Pulcherrima Strains Research Articles

Bioprospecting of Metschnikowia pulcherrima Strains, Isolated from a Vineyard Ecosystem, as Novel Starter Cultures for Craft Beer Production

Several studies in recent years have shown that the use of non-Saccharomyces yeasts, used both in single and in mixed fermentations with Saccharomyces cerevisiae, can help produce craft beers with distinctive compositional characteristics. The aim of this study was to evaluate the suitability of three Metschnikowia pulcherrima strains, isolated from Albanian vineyards, for use as starters in the brewing process. Because of its specific enzymatic activities (protease, β-glucosidase, and β-lyase) and its low production of hydrogen sulfide, M. pulcherrima 62 was selected as a starter culture for the production of craft beer. Specifically, the suitability of this yeast for use in sequential inoculation with S. cerevisiae S0-4 for the production of an American IPA-style beer and the main volatile compounds produced during fermentation were evaluated. The results show significant differences in the glycerol, isoamyl alcohol, and isoamyl acetate contents in beer obtained by sequential inoculum of M. pulcherrima 62 with S. cerevisiae S0-4 compared to beer obtained using S. cerevisiae S0-4 as a single starter. Therefore, these preliminary data support the candidacy of M. pulcherrima 62 as a new starter in the brewing process.

Assessing Metschnikowia pulcherrima as a potential probiotic yeast for animal feed

Abstract In response to escalating antimicrobial resistance fuelled by their extensive use in livestock farming, this study explores alternative strategies in animal health management. We investigated the probiotic potential of Metschnikowia pulcherrima strains 4 × 3, DH5, ICS1 and QRI1 for their suitability in poultry environments. Our research demonstrated that previously discovered antimicrobial M. pulcherrima strains exhibit promising probiotic behaviours, including biofilm production, auto-aggregation, cell surface hydrophobicity, adhesion to Caco-2 cells, antioxidant capacity, and resilience to gastric stresses. Notable findings include DH5 exhibiting the highest biofilm formation, 4 × 3 and DH5 showing rapid auto-aggregation, 4 × 3 and ICS1 displaying high cell surface hydrophobicity, and all strains demonstrating considerable adherence to Caco-2 cells. 4 × 3 also exhibited exceptional bile tolerance, while ICS1 showed robust survival under simulated gastrointestinal conditions. These traits suggest M. pulcherrima’s capacity to colonise the poultry gastrointestinal tract, promote animal health, and support more sustainable livestock practices as a potential alternative to conventional antibiotics.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Competition for Nitrogen Resources: An Explanation of the Effects of a Bioprotective Strain Metschnikowia pulcherrima on the Growth of Hanseniaspora Genus in Oenology.

As a biological alternative to the antimicrobial action of SO2, bioprotection has been proposed to winemakers as a means to limit or prevent grape musts microbial alteration. Competition for nitrogenous nutrients and for oxygen are often cited as potential explanations for the effectiveness of bioprotection. This study analyses the effect of a bioprotective M. pulcherrima strain on the growth of one H. valbyensis strain and one H. uvarum strain. Bioprotection efficiency was observed only against H. valbyensis inoculated at the two lowest concentrations. These results indicate a potential species-dependent efficiency of the bioprotective strain and a strong impact of the initial ratio between bioprotective and apiculate yeasts. The analysis of the consumption of nitrogen compounds revealed that leucine, isoleucine, lysine and tryptophan were consumed preferentially by all three strains. The weaker assimilation percentages of these amino acids observed in H. valbyensis at 24 h growth suggest competition with M. pulcherrima that could negatively affects the growth of the apiculate yeast in co-cultures. The slowest rate of O2 consumption of H. valbyensis strain, in comparison with M. pulcherrima, was probably not involved in the bioprotective effect. Non-targeted metabolomic analyses of M. pulcherrima and H. valbyensis co-culture indicate that the interaction between both strains particularly impact lysin and tryptophan metabolisms.

Isolation of local strains of the yeast Metschnikowia for biocontrol and lipid production purposes

The bioprospection of indigenous microorganism strains with biotechnological potential represents a prominent trend. Metschnikowia yeasts exhibit diverse capabilities, such as ethanol reduction in winemaking, biocontrol potential, and lipid production. In this work, local Metschnikowia strains were isolated from different fruits by their ability to produce pulcherrimic acid, a molecule that has been linked to biocontrol activity and that binds iron giving colored colonies. Five strains were selected, each from one of five distinct sources. All of them were identified as M. pulcherrima. All five were able inhibit other yeasts and one M. pulcherrima, called M7, inhibited the growth of Aspergillus nidulans. The selected strains accumulated lipid bodies in stationary phase. Certain non-conventional yeasts like Hanseniaspora vineae are very sensitive to biomass drying, but cell extracts from M. pulcherrima added to the growth media as a source of antioxidant lipids increased their tolerance to drying. All strains isolated showed good stress tolerance (particularly to heat) and have nutrient requirements similar to a commercial M. pulcherrima strain. In addition, the M7 strain had a good growth in sugarcane and beet molasses and behaved like Saccharomyces cerevisiae in a growth medium derived from agricultural waste, a persimmon hydrolysate. Therefore, the isolation of local strains of Metschnikowia able to grow in a variety of substrates is a good source of biocontrol agents.

Differences in the management of intracellular redox state between wine yeast species dictate their fermentation performances and metabolite production

The maintenance of the balance between oxidised and reduced redox cofactors is essential for the functioning of many cellular processes in all living organisms. While the electron transport chain plays a key role in maintaining this balance under respiratory conditions, its inactivity in the absence of oxygen poses a challenge that yeasts such as Saccharomyces cerevisiae overcome through the production of various metabolic end-products during alcoholic fermentation. In this study, we investigated the diversity occurring between wine yeast species in their management of redox balance and its consequences on the fermentation performances and the formation of metabolites. To this aim, we quantified the changes in NAD(H) and NADP(H) concentrations and redox status throughout the fermentation of 6 wine yeast species. While the availability of NADP and NADPH remained balanced and stable throughout the process for all the strains, important differences between species were observed in the dynamics of NAD and NADH intracellular pools. A comparative analysis of these data with the fermentation capacity and metabolic profiles of the strains revealed that Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans strains were able to reoxidise NADH to NAD throughout the fermentation, mainly by the formation of glycerol. These species exhibited good fermentation capacities. Conversely, Starmerella bacillaris and Metschnikowia pulcherrima species were unable to regenerate NAD as early as one third of sugars were consumed, explaining at least in part their poor growth and fermentation performances. The Kluyveromyces marxianus strain exhibited a specific behaviour, by maintaining similar levels of NAD and NADH throughout the process. This balance between oxidised and reduced redox cofactors ensured the consumption of a large part of sugars by this species, despite a low fermentation rate. In addition, the dynamics of redox cofactors affected the production of by-products by the various strains either directly or indirectly, through the formation of precursors. Major examples are the increased formation of glycerol by S. bacillaris and M. pulcherrima strains, as a way of trying to reoxidise NADH, and the greater capacity to produce acetate and derived metabolites of yeasts capable of maintaining their redox balance. Overall, this study provided new insight into the contribution of the management of redox status to the orientation of yeast metabolism during fermentation. This information should be taken into account when developing strategies for more efficient and effective fermentation.

Iron Competition as an Important Mechanism of Pulcherrimin-Producing Metschnikowia sp. Strains for Controlling Postharvest Fungal Decays on Citrus Fruit.

This study identified and tested fruit-isolated Metschnikowia yeasts against three major postharvest citrus pathogens, namely, Penicillium digitatum, Penicillium italicum, and Geotrichum citri-aurantii, and further evaluated the impact of FeCl3 on the biocontrol efficiency of pulcherrimin-producing M. pulcherrima strains. Based on the characterization of the pigmented halo surrounding the colonies and the analysis of the D1/D2 domain of 26S rDNA, a total of 46 Metschnikowia sp. were screened and identified. All 46 Metschnikowia strains significantly inhibited the hyphal growth of Penicillium digitatum, Penicillium italicum, and Geotrichum citri-aurantii, and effectively controlled the development of green mold, blue mold and sour rot of citrus fruit. The introduction of exogenous FeCl3 at certain concentrations did not significantly impact the pulcherriminic acid (PA) production of pigmented M. pulcherrima strains, but notably diminished the size of pigmented zones and the biocontrol efficacy against the three pathogens. Iron deficiency sensitivity experiments revealed that P. digitatum and P. italicum exhibited higher sensitivity compared to G. citri-aurantii, indicating that iron dependence varied among the three pathogens. These results suggested that M. pulcherrima strains, capable of producing high yields of PA, possessed great potential for use as biocontrol agents against postharvest citrus diseases. The biocontrol efficacy of these yeasts is mainly attributed to their ability to competitively deplete iron ions in a shared environment, with the magnitude of their pigmented halo directly correlating to their antagonistic capability. It is worth noting that the level of sensitivity of pathogens to iron deficiency might also affect the biocontrol effect of pulcherrimin-producing M. pulcherrima.

Bioprotection Efficiency of Metschnikowia Strains in Synthetic Must: Comparative Study and Metabolomic Investigation of the Mechanisms Involved.

Three Metschnikowia strains marketed as bioprotection yeasts were studied to compare their antimicrobial effect on a mixture of two Hanseniaspora yeast strains in synthetic must at 12 °C, mimicking pre-fermentative maceration by combining different approaches. The growth of the different strains was monitored, their nitrogen and oxygen requirements were characterised, and their metabolomic footprint in single and co-cultures studied. Only the M. fructicola strain and one M. pulcherrima strains colonised the must and induced the rapid decline of Hanseniaspora. The efficiency of these two strains followed different inhibition kinetics. Furthermore, the initial ratio between Metschnikowia and Hanseniaspora was an important factor to ensure optimal bioprotection. Nutrient consumption kinetics showed that apiculate yeasts competed with Metschnikowia strains for nutrient accessibility. However, this competition did not explain the observed bioprotective effect, because of the considerable nitrogen content remaining on the single and co-cultures. The antagonistic effect of Metschnikowia on Hanseniaspora probably implied another form of amensalism. For the first time, metabolomic analyses of the interaction in a bioprotection context were performed after the pre-fermentative maceration step. A specific footprint of the interaction was observed, showing the strong impact of the interaction on the metabolic modulation of the yeasts, especially on the nitrogen and vitamin pathways.

From bioprotective effects to diversification of wine aroma: Expanding the knowledge on Metschnikowia pulcherrima oenological potential

Microbial diseases are of major concern in vitiviniculture as they cause grape losses and wine alterations, but the prevention with chemical substances represents a risk to human health and agricultural ecosystem. A promising alternative is the biocontrol and bioprotection activity of non-Saccharomyces yeasts, such as Metschnikowia pulcherrima, which also presents positive oenological traits when used in multistarter fermentations. The aim of this study was to assess the impact of a selected M. pulcherrima strain in the post-harvest withering and vinification of Garganega grapes to produce the sweet ‘passito’ wine Recioto di Gambellara DOCG (Italy). M. pulcherrima was firstly inoculated on grape at the beginning of the withering process, and afterwards in must for multistarter sequential microfermentation trials with Saccharomyces cerevisiae. Microbiological, chemical, and sensory analyses were carried out to monitor the vinification of treated and control grapes. Grape bunches during withering were a suitable environment for the colonization by M. pulcherrima, which effectively prevented growth of molds. Differences in grape must composition were observed, and the diverse inoculation strategies caused noticeable variations of fermentation kinetics, main oenological parameters, wine aroma profile, and sensory perception. M. pulcherrima proved effective to protect grapes against fungal infections during withering and contribute to alcoholic fermentation generating wine with distinguished aromatic characteristics.

Biocontrol Efficacy of Metschnikowia spp. Yeasts in Organic Vineyards against Major Airborne Diseases of Table Grapes in the Field and in Postharvest.

The aim of this work was to evaluate the efficacy of two antagonistic yeasts, Metschnikowia pulcherrima strain MPR3 and M. fructicola strain NRRL Y-27328 (commercial product NOLI), applied in addition to the "on-farm biological treatments" (BIO) carried out during the production season, for the containment of powdery mildew and grey mould diseases on organic table grapes 'Italia'. The yeast strains were applied in the field three times, and their efficacy was evaluated during the production season and under postharvest conditions. Overall, M. pulcherrima MPR3 combined with BIO treatments reduced disease incidence caused by Erysiphe necator and disease incidence and severity caused by Botrytis cinerea with values between 67.8% and 86.2%, showing higher efficacy than BIO treatments applied alone and in combination with NOLI. Field treatments based on BIO+MPR3 maintained their performance also during fruit storage, protecting grape berries from grey mould development to a greater extent than the other treatments (disease reduction of about 98%). Thus, the presence of M. pulcherrima MPR3 seems to improve disease management both in the field and in postharvest environments, without negative impacts on grape microbial communities. These findings highlight the potential of M. pulcherrima MPR3 as a promising alternative strategy for disease control in organic vineyards and in postharvest, providing sustainable solutions to improve food quality and safety.

The Metschnikowia pulcherrima Clade as a Model for Assessing Inhibition of Candida spp. and the Toxicity of Its Metabolite, Pulcherrimin.

Candidiasis is one of the most frequent infections worldwide. In this study, the antimicrobial properties of six strains belonging to the Metschnikowia pulcherrima clade were evaluated against twenty Candida and Candida-related Filobasidiella neoformans var. bacillispora (syn. Cryptococcus neoformans) of different origins, employing the agar cross method. The toxic effect of pulcherrimin, a red metabolite that is responsible for the antimicrobial activities of Metschnikowia spp., was evaluated in various experimental models. The results of agar tests showed that the selected M. pulcherrima strains inhibited the growth of the Candida and non-Candida strains. However, inhibition was dependent on the strain and the environment. The presence of peptone, sodium silicate, and a higher incubation temperature decreased the antifungal action of the M. pulcherrima strains. Pulcherrimin showed cytotoxic and antiproliferative activity, with oxidative stress in cells leading to apoptosis. More research is needed on the mechanism of action of pulcherrimin on somatic cells.

Biotechnological strategies to reduce the doses of sulfur dioxide in white winemaking and avoid problems of browning

This communication studies some possible strategies to reduce or even to replace sulfur dioxide in winemaking. Specifically, the aim of this work was to study the protective effect against oxidation of a commercial inactivated dry yeast (IDY) with very high level of glutathione, and of a selected non-Saccharomyces yeast, Metschnikowia pulcherrima strain in comparison with sulfur dioxide.

Efficacy and mechanism of action of food isolated yeasts in the control of Aspergillus flavus growth on pistachio nuts

Due to its nutritional characteristics, dried fruit and in particular pistachio is considered an important component in the daily diet. Unfortunately, pistachio nuts can be contaminated during storage with a wide range of pathogenic fungi, including Aspergillus flavus. The present work has evaluated how the use of two yeast strains belonging to the species Wickerhamomyces anomalus and Metschnikowia pulcherrima can inhibit the in vitro and in vivo growth of A. flavus. Both yeast strains demonstrated a good in vitro antifungal activity attributable to a specific mechanism of action, although higher efficacy was evidenced by W. anomalus strain. The production of volatile organic compounds (VOCs) and lytic enzymes was hypothesized as the main mechanisms of action exerted by W. anomalus, while the nutritional competition for iron was assumed as the main biocontrol mechanism for M. pulcherrima. Moreover, from the results of the in vivo test carried out on artificially infected pistachio seeds, it was clear as M. pulcherrima strain showed the same efficacy of W. anomalus in inhibiting the growth and sporulation of A. flavus mold, despite W. anomalus was the most effective strain during in vitro assay. Altogether, these results indicate that both strains could be considered as potential biocontrol agents against A. flavus fungal growth, notwithstanding it is always important considering the tritrophic interaction (yeast-mold- host), which could play a crucial role in determining the final results.

The Oleaginous Yeast Metschnikowia pulcherrima Displays Killer Activity against Avian-Derived Pathogenic Bacteria.

Simple SummaryPathogenic bacteria in poultry and the widespread use of antibiotics to manage them are costly in terms of production, environmental risk and human health. Probiotic and other low-cost, non-antibiotic treatments offer attractive alternatives to antibiotic applications, but relatively few of these options exist. In this research, we investigated the potential of an otherwise-useful industrial yeast, Metschnikowia pulcherrima, for the active suppression of poultry pathogenic bacteria. We tested multiple strains of yeast against several important bacterial pathogens and found that the more inhibitory strains of yeast supressed bacterial growth and actively killed the most recalcitrant bacteria. Less aggressive yeast strains could increase the growth of some bacterial strains in some environments. The yeast produced novel molecules in response to the presence of the bacteria and we identified several potential mechanisms by which the yeast inhibited or killed bacteria. Together, these results point towards a useful application of a novel yeast for enhanced, antibiotic-free pathogen control.Metschnikowia pulcherrima is a non-conventional yeast with potential to be used in biotechnological processes, especially those involving low-cost feedstock exploitation and biocontrol applications. The combination of traits that supports these industrial applications in M. pulcherrima also makes it an attractive option to study in the context of livestock health. In this study, we examined the specific interactions between M. pulcherrima and multiple avian pathogenic bacteria. We tested individual bacteria–yeast interactions and bacterial combinations in both solid and liquid media and in variable nutrient environments. Across multiple isolates of M. pulcherrima, we observed different levels of antimicrobial activity, varying from supporting the growth of competing bacteria through suppression and bacterial killing, and we found that these responses varied depending on the bacterial strains and media. We identified multiple molecular routes, including proteins produced by M. pulcherrima strains, that acted to control these microbial interactions. Furthermore, protein screening revealed that M. pulcherrima strains were induced to produce proteins specifically when exposed to bacterial strains, suggesting that fine-tuned mechanisms allow M. pulcherrima to function as a potential lynchpin in a microbial community.

Chemical and Sensory Profiles of Merlot Wines Produced by Sequential Inoculation of Metschnikowia pulcherrima or Meyerzyma guilliermondii

Inoculation of selected non-Saccharomyces yeasts with Saccharomyces cerevisiae as means to produce Merlot wines with reduced ethanol contents was investigated. Fermentations of grape musts (25.4° Brix, pH 3.50, and 4.23 g/L titratable acidity) were conducted in stainless steel tanks inoculated with Metschnikowia pulcherrima strains P01A016 or NS-MP or Meyerozyma guilliermondii P40D002 with S. cerevisiae Syrah added after three days. After fermentation, wines with Mt. pulcherrima contained 13.8% (P01A016) or 13.9% (NS-MP) v/v ethanol, respectively, amounts which were lower than in wines with S. cerevisiae alone (14.9% v/v). Delayed inoculation of must with S. cerevisiae (day 3) or musts with My. guilliermondii contained elevated concentrations of ethyl acetate (145 and 148 mg/L, respectively), concentrations significantly higher than those with S. cerevisiae inoculated on day 0 or with either strain of Mt. pulcherrima. Descriptive sensory analysis revealed a significant effect due to panelist but not due to Mt. pulcherrima or My. guilliermondii. This research indicates the potential for commercial application of these yeasts towards the production of reduced alcohol wines but without imparting negative sensory attributes.

Successful biocontrol of major postharvest and soil-borne plant pathogenic fungi by antagonistic yeasts

Fungal pathogens are the main biotic burden of productivity for economically important crops under field, greenhouse or postharvest conditions. The discovery and development of new environmental-friendly solutions, such as application of living organisms and their derivatives to control plant diseases and pests, are of enormous interest. This study presents the results of a mass screening designed to detect yeast strains with antagonistic activity against postharvest pathogens (Alternaria alternata, Penicillium expansum and Botrytis cinerea) and soil-borne diseases (Verticillium dahliae and Fusarium oxysporum). In fact, this is the first study that focuses on screening the antagonistic potential of a wide variety of yeast genera (13) and species (30) against vascular wilts. The results from in vivo trials demonstrated that fungal infected tomato plants, grown under hydroponic or soil conditions, showed a significant reduction in disease severity after yeast treatment. Wickerhamomyces anomalus Wa-32 was able to antagonise both pathogens and reduce the disease severity up to 40% (V. dahliae) and 50% (F. oxysporum) in soil conditions. In addition, this strain became endophytic in tomato plants. The features of Wa-32 are of enormous interest since no effective antagonistic biocontrol product is available for the simultaneous control of these two fungal pathogens. Postharvest assays with wounded tomato fruits showed that several strains displayed very high biocontrol levels against P. expansum and B. cinerea (up to 86 and 97% reduction in disease severity, respectively) but none of them showed protection against A. alternata. The best protection against B. cinerea was again achieved with W. anomalus Wa-32 and two Metschnikowia pulcherrima strains (Mp-22 and Mp-30). However, the best antagonistic strains of P. expansum were Candida lusitaniae Cl-28, Candida oleophila Co-13, Debaryomyces hansenii Dh-67 and Hypopichia pseudoburtonii Hp-54. These biocontrol effects were also demonstrated in grapes and apples.

Volatile aroma composition and sensory profile of Shiraz and Cabernet Sauvignon wines produced with novelMetschnikowia pulcherrimayeast starter cultures

Background and Aims The use of non-Saccharomyces yeast strains as starter cultures for wine production has become increasingly popular, particularly due to their positive effect on wine composition, colour, aroma and flavour. Here, we characterise the volatile aroma composition and the sensory profile of Shiraz and Cabernet Sauvignon wines produced with novel active dry yeast preparations of Metschnikowia pulcherrima compared to that of reference strains. Methods and Results Winemaking treatments included an uninoculated fermentation, two reference Saccharomyces cerevisiae fermentations, and sequential fermentations inoculated with either M. pulcherrima AWRI1149 or M. pulcherrima AWRI3050 and S. cerevisiae. Amplicon-based internal transcribed spacer phylotyping was used to determine microbial population dynamics during fermentation. Wines were analysed for volatile composition and subjected to sensory analysis. The M. pulcherrima strains survived and dominated in both grape cultivars, and produced distinctive wine volatile profiles depending on the inoculation treatment. These differences in volatiles resulted in significant differences for several sensory attributes. Conclusions Wines made with active dry yeast preparations of M. pulcherrima AWRI1149 and M. pulcherrima AWRI3050 were characterised by increased intensity of desirable sensory attributes and by low scores for negative descriptors. Significance of the Study This work provides winemakers with additional yeast preparations that can shape sensory profile and wine style.

Native Vineyard Non-Saccharomyces Yeasts Used for Biological Control of Botrytis cinerea in Stored Table Grape.

Postharvest spoilage fungi, such as Botrytis cinerea, are considered the main cause of losses of fresh fruit quality and vegetables during storage, distribution, and consumption. The current control strategy is the use of SO2 generator pads whose application is now largely under observation. A high quantity of SO2 can be deleterious for fresh fruits and vegetables and it is not allowed in organic agriculture. For this reason, great attention has been recently focused on identifying Biological Control Agents (BCA) to implement biological approaches devoid of chemicals. In this direction, we carried out our study in isolating five different non-Saccharomyces yeast strains from local vineyards in the South of Italy as possible BCA. We performed both in vitro and in vivo assays in semi-commercial conditions on detached grape berries stored at 0 °C, simulating the temperature normally used during cold storage, and obtained relevant results. We isolated three M. pulcherrima strains and one L. thermotolerans strain able to largely antagonize the development of the B. cinerea, at both in vitro and in vivo conditions. In particular, we detected the ability of the three isolates of M. pulcherrima strains Ale4, N20/006, and Pr7 and the L. thermotolerans strain N10 to completely inhibit (100% in reduction) the mycelial growth of B. cinerea by producing fungistatic compounds. We found, using an extracellular lytic enzymes activity assay, that such activity could be related to lipid hydrolyzation, β-1,3-glucanase and pectinase activity, and pectinase and protease activity, depending on the yeasts used. Results from our in vitro assays allowed us to hypothesize for M. pulcherrima strains Ale4 and N20/006 a possible combination of both the production of soluble metabolites and volatile organic compounds to antagonize against B. cinerea growth. Moreover, in semi-commercial conditions, the M. pulcherrima strain N20/006 and L. thermotolerans strain N10 showed relevant antagonistic effect also at low concentrations (with a significantly reduction of ‘slip skin’ incidence of 86.4% and 72.7%, respectively), thus highlighting a peculiar property to use in commercial development for organic agriculture and the handling process.

Enological potential of non-Saccharomyces yeast strains of enological and brewery origin from Ukrainian Collection of Microorganisms

ABSTRACT Non-conventional wine yeasts are extensively studied as promising producers of hydrolytic enzymes and as potential starter cultures in winemaking due to their ability to improve organoleptic properties of wine. Thirty-six yeast strains of enological and brewery origin from the Ukrainian Collection of Microorganisms belonging to Torulaspora, Kloeckera, Candida, Metschnikowia, Pichia, and Zygosaccharomyces genera have been screened for the production of extracellular hydrolases, stress tolerance, fermentative activity, and other traits of enological interest. This study revealed the high incidence of lipolytic, proteolytic, and β-glucosidase activities among the yeasts, while no pectinase activity was detected. Esterase, cellulase and glucanase activities were found in a small proportion of yeasts (8.33–16.66%). Several Pichia anomala, Kloekera javanica, Pichia membranifaciens, and Metschnikowia pulcherrima strains demonstrated a wide range of hydrolytic activities. High tolerance to stress factors (ethanol, osmotic, and oxidative stress) present during alcoholic fermentation was detected in P. anomala and M. pulcherrima strains. Fermentative activity of several yeast strains was evaluated in microfermentations in a model semi-synthetic medium. Strain P. anomala UCM Y-216 was selected as the most promising culture for winemaking due to its hydrolytic activities, tolerance to stress factors and other valuable metabolic traits. This study represents the first step for selecting a non-conventional yeast strain of enological origin as a potential co-culture for winemaking.

Evaluating the effect of using non-Saccharomyces on Oenococcus oeni and wine malolactic fermentation

Interest in using non-Saccharomyces yeasts in winemaking has increased in recent years due to their ability to improve wine quality. However, little information has been published regarding the possible effect on malolactic fermentation (MLF), carried out mostly by Oenococcus oeni. The aim of this paper is therefore to evaluate the effect of the most representative non-Saccharomyces species on O. oeni and wine MLF. Different strains of Torulaspora delbrueckii, Metschnikowia pulcherrima, Hanseniaspora uvarum, Hanseniaspora vineae and Starmerella bacillaris (syn. Candida zemplinina) were used in sequential alcoholic fermentation with Saccharomyces cerevisiae. The resulting wines were inoculated with four O. oeni strains. The action of non-Saccharomyces affected the final wine composition and the later role of O. oeni. Some of its strains could not perform MLF in H. uvarum wine due to high SO2 concentrations. In some cases, MLF was inhibited in wines inoculated with S. bacillaris. All the H. uvarum and H. vineae strains notably increased acetic acid concentrations, thus threatening wine quality. The best conditions for MLF were provided by some T. delbruecckii and M. pulcherrima strains, which showed increased concentrations of mannoproteins – compounds described as MLF activators –, no production of SO2, and low consumption of L-malic acid. In conclusion, non-Saccharomyces yeasts have diverse effects on O. oeni and MLF depending on the species, with T. delbrueckii and M. pulcherrima being those that showed the best compatibility with MLF development.