Impairment of carotenoid biosynthesis through CAR1 gene mutation results in CoQ10, sterols, and phytoene accumulation in Rhodotorula mucilaginosa.

Red yeasts, mainly included in the genera Rhodotorula, Rhodosporidiobolus, and Sporobolomyces, are renowned biocatalysts for the production of a wide range of secondary metabolites of commercial interest, among which lipids, carotenoids, and other isoprenoids. The production of all these compounds is tightly interrelated as they share acetyl-CoA and the mevalonate pathway as common intermediates. Here, T-DNA insertional mutagenesis was applied to the wild type strain C2.5t1 of Rhodotorula mucilaginosa for the isolation of albino mutants with impaired carotenoids biosynthesis. The rationale behind this approach was that a blockage in carotenoid biosynthetic pathway could divert carbon flux toward the production of lipids and/or other molecules deriving from terpenoid precursors. One characterized albino mutant, namely, strain W4, carries a T-DNA insertion in the CAR1 gene coding for phytoene desaturase. When cultured in glycerol-containing medium, W4 strain showed significant decreases in cell density and fatty acids content in respect to the wild type strain. Conversely, it reached significantly higher productions of phytoene, CoQ10, and sterols. These were supported by an increased expression of CAR2 gene that codes for phytoene synthase/lycopene cyclase. Thus, in accordance with the starting hypothesis, the impairment of carotenoids biosynthesis can be explored to pursue the biotechnological exploitation of red yeasts for enhanced production of secondary metabolites with several commercial applications. KEY POINTS: • The production of lipids, carotenoids, and other isoprenoids is tightly interrelated. • CAR1 gene mutation results in the overproduction of phytoene, CoQ10, and sterols. • Albino mutants are promising tools for the production of secondary metabolites.

The role of microorganisms on biotransformation of brewers' spent grain.

Brewers' spent grain (BSG) is the most abundant by-product of brewing. Due to its microbiological instability and high perishability, fresh BSG is currently disposed of as low-cost cattle feed. However, BSG is an appealing source of nutrients to obtain products with high added value through microbial-based transformation. As such, BSG could become a potential source of income for the brewery itself. While recent studies have covered the relevance of BSG chemical composition in detail, this review aims to underline the importance of microorganisms from the stabilization/contamination of fresh BSG to its biotechnological exploitation. Indeed, the evaluation of BSG-associated microorganisms, which include yeast, fungi, and bacteria, can allow their safe use and the best methods for their exploitation. This bibliographical examination is particularly focused on the role of microorganisms in BSG exploitation to (1) produce enzymes and metabolites of industrial interest, (2) supplement human and animal diets, and (3) improve soil fertility. Emerging safety issues in the use of BSG as a food and feed additive is also considered, particularly considering the presence of mycotoxins.Key points• Microorganisms are used to enhance brewers' spent grain nutritional value.• Knowledge of brewers' spent grain microbiota allows the reduction of health risks. Graphical abstract.

Antimicrobial activity of gaseous Citrus limon var pompia leaf essential oil against Listeria monocytogenes on ricotta salata cheese.

Contamination by Listeria monocytogenes is a particularly challenging problem in the food industry due to the ability of the bacterium to develop under conditions normally used for food preservation. Here, we show that the gaseous phase of Citrus limon var pompia leaf essential oil (hereafter PLEO) exerts specific anti-Listeria activity on ricotta salata cheese stored at 5 °C. The synergic effect of gaseous PLEO treatment and refrigeration was first confirmed in vitro on L. monocytogenes strains treated for 3 h with gaseous PLEO and then stored at 5 °C. Ricotta cheese was then inoculated with L. monocytogenes strains and subjected to hurdle technology with different concentrations of gaseous PLEO. Cell counts revealed gaseous PLEO to exert a bactericidal effect on L. monocytogenes 20600 DSMZ and a bacteriostatic effect on a mix of L. monocytogenes strains. Scanning and transmission electron microscopy analyses of L. monocytogenes cells suggested that gaseous PLEO targets the bacterial cell wall and plasma membrane. Chemical analyses of the liquid and vapor phases of PLEO indicated linalyl acetate to be the predominant compound, followed by limonene and the two isomers of citral, whereas EO composition analysis, although generally in line with previous findings, showed the presence of linalyl acetate for the first time. Solid-phase microextraction coupled with gas chromatography confirmed the presence of all crude oil components in the headspace of the box.

Yeast biofilm in food realms: occurrence and control.

In natural environments, microorganisms form microbial aggregates called biofilms able to adhere to a multitude of different surfaces. Yeasts make no exception to this rule, being able to form biofilms in a plethora of environmental niches. In food realms, yeast biofilms may cause major problems due to their alterative activities. In addition, yeast biofilms are tenacious structures difficult to eradicate or treat with the current arsenal of antifungal agents. Thus, much effort is being made to develop novel approaches to prevent and disrupt yeast biofilms, for example through the use of natural antimicrobials or small molecules with both inhibiting and dispersing properties. The aim of this review is to provide a synopsis of the most recent literature on yeast biofilms regarding: (i) biofilm formation mechanisms; (ii) occurrence in food and in food-related environments; and (iii) inhibition and dispersal using natural compounds, in particular.

An analysis of codon bias in six red yeast species.

Red yeasts, primarily species of Rhodotorula, Sporobolomyces, and other genera of Pucciniomycotina, are traditionally considered proficient systems for lipid and terpene production, and only recently have also gained consideration for the production of a wider range of molecules of biotechnological potential. Improvements of transgene delivery protocols and regulated gene expression systems have been proposed, but a dearth of information on compositional and/or structural features of genes has prevented transgene sequence optimization efforts for high expression levels. Here, the codon compositional features of genes in six red yeast species were characterized, and the impact that evolutionary forces may have played in shaping this compositional bias was dissected by using several computational approaches. Results obtained are compatible with the hypothesis that mutational bias, although playing a significant role, cannot alone explain synonymous codon usage bias of genes. Nevertheless, several lines of evidences indicated a role for translational selection in driving the synonymous codons that allow high expression efficiency. These optimal synonymous codons are identified for each of the six species analyzed. Moreover, the presence of intragenic patterns of codon usage, which are thought to facilitate polyribosome formation, was highlighted. The information presented should be taken into consideration for transgene design for optimal expression in red yeast species.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.

The administration of L-cysteine and L-arginine inhibits biofilm formation in wild-type biofilm-forming yeast by modulating FLO11 gene expression.

Microbial biofilms are undesired in food manufacturing, drinking water distribution systems, and clinical realms. Yeast biofilms are particularly problematic because of the strong capacity of yeast cells to adhere to abiotic surfaces, cells, and tissues. Novel approaches have been developed over recent years to prevent the establishment of microbial biofilms, such as through the use of small molecules with inhibiting and dispersing properties. Here, we studied the inhibitory activity of 11 different amino acids on the biofilm formation ability of three wild-type Saccharomyces cerevisiae strains and the reference strain ∑1278b. Subsequent evaluation of different concentrations of the two most effective amino acids, namely, arginine and cysteine, revealed that they acted in different ways. Arginine prevented biofilm formation by reducing FLO11 gene expression; its addition did not affect cell viability and was even found to enhance cell metabolism (vitality marker) as determined by phenotype microarray (PM) analysis. On the contrary, the addition of cysteine reduced both cell viability and vitality as well as FLO11 expression. Thus, the use of cysteine and arginine as agents against biofilm formation can be diversified depending on the most desired action towards yeast growth.

CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

Genotyping, identification and multifunctional features of yeasts associated to Bosana naturally black table olive fermentations.

Directly brined black table olives of Bosana variety are a traditional food product of Sardinia island (Italy), spontaneously fermented by yeasts among other microorganisms. However, as far as we know, the identification, biotechnological and probiotic potential of this yeast community has not been investigated yet. In this work, a total of 72 yeast isolates previously obtained from Bosana olive brines were first genotyped by Random Amplified Polymorphic DNA (RAPD-PCR) analysis with primer M13, and then identified by sequencing of D1/D2 domains of rDNA 26S gene. The dominant species were Wickerhamomyces anomalus and Nakazawaea molendini-olei, albeit Candida diddensiae, Candida boidinii, Zygotorulaspora mrakii, and Saccharomyces cerevisiae were also present in lower proportions. For the different biotypes of yeasts obtained, the multivariate analysis of their technological (esterase, lipase and ß-glucosidase activities, growth in presence of oleuropein, resistance and susceptibility to NaCl) and probiotic (removal of cholesterol, gastric and pancreatic digestions, biofilms assays alone and in co-culture with Lactobacillus pentosus) features, showed that W. anomalus Wa1 exhibited the best technological characteristics, while S. cerevisiae Sc24 and C. boidinii Cb60 showed promising probiotic features. Therefore, they may have potential application as multifunctional starters, alone or in combination with lactic acid bacteria, during olive processing, albeit further studies are necessary to validate these results.

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with ß-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has ß-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.

A putative phospholipase C is involved in Pichia fermentans dimorphic transition.

BACKGROUND: Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi. METHODS: Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition. RESULTS: Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18. CONCLUSION: Phospholipase C is part of a putative "methionine sensing machinery" that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition. GENERAL SIGNIFICANCE: Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.

Red yeasts and carotenoid production: outlining a future for non-conventional yeasts of biotechnological interest.

Carotenoids are one of the most common classes of pigments that occur in nature. Due to their biological properties, they are widely used in phytomedicine and in the chemical, pharmaceutical, cosmetic, food and feed industries. Accordingly, their global market is continuously growing, and it is expected to reach about US$1.4 billion in 2018. Carotenoids can be easily produced by chemical synthesis, although their biotechnological production is rapidly becoming an appealing alternative to the chemical route, partly due to consumer concerns against synthetic pigments. Among the yeasts, and apart from the pigmented species Phaffia rhodozyma (and its teleomorph Xanthophyllomyces dendrorhous), a handful of species of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus are well known carotenoid producers. These are known as 'red yeasts', and their ability to synthesize mixtures of carotenoids from low-cost carbon sources has been broadly studied recently. Here, in agreement with the renewed interest in microbial carotenoids, the recent literature is reviewed regarding the taxonomy of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus, the stress factors that influence their carotenogenesis, and the most advanced analytical tools for evaluation of carotenoid production. Moreover, a synopsis of the molecular and "-omic" tools available for elucidation of the metabolic pathways of the microbial carotenoids is reported.

TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a ß-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding ß-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

Non-Saccharomyces yeast derivatives: Characterization of novel potential bio-adjuvants for the winemaking process.

Winemakers have access to a diverse range of commercially available Inactivated Dry Yeast Based products (IDYB) from various companies and brand names. Among these, thermally inactivated dried yeasts (TIYs) are utilized as yeast nutrients during alcoholic fermentation, aiding in the rehydration of active dry yeasts and reducing ochratoxin A levels during wine maturation and clarification. While IDYB products are generally derived from Saccharomyces spp., this study investigates into the biodiversity of those deriving from non-Saccharomyces for potential applications in winemaking. For that S. cerevisiae and non-Saccharomyces TIYs were produced, characterized for nitrogen and lipid content using FT-NIR spectroscopy, and applied in a wine-like solution (WLS) for analyzing and quantifying released soluble compounds. The impact of TIYs on oxygen consumption was also assessed. Non-Saccharomyces TIYs exhibited significant diversity in terms of cell lipid composition, and amount, composition, and molecular weight of polysaccharides. Compared to that of S. cerevisiae, non-Saccharomyces TIYs released notably higher protein amounts and nHPLC-MS/MS-based shotgun proteomics highlighted the release of cytosolic proteins, as expected due to cell disruption during inactivation, along with the presence of high molecular weight cell wall mannoproteins. Evaluation of antioxidant activity and oxygen consumption demonstrated significant differences among TIYs, as well as variations in GSH and thiol contents. The Principal Component Analysis (PCA) results suggest that oxygen consumption is more closely linked to the lipid fraction rather than the glutathione (GSH) content in the TIYs. Overall, these findings imply that the observed biodiversity of TIYs could have a significant impact on achieving specific oenological objectives.

Impact of Thermally Inactivated Non-Saccharomyces Yeast Derivatives on White Wine.

While a recent characterization of non-Saccharomyces thermally inactivated yeasts (TIYs) in a wine-like solution highlighted the release of oenologically relevant compounds and different oxygen consumption rates and antioxidant activity, here the impact of TIYs derived from Saccharomycodes ludwigii (SL), Metschnikowia pulcherrima (MP), Torulaspora delbrueckii (TD), and Saccharomyces cerevisiae (SC), as the reference strain, was evaluated in white wine. Wine treatment with TIYs resulted in an increase in polysaccharide concentration compared to the untreated wine, with SL-TIY exhibiting the highest release. Additionally, all TIYs, particularly SL-TIY, improved protein stability by reducing heat-induced haze formation. The addition of TIYs also demonstrated an effect on color parameters through phenolic compound adsorption, preventing potential browning phenomena. All TIYs significantly impacted the wine's volatile profile. Overall, it was shown that an improvement in wine quality and stability may be obtained by using TIYs in the winemaking process.

Assessment of ß-carotene content, cell physiology and morphology of the yellow yeast Rhodotorula glutinis mutant 400A15 using flow cytometry.

Flow cytometry was used to assess ß-carotene content, cell membrane permeability, cell size and granularity in Rhodotorula glutinis mutant 400A15 grown under different oxygen transfer coefficients (k L a) and carbon to nitrogen ratios (C/N). A Doehlert distribution was used in order to select the best conditions that induced the highest carotenoids production. The highest ß-carotene content (0.79 mg g(-1) DCW) at the lowest k L a and C/N (5 × 10(-3) s(-1) and 11.3 respectively). Under these conditions, the biomass concentration attained 18.60 g L(-1). The highest ratio of cells with permeabilised membranes (2.6 %), and the highest cell size and granularity were also obtained under these conditions. It was observed that C/N showed a stronger influence than the k L a on the measured cell parameters.

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine.

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations (S. cerevisiae EC1118 and L. thermotolerans 101), this non-Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans. However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases.

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant.

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in ß-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2) = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l(-1)) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

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 (>80% survival after 1 h at pH 2), osmostress (growth at up to 6.5% NaCl) and lyophilization (survival rate > 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.