source, were then characterized for functional and biotechnological properties. Four of them, ascribed to Propionibacterium freudenreichii species, and harboring resistance to bile salts (growth at 0.7–1.56 mM of unconjugated bile salts), acid stress (&gt;80% survival after 1 h at pH 2), osmostress (growth at up to 6.5% NaCl) and lyophilization (survival rate &gt; 80%), were selected and inoculated in scotta. On this substrate the four isolates reached cell densities ranging from 8.11 ± 0.14 to 9.45 ± 0.06 Log CFU mL−1 and proved capable of producing different vitamin B9 vitamers after 72 h incubation at 30°C. In addition, the semi-quantitative analysis following the metabolomics profiling revealed a total production of cobalamin derivatives (vitamin B12) in the range 0.49–1.31 mg L−1, thus suggesting a full activity of the corresponding biosynthetic pathways, likely involving a complex interplay between folate cycle and methylation cycle required in vitamin B12 biosynthesis. These isolates appear interesting candidates for further ad-hoc investigation regarding the production of pro-bioactive scotta

Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections

Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs