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.

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.

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.

A set of haploid strains available for genetic studies of Saccharomyces cerevisiae flor yeasts.

Flor yeasts of Saccharomyces cerevisiae have been extensively studied for biofilm formation, however the lack of specific haploid model strains has limited the application of genetic approaches such as gene knockout, allelic replacement and Quantitative Trait Locus mapping for the deciphering of the molecular basis of velum formation under biological ageing. The aim of this work was to construct a set of flor isogenic haploid strains easy to manipulate genetically. The analysis of the allelic variations at 12 minisatellite loci of 174 Saccharomyces cerevisiae strains allowed identifying three flor parental strains with different phylogenic positions. These strains were characterized for sporulation efficiency, growth on galactose, adherence to polystyrene, agar invasion, growth on wine and ability to develop a biofilm. Interestingly, the inability to grow on galactose was found associated with a frameshift in GAL4 gene that seems peculiar of flor strains. From these wild flor strains, isogenic haploid strains were constructed by deleting HO gene with a loxP-KanMX-loxP cassette followed by the removal of the kanamycin cassette. Haploid strains obtained were characterized for their phenotypic and genetic properties and compared with the parental strains. Preliminary results showed that the haploid strains represent new tools for genetic studies and breeding programs on biofilm formation.

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.

Novel starters for old processes: use of Saccharomyces cerevisiae strains isolated from artisanal sourdough for craft beer production at a brewery scale.

The deliberate inoculation of yeast strains isolated from food matrices such as wine or bread, could allow the transfer of novel properties to beer. In this work, the feasibility of the use of baker's yeast strains as starters for craft beer production has been evaluated at laboratory and brewery scale. Nine out of 12 Saccharomyces cerevisiae strains isolated from artisanal sourdoughs metabolized 2 % maltose, glucose and trehalose and showed growth rates and cell populations higher than those of the brewer's strain Safbrew-S33. Analysis of allelic variation at 12 microsatellite loci clustered seven baker's strains and Safbrew-S33 in the main group of bread isolates. Chemical analyses of beers produced at a brewery scale showed significant differences among the beers produced with the baker's strain S38 or Safbrew-S33, while no significant differences were observed when S38 or the brewer's strain Safbrew-F2 was used for re-fermentation. The sensory profile of beers obtained with S38 or the brewer's yeasts did not show significant differences, thus suggesting that baker's strains of S. cerevisiae could represent a reservoir of biodiversity for the selection of starter strains for craft beer production.

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.

Microbial community assembly and chemical dynamics of raw brewers' spent grain during inoculated and spontaneous solid-state fermentation.

Solid-state fermentation (SSF) carried out by microbial bioinoculants is an environmentally friendly technology for the sustainable recovery and valorization of agri-food wastes. Particularly, mesophilic SSF processes allows the production of bio-organic fertilizers enriched with beneficial soil microorganisms. However, the establishment of microbial consortia and the interaction with native waste microbiota still require thoughtful investigations. Here, raw brewers' spent grain (BSG), the main waste from the brewing industry, was subjected to two mesophilic SSF processes (maximum temperature of 35 °C) carried out by a multi-kingdom microbial bioinoculant and the BSG spontaneous microbiota. After 90 days, both SSF processes led to stable organic soil amendments, as indicated by the C:N ratio (10.00 ± 1.4), pH (6.66 ± 0.09), and DOC (8.45 ± 1.2 mg/g) values. Additionally, the fermented BSG showed a high nitrogen content (42.2 ± 3.4 mg/Kg) and biostimulating activities towardLepidium sativumseeds. The monitoring of microbial communities by high-throughput sequencing of 16S and ITS rRNA indicated that BSG samples were enriched in microbial genera with interesting agronomic applications (i.e.,Devosia, Paenibacillum, Trichoderma, Mucor, etc.). Microbial cross-kingdom network analyses suggested that the microbial assembly of BSG was significantly influenced by the bioinoculant, despite the inoculated microbial genera being able to persist in BSG samples only the first week of SSF. This suggests that the study of microbial interactions between exogenous microbial inoculants and waste resident microbiota is required to optimize SSF processes aimed at the recovery and valorization of unprocessed wastes.

Polyphenolic Content and Antimicrobial Effects of Plant Extracts as Adjuncts for Craft Herbal Beer Stabilization.

Extracts from locally grown aromatic plants can enhance the geographical characteristics and microbial stability of craft beers, which are often not pasteurized or filtered. Here, the chemical and antimicrobial properties of aqueous extracts from leaves of Myrtus communis L., Pistacia lentiscus L., Artemisia arborescens L., and floral wastes of Crocus sativus L., all cultivated in Sardinia (Italy), were assessed. P. lentiscus extract had the highest polyphenol content (111.20 mg GAE/g), followed by M. communis (56.80 mg GAE/g), C. sativus (32.80 mg GAE/g), and A. arborescens (8.80 mg GAE/g). Notably, only the M. communis extract demonstrated significant inhibitory activity against pathogenic and spoilage microorganisms, with minimum inhibitory concentrations of 0.18, 0.71, and 1.42 mg GAE/mL against Staphylococcus aureus, Lactiplantibacillus plantarum, and Lacticaseibacillus casei, respectively. Additionally, it reduced the growth of Levilactobacillus brevis and Fructilactobacillus lindneri at concentrations of 0.35 and 0.71 mg GAE/mL, respectively. Based on its significant antimicrobial activity, the M. communis extract was further characterized using high-resolution mass spectrometry, revealing high abundances of nonprenylated phloroglucinols, flavonoid derivatives (myricetin), and quinic acids. Lastly, adding M. communis extract (2.84 mg GAE/mL) to commercial beer effectively prevented the growth of L. brevis and F. lindneri, showing its potential to avoid beer's microbial spoilage.

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.

FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis.

Identification of differentially expressed genes associated with changes in the morphology of Pichia fermentans on apple and peach fruit.

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization (SSH) strategies, that is, rapid subtraction hybridization (RaSH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies.

Microbial and chemical dynamics of brewers' spent grain during a low-input pre-vermicomposting treatment.

The eco-sustainability of industrial processes relies on the proper exploitation of by-products and wastes. Recently, brewers' spent grain (BSG), the main by-product of brewing, was successfully recycled through vermicomposting to produce an organic soil conditioner. However, the pre-processing step there applied (oven-drying) resulted in high costs and the suppression of microbial species beneficial for soil fertility. To overcome these limitations, a low-input pre-processing step was here applied to better exploit BSG microbiota and to make BSG suitable for vermicomposting. During 51 days of pre-treatment, the bacterial and fungal communities of BSG were monitored by denaturing gradient gel electrophoresis (DGGE). Chemical (carbon, nitrogen, ammonium, nitrate content, dissolved organic carbon) and biochemical (dehydrogenase activity) parameters were also evaluated. Mature vermicompost obtained from pre-processed BSG was characterized considering its legal requirements (e.g., absence of pathogens and mycotoxins, lack of phytotoxicity on seeds), microbiota composition, and chemical properties. Results obtained showed that throughout the pre-process, the BSG microbiota was enriched in bacterial and fungal species of significant biotechnological and agronomic potential, including lactic acid bacteria (Weissella, Pediococcus), plant growth-promoting bacteria (Bacillus, Pseudomonas, Pseudoxhantomonas), and biostimulant yeasts (Pichia fermentans, Trichoderma reesei, Beauveria bassiana). Pre-processing increased the suitability of BSG for earthworms' activity to produce high-quality mature vermicompost.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

Air-liquid biofilm formation is dependent on ammonium depletion in a Saccharomyces cerevisiae flor strain.

Air-liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air-liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air-liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air-liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains.

Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins.

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows ß-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25-2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.