Search Results (61)

Antifungal killer toxins are cytotoxic proteins that have the potential to combat the growing threat of fungi to human health and agriculture. A lack of empirical tertiary structures has limited understanding of their mechanisms of action and their ability to target pathogens. In this study, AlphaFold and molecular dynamics simulations were used to generate tertiary structure models of all canonical Saccharomyces killer toxins and to place them in the context of historical empirical data. These models enabled the prediction of functional domains and posttranslational modifications, including proteolytic cleavage sites and disulfide bonds. They also revealed unexpected homology between Saccharomyces killer toxins, suggesting that all but K28 are likely ionophores. Structural homology to the well-studied killer toxins K1 and K2 enabled the prediction of the antifungal and immunity mechanisms of K1L, K21, K45, K74, and KHS. The understudied killer toxins Klus, KHR, and K62 were found to have homology to bacterial and plant toxins, including members of the aerolysin family and antifungal lectins. These structural similarities provide clues for the mechanisms of killer toxin carbohydrate binding, oligomerization, and membrane attack. This modeling approach will help guide the continued use of the model yeast S. cerevisiae to study killer toxins in the context of the wealth of functional data gathered in the decades since their first discovery. Full article

Spontaneously fermented wines are a habitat for many Saccharomyces and non-Saccharomyces strains that are typical for a given region. The isolates obtained can serve as regional starter cultures for winemaking. The aim of this study was to isolate, identify and evaluate the oenological properties and fermentation suitability of selected yeast isolates obtained from Polish spontaneously fermented grape wines. The isolated yeasts were genetically identified and characterised in terms of ethanol tolerance, enzymatic activities, H₂S production, and preliminary killer activity. In small-scale fermentations conducted in CDGJ medium and grape juice, the fermentation rate, pH, number of yeast, content of sugars, ethanol, organic acids and volatile compounds were determined. Genetic identification revealed the species: Saccharomyces cerevisiae, S. paradoxus, Metschnikowia pulcherrima, M. ziziphicola, Hanseniaspora uvarum, and Pichia kluyveri. Non-Saccharomyces and Saccharomyces strains grew poorly in the presence of 4–6% (v/v) and 14–16% (v/v) ethanol, respectively. The yeasts had varied enzymatic activities in API ZYM tests, and production of H₂S, but did not exhibit killer activity. The monocultures showed differences in fermentation rates. The best growth was recorded for all strains during grape juice fermentation, up to 10⁹ cfu/mL, producing ethanol and glycerol in the range of 53.92–86.54 g/L and 0.0–4.48 g/L. Yeasts produced characteristic volatile compounds, e.g., esters: 2-phenylethyl acetate and ethyl decanoate. The monocultures of isolated yeasts can be used in fermentation of grape must, yielding wines with diverse characteristics in terms of ethanol, organic acids and volatile compounds. Full article

Currently, there is little scientific data to support the importance of selecting non-Saccharomyces yeasts from different wineries in the Protected Designation of Origin (PDO) in Andalusia, southern Spain, and how this group of yeasts can affect the sensory properties of wine. Therefore, this research aimed to study some specific microbiological properties and the metabolites they could produce in order to evaluate the oenological potential of two non-Saccharomyces yeast strains isolated from a region of Andalusia (Córdoba, Spain), Hanseniaspora uvarum TJ-27 and Lachancea thermotolerans T-9, isolated from musts with high sugar content. Of 80 yeast isolates selected, these two strains were chosen for their notable β-glucosidase activity (observed in up to 40% of isolates), cellulase activity (present in 24%), and killer phenotype (found in 40%). In this study, strains that displayed characteristics associated with aroma release were selected. Fermentation assays using a high-sugar synthetic medium revealed that neither H. uvarum TJ-27 nor L. thermotolerans T-9 was able to complete alcoholic fermentation independently, achieving ethanol yields of only 5–6% v/v, indicating the need for subsequent fermentation by Saccharomyces cerevisiae. The originality of this study provides insight into the metabolites contributed by these strains to the wines produced. The best results were obtained when both strains were inoculated together. Furthermore, volatilome analysis showed elevated levels of key compounds such as isoamyl alcohols and 2,3-butanediol. These findings highlight the practical potential of using selected non-Saccharomyces strains from Andalusia to improve fermentation results and wine quality. The novelty of this study lies mainly in confirmation within region-specific isolates. Full article

Background/Objectives: Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, typically is asymptomatic in immunocompetent individuals but causes severe complications in immunocompromised subjects and during pregnancy. Current treatments such as pyrimethamine and sulfadiazine are effective for acute infections but cannot eliminate encysted bradyzoites and have significant side effects. The antimicrobial killer peptide (KP) has interesting therapeutic potential, but its intracellular delivery is challenging; hyaluronate-based nanoparticles loaded with KP (KP-NPs) were evaluated to target T. gondii-infected cells that overexpress CD44. Methods: KP-NPs made of chitosan and hyaluronate were produced by microfluidics and were characterized for size, surface charge, encapsulation efficiency, and stability under stress conditions. After excluding their toxicity, their activity was tested in vitro against Candida albicans and T. gondii as free tachyzoite or in infected human foreskin fibroblasts (HFFs). Results: KP was efficiently encapsulated in nanoparticles and protected from harsh acidic conditions at high temperature. Preliminary in vitro testing against C. albicans showed that, at the lowest candidacidal concentration of KP (2.5 μg/mL), KP-NPs killed 90.97% of yeast cells. KP itself proved to be non-toxic for HFFs as host cells and effective against T. gondii. Comparable results were obtained for KP-NPs and blank nanoparticles (BLK-NPs), with no observed toxicity to host cells, confirming that encapsulation did not alter peptide efficacy. The parasiticidal effect of KP alone, as well as KP-NPs at 250 µg/mL and BLK-NPs, was confirmed through tests on free T. gondii tachyzoites. Reduction rates for the number of infected cells ranged from 66% to 90% with respect to control, while the reduction in the number of intracellular tachyzoites ranged from 66% to 80%. Interestingly, KP alone was not effective against intracellular tachyzoite, while KP-NPs maintained an efficacy comparable to the extracellular model, suggesting that particles helped the internalization of the peptide. Conclusions: Encapsulation of KP into hyaluronate/chitosan nanoparticles does not alter its activity and improves its efficacy against the intracellular parasite. Notably, BLK-NPs appeared to exhibit efficacy against the parasite on its own, without the presence of KP. Full article

This study evaluated the oenological potential of two non-Saccharomyces yeast strains, Hanseniaspora opuntiae TR-5 and Zygosaccharomyces bailii L-25, isolated from extreme winemaking environments in southern Spain. Out of 156 yeast isolates screened from high-sugar musts and flor yeast biofilms, strains were selected based on their β-glucosidase activity, killer phenotype, and ethanol production, traits associated with aroma release and microbial competition. Fermentation trials on sugar-rich synthetic medium showed that both H. opuntiae and Z. bailii achieved ethanol yields of 10% v/v and residual sugars at 4 g/L. Co-culture and sequential inoculation, with H. opuntiae introduced first and Z. bailii added on day four, resulted in complete alcoholic fermentation and a reduction in undesirable acetoin levels compared to single-strain fermentations. These findings highlight the practical potential of using selected non-Saccharomyces strains in sequential fermentations to improve aroma complexity, fermentation reliability, and sensory quality in wines, even in the absence of Saccharomyces cerevisiae. The application of these strains offers a novel approach for precision oenology and varietal expression in challenging musts. Full article

Bioprotection in winemaking refers to the use of naturally occurring microorganisms—mainly non-Saccharomyces yeasts—to inhibit the growth of spoilage microbes and reduce the need for chemical preservatives like sulfur dioxide (SO₂). Numerous studies have demonstrated the benefits of non-Saccharomyces as bioprotectants. However, the use of Saccharomyces cerevisiae as a bioprotectant has been studied very little. Furthermore, it can offer many advantages for the production of sulfite-free wines. To test if S. cerevisiae could be used in bioprotection, we compared the ability of different strains to inhibit the growth of Brettanomyces bruxellensis and Hanseniaspora uvarum. Among the strains tested, the S. cerevisiae Sc54 strain isolated from the vineyard of the Bekaa plain was selected. To investigate its mechanisms of action, we analyzed its metabolite production, including acetic acid and ethanol. Taking into account the low levels of these metabolites and the lack of similar inhibition patterns in media supplemented with acetic acid and ethanol, it appears that other factors contribute to its antagonistic properties. Nutrient competition was ruled out as a factor, as the growth inhibition of B. bruxellensis and H. uvarum occurred rapidly within the first 24 h of co-culture. In this study, we explored the role of the S. cerevisiae killer toxin (Sc54Kt) as a bioprotective agent against H. uvarum and B. bruxellensis spoilage yeasts. Purification procedures with ethanol allowed the extraction of Sc54Kt, yielding two concentrations (0.185 and 0.5 mg/mL). Remarkably, semi-purified Sc54Kt exhibited inhibitory effects at both concentrations under winemaking conditions, effectively controlling the growth and metabolic activity of the target spoilage yeasts. Overall, these findings demonstrate that S. cerevisiae Sc54 not only exerts a strong bioprotective effect but also contributes to improving the quality of wine. The results suggest that S. cerevisiae Sc54 is a promising bioprotective agent for mitigating spoilage yeasts in winemaking, offering a natural and effective alternative to conventional antimicrobial strategies. Full article

Botryosphaeriaceae species were recently found to be responsible for heavy mango crop losses worldwide. In 2020, mango fruit samples showing fruit decay symptoms were collected from Glenn, Kent, Irwin, Palmer, Brokaw 2, and Gomera 3 accessions in 4 orchards located in Sicily (Italy). A molecular analysis of the ITS and tub2 regions performed on 41 representative isolates allowed for the identification of mainly Neofusicoccum parvum and occasionally Botryosphaeria dothidea (1/41) as the causal agents of fruit decay. Pathogenicity proofs were satisfied for both fungal pathogens. Ripe and unripe Gomera 3 mango fruits were used to compare the virulence among the N. parvum isolates. Postharvest experiments performed on Gomera 3 fruits and by using different biocontrol agents (BCAs) showed that the performance of treatments in reducing fruit decay depends on N. parvum virulence. The data show that unregistered Wickerhamomyces anomalus WA-2 and Pichia kluyveri PK-3, followed by the trade bioformulate Serenade™ (Bacillus amyloliquefaciens QST713), were the most effective in managing mango fruit rot. This paper shows, for the first time, the potential of different BCAs, including Trichoderma spp., for the controlling of postharvest decay caused by N. parvum on mango fruits. Full article

This paper explores the broad probiotic and functional properties of two non-Saccharomyces strains (MI120 and MI125) and one Saccharomyces cerevisiae BB06 strain (as a reference probiotic). Torulaspora delbrueckii MI120 and Starmerella bacillaris MI125 were identified via 5.8S rDNA sequencing. All the strains survived well in simulated gastrointestinal conditions and had strong antioxidant activity (>68%). S. bacillaris MI125 excelled in antimicrobial activity against Gram-positive and Gram-negative bacteria. S. bacillaris MI125 and Sacch. cerevisiae BB06 resisted all the tested antibiotics. No strain displayed hemolytic behavior. The freeze-dried yeast strains achieved survival rates between 76.62% and 93.38%. Based on our physiological characterization analysis (carbon assimilation, ethanol tolerance, acetic acid and H₂S production, temperature and low pH tolerance, enzymatic pattern, and killer phenotype), all the strains showed interesting attributes, with Sacch. cerevisiae BB06 fermenting vigorously in malt extract medium. Beer fermented with T. delbrueckii MI120 had the highest phenolic content (96.02 μg GAE·mL⁻¹) and antioxidant activity (90.43%), matching commercial Sacch. cerevisiae US-05 in sensory traits such as taste and aroma. However, after two months in bottled beer, the yeast viability decreased to 2–3 log CFU·mL⁻¹. The pilot brewing and the assessments of the strains’ technological, physico-chemical, and sensorial properties confirmed their suitability for industrial brewing. Overall, T. delbrueckii MI120 emerges as a promising brewing strain, and S. bacillaris MI125 is a potential probiotic. Full article

Insect symbionts and their metabolites are complex and diverse and are gradually becoming an important source of new medical materials. Some culturable symbionts from insects produce a variety of active compounds with medical potential. Among them, fatty acids, antibacterial peptides, polyene macrolides, alkaloids, and roseoflavin can inhibit the growth of human pathogenic bacteria and fungi; lipases, yeast killer toxins, reactive oxygen species, pyridines, polyethers, macrotetrolide nactins, and macrolides can kill human parasites; and peptides and polyketides can inhibit human tumors. However, due to difficulty in the culture of symbionts in vitro, difficulty in targeting bacteria to specific sites in the human body, the limited capability of symbionts to produce active metabolites in vitro, inconsistent clinical research results, adverse reactions on humans, and the development of antibiotic resistance, the application of insect symbionts and their metabolites in the medical field remains in its infancy. This paper summarizes the medical potential of insect symbionts and their metabolites and analyzes the status quo and existing problems with their medical application. Possible solutions to these problems are also proposed, with the aim of hastening the utilization of insect symbionts and their metabolites in the medical field. Full article

The spoilage of wine caused by Brettanomyces bruxellensis and Hanseniaspora uvarum poses a significant challenge for winemakers, necessitating the development of effective and reliable strategies to control the growth of these yeasts, such as grape must bioprotection. Despite evidence that certain microorganisms can inhibit the growth of Brettanomyces bruxellensis and Hanseniaspora uvarum, the specific mechanisms driving this inhibition remain unclear. The primary objective of this study is to elucidate the underlying mechanisms responsible for this inhibitory effect. We analyzed one Metschnikowia pulcherrima (Mp2) and two Lachancea thermotolerans (Lt29 and Lt45) strains, all of which demonstrated significant killing and inhibitory effects on Brettanomyces bruxellensis (B1 and B250) and Hanseniaspora uvarum (Hu3137) in synthetic must at pH 3.5 and 22 °C. The effectiveness of these two strains exhibited varying inhibition kinetics. The strains were monitored for growth and metabolite production (L-lactic acid, ethanol, and acetic acid) in both single and co-cultures. The low levels of these metabolites did not account for the observed bioprotective effect, indicating a different mechanism at play, especially given the different growth profiles observed with added L-lactic acid and ethanol compared to direct bioprotectant addition. Following the production, purification, and quantification of killer toxins, different concentrations of toxins were tested, showing that the semi-purified Mp2Kt, Lt29Kt, and Lt45Kt toxins controlled the growth of both spoilage yeasts in a dose-dependent manner. These bioprotectant strains also showed compatibility with Saccharomyces cerevisiae in co-cultures, suggesting their potential use alongside commercial starter cultures. Full article

Non-Saccharomyces yeasts have the potential to ameliorate wine ethanol levels, but such fit-for-purpose yeast strains are still lacking. Seventy-one indigenous non-Saccharomyces yeasts isolated from spontaneous fermentations of four wine regions in China (Ningxia, Xinjiang, Gansu, and Shaanxi) were screened for ethanol formation and were characterized for major metabolite profiles in synthetic grape juice fermentation to obtain non-Saccharomyces yeasts with low ethanol yields. Four Hanseniaspora strains with less volatile acidity production were primarily selected, and their ethanol yield was reduced by 22–32% compared to S. cerevisiae. These strains were further evaluated for oenological properties, namely ethanol and temperature tolerance, H₂S production, and killer activities against S. cerevisiae. Strain HuC-3-2 was then subjected to Atmospheric Room Temperature Plasma (ARTP) mutagenesis, and a mutant (HuC32-2-72) with rapid growth and optimized ethanol-reducing capability was obtained. The best-performing strains were further characterized in sequential fermentations with S. cerevisiae in Merlot juice, and resulted in a 1.4% v/v decrease in ethanol yield. Comprehensive analysis of yeast populations and the production of key metabolites highlighted important carbon sinks, as well as glycerol formation, partially accounting for the ethanol reduction. In addition to ethanol amelioration, the Hanseniaspora strains also led to alterations in many metabolites, including volatile compounds and some organic acids, which can further modulate wine aroma and flavor. Full article

Yeasts produce numerous antimicrobial agents such as killer toxins, volatile organic compounds (VOCs), and other secondary metabolites, establishing themselves in developing natural and sustainable biocontrol strategies for agriculture and food preservation. This study addressed the biocontrol potential of yeasts, isolated from spontaneous fermentations of rosehips (Rosa canina L.) and rowanberries (Sorbus aucuparia L.), focusing on their killer phenotypes and VOCs production. Yeasts were isolated using spontaneous fermentations with Hanseniaspora uvarum and Metschnikowia pulcherrima identified as the dominant species, comprising approximately 70% of the yeast population. Among 163 isolated strains, 20% demonstrated killing activity, with Saccharomyces cerevisiae exhibiting the strongest killing efficiency, as well as Pichia anomala and M. pulcherrima showing broad-spectrum antagonistic activity. This study identified dsRNA-encoded killer phenotypes in S. cerevisiae, S. paradoxus, and Torulaspora delbrueckii, revealing multiple distinct killer toxin types. The biocontrol potential of wild berry-inhabiting yeasts was demonstrated in a real food system, grape juice, where the S. cerevisiae K2-type killer strain significantly reduced fungal contaminants. The selected H. uvarum, M. pulcherrima, S. cerevisiae, and S. paradoxus yeast strains representing both berries were applied for VOC analysis and identification by gas chromatography-linked mass spectrometry. It was revealed that the patterns of emitted volatiles are yeast species-specific. Statistically significant differences between the individual VOCs were observed among killing phenotype-possessing vs. non-killer S. paradoxus yeasts, thus revealing the involvement of killer systems in multi-level biocontrol enablement. The performed studies deepen our understanding of potential yeast biocontrol mechanisms, highlight the importance of produced antimicrobials and volatiles in ensuring antagonistic efficacy, and prove the relevance of isolated biocontrol yeasts for improving food safety. Full article

The control of spoilage yeasts in wines is crucial to avoid organoleptic deviations in wine production. Traditionally, sulfur dioxide (SO₂) was used to control them; nevertheless, SO₂ influence on human health and its use is criticized. Biocontrol emerges as an alternative in wine pre-fermentation, but there is limited development in its applicability. Managing kinetics is relevant in the microbial interaction process. pH was identified as a factor affecting the interaction kinetics of Wickerhamomyces anomalus killer biocontrol on Zygosaccharomyces rouxii. Mathematical modeling allows insight into offline parameters and the influence of physicochemical factors in the environment. Incorporating submodels that explain manipulable factors (pH), the process can be optimized to achieve the best-desired outcomes. The aim of this study was to model and optimize, using a constant and a variable pH profile, the interaction of killer biocontrol W. anomalus vs. Z. rouxii to reduce the spoilage population in pre-fermentation. The evaluated biocontrol was W. anomalus against the spoilage yeast Z. rouxii in wines. The kinetic interactions of yeasts were studied at different pH levels maintained constant over time. The improved Ramón-Portugal model was adopted using the AMIGO2 toolbox for Matlab. A static optimization of a constant pH profile was performed using the Monte Carlo method, and a dynamic optimization was carried out using a method based on Fourier series and orthogonal polynomials. The model fit with an adjusted R² of 0.76. Parametric analyses were consistent with the model behavior. Variable vs. constant optimization achieved a lower initial spoilage population peak (99% less) and reached a lower final population (99% less) in a reduced time (100 vs. 140 h). These findings reveal that control with a variable profile would allow an early sequential inoculation of S. cerevisiae. The models explained parameters that are difficult to quantify, such as general inhibitor concentration and toxin concentration. Also, the models indicate higher biocontrol efficiency parameters, such as toxin emission or sensitivity to it, and lower fitness of the contaminant, at pH levels above 3.7 during biocontrol. From a technological standpoint, the study highlights the importance of handling variable profiles in the controller associated with the pH management actuators in the process without incurring additional costs. Full article

In the context of the growing concern regarding the appearance and spread of emerging pathogens with high resistance to chemically synthetized biocides, the development of new agents for crops and human protection has become an emergency. In this context, the yeasts present a huge potential as eco-friendly agents due to their widespread nature in various habitats and to their wide range of antagonistic mechanisms. The present review focuses on some of the major yeast antimicrobial mechanisms, their molecular basis and practical applications in biocontrol and biomedicine. The synthesis of killer toxins, encoded by dsRNA virus-like particles, dsDNA plasmids or chromosomal genes, is encountered in a wide range of yeast species from nature and industry and can affect the development of phytopathogenic fungi and other yeast strains, as well as human pathogenic bacteria. The group of the “red yeasts” is gaining more interest over the last years, not only as natural producers of carotenoids and rhodotorulic acid with active role in cell protection against the oxidative stress, but also due to their ability to inhibit the growth of pathogenic yeasts, fungi and bacteria using these compounds and the mechanism of competition for nutritive substrate. Finally, the biosurfactants produced by yeasts characterized by high stability, specificity and biodegrability have proven abilities to inhibit phytopathogenic fungi growth and mycelia formation and to act as efficient antibacterial and antibiofilm formation agents for biomedicine. In conclusion, the antimicrobial activity of yeasts represents a direction of research with numerous possibilities of bioeconomic valorization as innovative strategies to combat pathogenic microorganisms. Full article

M. bicuspidata var. bicuspidata is a pathogenic yeast which can affect aquacultured and marine-cultured animals such as brine shrimp, ridgetail white prawn, chinook salmon, giant freshwater prawn, the Chinese mitten crab, marine crab, the mud crab, the mangrove land crab, the Chinese grass shrimp, sea urchins, sea urchins, Daphnia dentifera and even snails, causing a milky disease, and it has caused big economic losses in aquacultural and marine-cultural industries in the past. However, the detailed mechanisms and the reasons for the milky disease in the diseased aquatic animals are still completely unknown. So far, only some antimycotics, killer toxins and Massoia lactone haven been found to be able to actively control and kill its growth. The ecofriendly, green and renewable killer toxins and Massoia lactone have high potential for application in controlling the milky disease. Full article