Agro-ecosystem of honeybees as source for native probiotic yeasts.

Probiotic microorganisms are used to improve the health and wellness of people and the research on this topic is of current relevance and interest. Fifty-five yeasts, coming from honeybee's ecosystem and belonging to Candida, Debaryomyces, Hanseniaspora, Lachancea, Metschnikowia, Meyerozyma, Starmerella and Zygosacchromyces genera and related different species, were evaluated for the probiotic traits. The resistance to gastrointestinal conditions, auto-aggregation, cell surface hydrophobicity or biofilm formation abilities as well as antimicrobial activity against common human pathogenic bacteria were evaluated. The safety analysis of strains was also carried out to exclude any possible negative effect on the consumer's health. The influence of proteinase treatment of living yeasts and their adhesion to Caco-2 cells were also evaluated. The greatest selection occurred in the first step of survival at the acidic pH and in the presence of bile salts, where more than 50% of the strains were unable to survive. Equally discriminating was the protease test which allowed the survival of only 27 strains belonging to the species Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia pulcherrima, Metschnikowia ziziphicola, Meyerozyma caribbica, Meyerozyma guilliermondii, Pichia kluyveri, Pichia kudriavzevii and Pichia terricola. An integrated analysis of the results obtained allowed the detection of seven yeast strains with probiotic aptitudes, all belonging to the Meyerozyma genus, of which three belonging to M. guillermondii and four belonging to M. caribbica species.

Recycled Brewer's Spent Grain (BSG) and Grape Juice: A New Tool for Non-Alcoholic (NAB) or Low-Alcoholic (LAB) Craft Beer Using Non-Conventional Yeasts.

Non-alcoholic beer (NAB) and low-alcoholic beer (LAB) are taking over the market with growing sales. Sustainable recycling and valorization of exhausted brewer's spent grain (BSG) coming from craft beer is a relevant issue in the brewing process. In this work, recycled BSG and BSG + GJ (supplemented with 10% grape juice) were used as a wort substrate to inoculate Lachancea thermotolerans, Wickeramhomyces anomalus, Torulaspora delbruecki and Pichia kluyveri non-conventional yeasts to produce NABLAB craft beer. Results showed that wort composed of only recycled BSG produced appreciated NAB beers (ethanol concentration from 0.12% to 0.54% v/v), while the addition of 10% grape juice produced LAB beers (ethanol concentration from 0.82 to 1.66% v/v). As expected, volatile compound production was highest with the addition of grape juice. L. thermotolerans showed lactic acid production, characterizing both worts with the production of ethyl butyrate and isoamyl acetate. T. delbrueckii exhibited relevant amounts of hexanol, phenyl ethyl acetate and ß-phenyl ethanol (BSG + GJ). W. anomalus and P. kluyveri showed consistent volatile production, but only in BSG + GJ where fermentation activity was exhibited. The overall results indicated that reused BSGs, non-conventional yeasts and grape juice are suitable bioprocesses for specialty NABLAB beer.

Yeast communities related to honeybees: occurrence and distribution in flowers, gut mycobiota, and bee products.

Honeybee (Apis mellifera) is an important agricultural pollinator and a model for sociality. In this study, a deep knowledge on yeast community characterizing the honeybees' environmental was carried out. For this, a total of 93 samples were collected: flowers as food sources, bee gut mycobiota, and bee products (bee pollen, bee bread, propolis), and processed using culture-dependent techniques and a molecular approach for identification. The occurrence of yeast populations was quantitatively similar among flowers, bee gut mycobiota, and bee products. Overall, 27 genera and 51 species were identified. Basidiomycetes genera were predominant in the flowers while the yeast genera detected in all environments were Aureobasidium, Filobasidium, Meyerozyma, and Metschnikowia. Fermenting species belonging to the genera Debaryomyces, Saccharomyces, Starmerella, Pichia, and Lachancea occurred mainly in the gut, while most of the identified species of bee products were not found in the gut mycobiota. Five yeast species, Meyerozyma guilliermondii, Debaryomyces hansenii, Hanseniaspora uvarum, Hanseniaspora guilliermondii, and Starmerella roseus, were present in both summer and winter, thus indicating them as stable components of bee mycobiota. These findings can help understand the yeast community as a component of the bee gut microbiota and its relationship with related environments, since mycobiota characterization was still less unexplored. In addition, the gut microbiota, affecting the nutrition, endocrine signaling, immune function, and pathogen resistance of honeybees, represents a useful tool for its health evaluation and could be a possible source of functional yeasts. KEY POINTS: • The stable yeast populations are represented by M. guilliermondii, D. hansenii, H. uvarum, H. guilliermondii, and S. roseus. • A. pullulans was the most abondance yeast detective in the flowers and honeybee guts. • Aureobasidium, Meyerozyma, Pichia, and Hanseniaspora are the main genera resident in gut tract.

Consortium of selected yeasts to produce healthy soy fermented beverage: Evaluation of microbial evolution, analytical, sensorial, and functional features.

Currently, an increasing number of intolerant and vegan consumers are driving the market towards plant-based milk alternatives. Here, selected probiotic yeasts, belonging to the Candida zeylanoides, Kluyveromyces lactis and Debaryomyces hansenii species, previously characterized for their aptitude to ferment animal milk, were tested in soy milk. Trials at different fermentation times with the developed yeast consortium (Yc) coinoculated with a lactic bacterium commercial strain were carried out. Yc showed good fermentation performance, conferring distinctive analytical and aromatic properties to the resulted soy fermented beverage, a product similar to an industrial kefir. Analytical determinations did not show significant variations between the end of fermentation and cold storage (4 weeks at 4 °C), indicating full stability. Phenol amounts and antioxidant activity were significantly increased in soy fermented beverage fermented by Yc. All yeasts remained viable until the end of storage with a final concentration of approximately 8 Log CFU/ml, a value suitable for a probiotic commercial claim. Overall, the results suggest that Yc is a promising multistarter candidate for functional soy products.

Biocontrol Using Torulaspora delbrueckii in Sequential Fermentation: New Insights into Low-Sulfite Verdicchio Wines.

Torulaspora delbrueckii has attracted renewed interest in recent years, for its biotechnological potential linked to its ability to enhance the flavor and aroma complexity of wine. Sequential fermentations with a selected native strain of T. delbrueckii (DiSVA 130) and low-sulfite native strain of Saccharomyces cerevisiae (DiSVA 709) were carried out to establish their contribution in biocontrol and the aroma profile. A first set of trials were conducted to evaluate the effect of the sulfur dioxide addition on pure and T. debrueckii/S. cerevisiae sequential fermentations. A second set of sequential fermentations without SO2 addition were conducted to evaluate the biocontrol and aromatic effectiveness of T. delbrueckii. Native T. delbrueckii showed a biocontrol action in the first two days of fermentation (wild yeasts reduced by c.a. 1 log at the second day). Finally, trials with the combination of both native and commercial T. delbrueckii/S. cerevisiae led to distinctive aromatic profiles of wines, with a significant enhancement in isoamyl acetate, phenyl ethyl acetate, supported by positive appreciations from the tasters, for ripe and tropical fruits, citrus, and balance. The whole results indicate that native T. delbrueckii could be a potential biocontrol tool against wild yeasts in the first phase of fermentation, contributing to improving the final wine aroma.

Biocontrol and Probiotic Function of Non-Saccharomyces Yeasts: New Insights in Agri-Food Industry.

Fermented food matrices, including beverages, can be defined as the result of the activity of complex microbial ecosystems where different microorganisms interact according to different biotic and abiotic factors. Certainly, in industrial production, the technological processes aim to control the fermentation to place safe foods on the market. Therefore, if food safety is the essential prerogative, consumers are increasingly oriented towards a healthy and conscious diet driving the production and consequently the applied research towards natural processes. In this regard, the aim to guarantee the safety, quality and diversity of products should be reached limiting or avoiding the addition of antimicrobials or synthetic additives using the biological approach. In this paper, the recent re-evaluation of non-Saccharomyces yeasts (NSYs) has been reviewed in terms of bio-protectant and biocontrol activity with a particular focus on their antimicrobial power using different application modalities including biopackaging, probiotic features and promoting functional aspects. In this review, the authors underline the contribution of NSYs in the food production chain and their role in the technological and fermentative features for their practical and useful use as a biocontrol agent in food preparations.

Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts.

Three killer toxins that were previously investigated, one excreted by Kluyveromyces wickerhamii and two by different strains of Wickerhamomyces anomalus, were produced at the pilot scale, lyophilized and characterized, and the formulates were assessed for their zymocidial effect against Brettanomyces bruxellensis spoilage yeast. A comparative analysis allowed the evaluation of the minimum inhibitory concentration (MIC) against a sensitive strain. Fungicidal and fungistatic concentrations were used to evaluate the cytocidal effect using a cytofluorimetric approach that confirmed the lethal effect of all lyophilized formulates against B. bruxellensis spoilage yeasts. Moreover, the potential killer toxins' cytotoxicity against human intestinal cells (Caco-2) were evaluated to exclude any possible negative effect on the consumers. Finally, the effective lethal effect of all three lyophilized killer toxins toward B. bruxellensis sensitive strain were tested. The results indicated that all of them acted without dangerous effects on the human epithelial cells, opening the way for their possible commercial application. In particular, D15 showed the lowest MIC and the highest activity, was evaluated also in wine, revealing a strong reduction of Brettamonyces yeast growth and, at the same time, a control of ethyl phenols production.

Lentil Fortification and Non-Conventional Yeasts as Strategy to Enhance Functionality and Aroma Profile of Craft Beer.

During the last few years, consumer demand has been increasingly oriented to fermented foods with functional properties. This work proposed to use selected non-conventional yeasts (NCY) Lachanceathermotolerans and Kazachstaniaunispora in pure and mixed fermentation to produce craft beer fortified with hydrolyzed red lentils (HRL). For this, fermentation trials using pils wort (PW) and pils wort added with HRL (PWL) were carried out. HRL in pils wort improved the fermentation kinetics both in mixed and pure fermentations without negatively affecting the main analytical characters. The addition of HRL determined a generalized increase in amino acids concentration in PW. L. thermotolerans and K. unispora affected the amino acid profile of beers (with and without adding HRL). The analysis of by-products and volatile compounds in PW trials revealed a significant increase of some higher alcohols with L. thermotolerans and ethyl butyrate with K. unispora. In PWL, the two NCY showed a different behavior: an increment of ethyl acetate (K. unispora) and ß-phenyl ethanol (L. thermotolerans). Sensory analysis showed that the presence of HRL characterized all beers, increasing the perception of the fruity aroma in both pure and mixed fermentation.

Biocontrol of Non-Saccharomyces Yeasts in Vineyard against the Gray Mold Disease Agent Botrytis cinerea.

BACKGROUND: Botrytis cinerea (B. cinerea) is responsible for grape infection and damage to the winemaking and table grape sectors. Although anti-Botrytis chemicals are available, they are considered unsustainable for resistance phenomenon and adverse effects on the environment and human health. Research is focused on developing alternative approaches, such as exploiting biological control agents (BCAs). In this context, 19 yeasts of the genera Cryptococcus, Aureobasidium, Metschnikowia, Kluyveromyces and Wickerhamomyces were tested as antimicrobial agents against B. cinerea development. METHODS: A combination of in vitro tests based on dual-culture methods, volatile organic compound production assay, laboratory tests on grape berries (punctured and sprayed with yeasts) and field experiments based on yeast treatments on grapes in vineyards allowed the selection of two potential BCAs. RESULTS: M. pulcherrima DiSVA 269 and A. pullulans DiSVA 211 exhibited the best ability to contain the development of B. cinerea, showing the severity, the decay and the McKinney index lower than a commercial biological formulation consisting of a mixture of two different A. pullulans strains, which were used as positive controls. CONCLUSIONS: The results indicated that the selected strains were effective BCA candidates to counteract B. cinerea in the field, applying them in the partial or total replacement of conventional treatments.

Yeast Interactions and Molecular Mechanisms in Wine Fermentation: A Comprehensive Review.

Wine can be defined as a complex microbial ecosystem, where different microorganisms interact in the function of different biotic and abiotic factors. During natural fermentation, the effect of unpredictable interactions between microorganisms and environmental factors leads to the establishment of a complex and stable microbiota that will define the kinetics of the process and the final product. Controlled multistarter fermentation represents a microbial approach to achieve the dual purpose of having a less risky process and a distinctive final product. Indeed, the interactions evolved between microbial consortium members strongly modulate the final sensorial properties of the wine. Therefore, in well-managed mixed fermentations, the knowledge of molecular mechanisms on the basis of yeast interactions, in a well-defined ecological niche, becomes fundamental to control the winemaking process, representing a tool to achieve such objectives. In the present work, the recent development on the molecular and metabolic interactions between non-Saccharomyces and Saccharomyces yeasts in wine fermentation was reviewed. A particular focus will be reserved on molecular studies regarding the role of nutrients, the production of the main byproducts and volatile compounds, ethanol reduction, and antagonistic actions for biological control in mixed fermentations.

Starmerella bombicola and Saccharomyces cerevisiae in Wine Sequential Fermentation in Aeration Condition: Evaluation of Ethanol Reduction and Analytical Profile.

In the last few decades, the increase of ethanol in wine, due to global climate change and consumers' choice is one of the main concerns in winemaking. One of the most promising approaches in reducing the ethanol content in wine is the use of non-Saccharomyces yeasts in co-fermentation or sequential fermentation with Saccharomyces cerevisiae. In this work, we evaluate the use of Starmerella bombicola and S. cerevisiae in sequential fermentation under aeration condition with the aim of reducing the ethanol content with valuable analytical profile. After a preliminary screening in synthetic grape juice, bench-top fermentation trials were conducted in natural grape juice by evaluating the aeration condition (20 mL/L/min during the first 72 h) on ethanol reduction and on the analytical profile of wines. The results showed that S. bombicola/S. cerevisiae sequential fermentation under aeration condition determined an ethanol reduction of 1.46% (v/v) compared with S. cerevisiae pure fermentation. Aeration condition did not negatively affect the analytical profile of sequential fermentation S. bombicola/S. cerevisiae particularly an overproduction of volatile acidity and ethyl acetate. On the other hand, these conditions strongly improved the production of glycerol and succinic acid that positively affect the structure and body of wine.

Purification and Characterization of WA18, a New Mycocin Produced by Wickerhamomyces anomalus Active in Wine Against Brettanomyces bruxellensis Spoilage Yeasts.

Wickerhamomyces anomalus strain 18, isolated from a natural underground cheese ripening pit, secretes a mycocin named WA18 that inhibits wine spoilage yeasts belonging to Brettanomyces bruxellensis species, with a broad-spectrum of activity. WA18 was purified, and the purified protein was digested with specific restriction enzymes (lysine K and arginine R cut sites). The LC-MS and LC-MS/MS analysis after enzymatic digestions revealed a molecular weight of 31 kDa. Bioinformatics processing and database research of digested pure killer protein showed 99% identity with a UDP-glycosyltransferase protein. Competitive inhibition assay of killer activity by cell-wall polysaccharides suggests that branched glucans represent the first receptor site of the toxin on the envelope of the sensitive target. The WA18 partially purified crude extract (PPCE) showed high stability of antimicrobial activity at the physicochemical conditions suitable for the winemaking process. Indeed, in wine WA18 was able to counteract B. bruxellensis and control the production of ethyl phenols. In addition, the strain WA18 was compatible with Saccharomyces cerevisiae in co-culture conditions with a potential application together with commercial starter cultures. These data suggest that WA18 mycocin is a promising biocontrol agent against spoilage yeasts in winemaking, particularly during wine storage.

Reduction of Sulfur Compounds through Genetic Improvement of Native Saccharomyces cerevisiae Useful for Organic and Sulfite-Free Wine.

Sulfites and sulfides are produced by yeasts in different amounts depending on different factors, including growth medium and specific strain variability. In natural must, some strains can produce an excess of sulfur compounds that confer unpleasant smells, inhibit malolactic fermentation and lead to health concerns for consumers. In organic wines and in sulfite-free wines the necessity to limit or avoid the presence of sulfide and sulfite requires the use of selected yeast strains that are low producers of sulfur compounds, with good fermentative and aromatic aptitudes. In the present study, exploiting the sexual mass-mating spores' recombination of a native Saccharomyces cerevisiae strain previously isolated from grape, three new S. cerevisiae strains were selected. They were characterized by low sulfide and sulfite production and favorable aromatic imprinting. This approach, that occurs spontaneously also in nature, allowed us to obtain new native S. cerevisiae strains with desired characteristics that could be proposed as new starters for organic and sulfite-free wine production, able to control sulfur compound production and to valorize specific wine types.

Potential Probiotic Yeasts Sourced from Natural Environmental and Spontaneous Processed Foods.

In the last decades, there has been a growing interest from consumers in their food choices. Organic, natural, less processed, functional, and pre-probiotic products were preferred. Although, Saccharomyces cerevisiae var. boulardii is the most well-characterized probiotic yeast available on the market, improvement in probiotic function using other yeast species is an attractive future direction. In the present study, un-anthropized natural environments and spontaneous processed foods were exploited for wild yeast isolation with the goal of amplifying the knowledge of probiotic aptitudes of different yeast species. For this purpose, 179 yeast species were isolated, identified as belonging to twelve different genera, and characterized for the most important probiotic features. Findings showed interesting probiotic characteristics for some yeast strains belonging to Lachancea thermotolerans, Metschnikowia ziziphicola, Saccharomyces cerevisiae, and Torulaspora delbrueckii species, although these probiotic aptitudes were strictly strain-dependent. These yeast strains could be proposed for different probiotic applications, such as a valid alternative to, or in combination with, the probiotic yeast S. cerevisiae var. boulardii.

The impact of fungicide treatments on yeast biota of Verdicchio and Montepulciano grape varieties.

Yeast species that colonize the surface of grape berries at harvest time play an important role during the winemaking process. In this study, the use of culturable microbial techniques permitted a quantitative and qualitative inventory of the different yeast species present on the grape berry surfaces of Montepulciano and Verdicchio varieties when treated with conventional and organic fungicides. The results show that the most widespread yeast species at harvest time were Aureobasidium pullulans and Hanseniaspora uvarum, which are considered normal resident species and independent of the grape varieties and treatments applied. Specific differences when comparing the grape varieties were observed in species and were detected at a lower frequency; Pichia spp. were prevalent in Verdicchio, whereas Lachancea thermotolerans and Zygoascus meyerae were found in Montepulciano. In both vineyards, the farming treatments improved the competitiveness of A. pullulans, which was probably due to its reduced susceptibility to treatments that improved the competition toward other fungi. In contrast, the fermenting yeast H. uvarum was negatively affected by fungicide treatments and showed a reduced presence if compared with untreated grapes. Organic treatments directly impacted the occurrence of Issachenkia terricola in Montepulciano grapes and Debaryomyces hansenii and Pichia membranifaciens in Verdicchio. Conversely, a negative effect of organic treatments was found toward Metschnikowia pulcherrima and Starmerella bacillaris. Overall, the data suggest that the yeast community colonizing the grape berry surface was influenced by both grape variety and farming treatments, which characterized the yeast biota of spontaneous must fermentation.

The Influence of Fungicide Treatments on Mycobiota of Grapes and Its Evolution during Fermentation Evaluated by Metagenomic and Culture-Dependent Methods.

The present study evaluated the impact of organic and conventional fungicide treatments compared with untreated samples (no fungicides were used) on the grape berry yeast community of the Montepulciano variety. The yeast dynamics during the spontaneous fermentation using culture-dependent and -independent methods was also evaluated. Results showed a reduction of yeast biodiversity by conventional treatments determining a negative influence on fermenting yeasts in favor of oxidative yeasts such as Aerobasidium pullulans. Starmerella bacillaris was significantly more present in organic samples (detected by next generation sequencing (NGS)), while Hanseniaspopa uvarum was significantly less present in untreated samples (detected by the culture-dependent method). The fermenting yeasts, developed during the spontaneous fermentation, were differently present depending on the fungicide treatments used. Culture-dependent and -independent methods exhibited the same most abundant yeast species during the spontaneous fermentation but a different relative abundance. Differently, the NGS method was able to detect a greater biodiversity (lower abundant species) in comparison with the culture-dependent method. In this regard, the methodologies used gave a different picture of yeast dynamics during the fermentation process. The results indicated that the fungal treatments can influence the yeast community of grapes leading must fermentation and the final composition of wine.

Brettanomyces Spoilage in Albanian Wines Assessed by Culture-Dependent and Culture-Independent Methods.

In the Albanian winemaking industry, there is little awareness of the potential detrimental effect of Brettanomyces in wines. The aim of this study was to detect and quantify Brettanomyces cells in 22 Albanian bottled wines, representing all the viticultural areas of Albania. A combined approach, including culture-dependent (viable plate counting) and culture-independent (qPCR) methods, was applied. Spoilage indicators (ethylphenols and total and volatile acidity), as well as the primary factors known to influence the growth of Brettanomyces in wine (pH, SO2 , and ethanol concentration), were also investigated. Brettanomyces was detected in only five (one Merlot, four Sheshi i Zi) out of 22 samples analyzed using viable counting, with loads ranging from 1.30 ± 0.03 log CFU/mL to 3.99 ± 0.00 log CFU/mL, whereas it was never detected in the Kallmet samples. When qPCR was applied, Brettanomyces cells were detected and quantified in all of the samples with a generally low load ranging from 0.47 ± 0.13 to 3.99 ± 0.01 log cells/mL. As a general trend, the loads of spoilage by this yeast were low (≤1.92 log cells/mL), with the exception of five samples that were also positive by plate counting. A positive correlation between the growth of this spoilage yeast on Dekkera/Brettanomyces differential media and its detection at high levels by qPCR was observed. A significant positive correlation between Brettanomyces and the concentration of ethylphenols and volatile acidity was also found. In summary, the results of this study demonstrated the low incidence of Brettanomyces spoilage yeasts in Albanian red wines. PRACTICAL APPLICATION: The awareness of Brettanomyces spoilage in the Albanian winemaking industry is very low. This study represents the first contribution to understand the extent of this spoilage yeast in Albanian autochthonous cultivars, which tend to have high economic value, to ensure product quality and safety. qPCR is confirmed to be a very sensitive method to rapidly detect Brettanomyces spoilage in wine samples.

Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content.

Nowadays, consumers require fermented alcoholic beverages with particular and enhanced flavour profiles while avoiding the health concerns due to high ethanol content. Here, the use of Torulaspora delbrueckii was evaluated for beer production, in both pure and in mixed cultures with a Saccharomyces cerevisiae starter strain (US-05). The yeast interactions were also evaluated. In mixed fermentations with S. cerevisiae, the main analytical characters from T. delbrueckii were comparable with those of the S. cerevisiae starter strain, but the beers were characterized by a distinctive overall analytical and aromatic profile. Indeed, there were interactions between S. cerevisiae and T. delbrueckii, with enhanced ethyl hexanoate (0.048 mg l(-1)) and ethyl octaonate (0.014 mg l(-1)) levels at the 1:20 and 1:10 inoculation ratios, respectively; while phenyl ethyl acetate increased in all mix combinations. The presence of T. delbrueckii resulted in reduced ß-phenyl ethanol and isoamyl acetate levels, which are responsible for floral and fruity aromas, respectively. Beer produced with T. delbrueckii pure cultures had a low alcohol content (2.66%; v/v), while also showing a particularly analytical and aromatic profile.