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 safety, quality and diversify of products should be reached limiting or avoiding the addition of antimicrobials or synthetic additives using the biological approach. In this paper, following the recent re-evaluation of non-Saccharomyces yeasts (NSYs) has been re-viewed the bio-protectant and biocontrol activity with a particular focus on their antimicrobial power, probiotic features and promoting functional aspects. In this review, the authors would underline the contribution of NSYs in all food production chain and its role in the technological and fermentative features for their practical and useful use as biocontrol agent in food preparations.

Wine is a processed beverage that offers high nutritional and health benefits. It is produced from grape must, which undergoes fermentation by yeasts (and sometimes lactic acid bacteria) to create a product that is highly appreciated by consumers worldwide. However, if only one type of yeast, specifically Saccharomyces cerevisiae, were used in the fermentation process, the resulting wine would lack aroma and flavor and may be rejected by consumers. To produce wine with a desirable taste and aroma, non-Saccharomyces yeasts are necessary. These yeasts contribute volatile aromatic compounds that significantly impact the wine’s final taste. They promote the release of primary aromatic compounds through a sequential hydrolysis mechanism involving several glycosidases unique to these yeasts. This review will discuss the unique characteristics of these yeasts (Schizosaccharomyces pombe, Pichia kluyveri, Torulaspora delbrueckii, Wickerhamomyces anomalus, Metschnikowia pulcherrima, Hanseniaspora vineae, Lachancea thermotolerans, Candida stellata, and others) and their impact on wine fermentations and co-fermentations. Their existence and the metabolites they produce enhance the complexity of wine flavor, resulting in a more enjoyable drinking experience.

Cocoa beans of the CCN-51 clone harvested in the Tumaco region (Nariño, Colombia) were fermented following four different treatments: (1) spontaneously (at room temperature for 120 h) in traditional conditions (Control); (2) traditional processing with a pre-drying (sun-dried for 24 h) treatment (PD); (3) with fermentation (for 120 h) after inoculation of a yeast starter culture (YS); and (4) including both treatments, pre-drying and yeast inoculation (PD+YS). Chemical composition, fatty acids, total polyphenol, methylxanthines (theobromine and caffeine) and lactic acid content of cocoa beans were determined. Chemical and fatty acid composition and theobromine content were not affected by the different fermentation treatment applied. When analysing total polyphenol content, YS and PD+YS treatments significantly increased the content of these compounds at the end of the fermentation process, while PD treatment decreased the caffeine content of cocoa beans. Finally, lactic acid content decreased as a consequence of both inoculation of yeast starter and mainly the pre-drying treatment. In conclusion, solar pre-drying in the open air and the inoculation of a yeast starter treatments could improve the final quality of coca beans.

Black yeasts are a highly specified group of fungi, which are characterized by a high resistance against stress factors. There are several factors enabling the cells to survive harsh environmental conditions. One aspect is the pigmentation, besides the melanin black yeasts often display a highly diverse carotenoid spectrum. Determination and characterization of carotenoids depend on an efficient extraction and separation, therefore especially for black yeast, characterized by thick cell walls specific protocols are needed to ensure analyses regarding stress responses in these fungi. Here we present both, a method to extract and analyze carotenoids and the unusual carotenoid composition of the black yeast Knufia petriola A95. Mechanical treatment combined with an acetonitrile extraction gave us very good extraction rates with a high reproducibility. The presented extraction and elution protocol allows the separation of the main carotenoids (7) in K. petricola A95 and should be suitable for the detection of additional carotenoids in other species. K. petricola A95 displays an unusual carotenoid composition, with mainly didehydrolycopene, torulene and lycopene. The pigment composition varied in dependency to oxidative stress but remained relatively constant if the cells were cultivated under low temperature. Black yeasts are a promising source for carotenoid production and other substances. To unravel the potential of these fungi new methods and studies are needed. The established protocol allows the determination carotenoid composition in black yeasts. Oxidative stress results in an adaptation in pigment composition in K. petricola A95. Future experiments have to be carried out to determine if didehydrolycopene functions as a protective agent itself or if it serves as a precursor for antioxidative pigments like torulene and torularhodin, which could be produced after induction under stress conditions.

The multidrug-resistant fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profile. C. auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from host immune responses. In this study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex and C. auris. Subsequently, we evaluated the effects of physicochemical factors on the fungal aggregation competence. The results demonstrated that cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates exhibited high aggregation after 2 h of incubation at 37°C. The aggregation was not impacted by pH, temperature, -mercaptoethanol (a protein-denaturing agent) and EDTA (a chelator agent). Conversely, proteinase K, trypsin and sodium dodecyl sulfate significantly diminished the aggregation. Collectively, our results demonstrated that the aggregation ability in these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation, since the presence of proteases and anionic detergent affected the aggregation ability. However, further studies are necessary to better elucidate this phenomenon.

We studied the capacity of glutathione and a specific inactivated dry yeast rich in glutathione to protect white grape must from browning compared to using sulphur dioxide. The results indicate that sulphur dioxide drastically reduces the oxygen consumption rate, protects hydroxycinnamic acids from oxidation and prevents grape must against browning even in the presence of laccase. Glutathione, pure or in the form of a specific inactivated dry yeast rich in glutathione, also reduces the oxygen consumption rate but to a lesser extent than sulphur dioxide. It favours the formation of GRP from hydroxycinnamic acids and effectively protects grape must against browning in healthy grape conditions. Nevertheless, in the presence of laccase it was not effective enough. Therefore, both forms of glutathione can be considered as interesting alternative tools to sulphur dioxide for preventing browning in white grape must, but only when the grapes are healthy.

Torulaspora delbrueckii showed renewed interest in recent years in the fermentation of wine, for its biotechnological potential linked to the ability to enhance flavor and aroma and it probably is the non-Saccharomyces yeast currently widely used in winemaking. On the base of this, 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 aroma profile. A first set trials, carried out in winery, were set up to establish 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 in the same conditions, to evaluate the biocontrol and aromatic effectiveness of the T. delbrueckii native strain and a commercial one. The effective biocontrol action of native T. delbrueckii inoculated in sequential fermentation was shown, indeed without SO₂ addition the presence of native T. delbrueckii revealed an effective fungistatic action in the first two days of fermentation. Moreover, the native T. delbrueckii strain seems to have fermentative performances comparable to those of T. delbrueckii commercial strain showing a more evident biocontrol action (wild yeasts reduced by c.a. 1 Log at 2^nd day) and its presence did not negatively affect S. cerevisiae fermentation activity. Finally, the combination of both native and commercial T. delbrueckii/S. cerevisiae trials led distinctive aromatic profile of wines with a significant enhancement of 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 the proposed strain could be a potential biocontrol tool toward wild yeasts in the first phase of fermentation also contributing to improve and differentiate the final aroma wine.

Sparkling wine producing and consumption are in constant increase in the last decade and show no sign of slowing. Prior consumption, origin, grape variety, occasion, price and sensorial perception are factors that typically influence wine consumers’ purchasing and consumption behavior. The presence of volatile compounds in sparkling wines, with direct impact on their organoleptic characteristics, is influenced by several factors, such as the base-wine characteristics, grapes cultivar conditions, the used yeasts, the aging stage or wine-making practices. For this study, five sparkling wines variants from Muscat Ottonel grape variety were obtained. For the experimental samples, the grapes were processed by applying secondary fermentation in bottles. The base-wine was obtained through reverse osmosis and had a predetermined alcoholic concentration (10.5 % vol.). This study aimed to analyze the influence of different commercial yeasts (IOC FIZZ™, IOC DIVINE™, LEVULIA CRISTAL™, IOC 18-2007™) on the volatile composition of experimental sparkling wines. The obtained sparkling wines were characterized by the physical-chemical parameters (according to OIV methods of analysis) and volatile fraction (using gas-chromatography). Data showed an important impact on the concentration of the volatile compounds (p < 0.05), depending on the type of inoculated yeast for the second fermentation and only a minor influence on the physical-chemical parameters was registered.

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomyces cerevisiae ale x Saccharomyces eubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus x Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae x S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20°C, one triple S. cerevisiae x S. eubayanus x S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2L wort fermentation trials at 12°C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for design of de novo lager beer hybrids and may help to diversify aroma profiles in lager beers.

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowia borealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metchnikowiacea, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 18 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications.

Abstract: To reduce the indiscriminate use of pesticides and extend the postharvest shelf life of peach fruit (cv. Baihua) from southeast China, microbial antagonism of indigenous yeasts was mainly studied and applied in construction of composite film. After isolation, purification, cultivation and identification, a total of 14 yeast strains from 9 genera were screened out from the surface of peaches. By experimental analysis of in vitro inhibition zone and in vivo colonizing capacity, Candida oleophila sp-ELPY12B and Cryptococcus laurentii sp-ELPY15A, which have conservative structure of D1/D2 domain sequences and were considered as new species by phylogenetic analysis, were finally chosen as fungicides against the major pathogens. In combination of Na-alginate film (0.4 % glycerin as plasticizer and 0.1 % Tween-80 as emulsifier), the preservative effects of composite-treated groups (1 × 108 CFU mL−1 of Candida oleophila sp-ELPY12B and Cryptococcus laurentii sp-ELPY15A) showed best antifungal effects, which significantly delayed the postharvest preservation period about 6 - 7 d under ambient temperature of 25 ± 3°C and relative humidity of 50 - 70%.

Here we propose the concept of an electro–microbial route to uncouple food production from photosynthesis, thereby enabling production of nutritious food in space without the need to grow plant-based crops. In the proposed process, carbon dioxide is fixed into ethanol using either chemical catalysis or microbial carbon fixation, and the ethanol created is used as a carbon source for yeast to synthesize food for human or animal consumption. The process depends upon technologies that can utilize electrical energy to fix carbon into ethanol and uses an optimized strain of the yeast Saccharomyces cerevisiae to produce high quality, food grade single cell protein using only ethanol, urea, phosphate, and inorganic salts as inputs. Unlike crops using photosynthesis that require months to mature and are challenging to grow under the conditions found in space, the electro–microbial process could generate significant quantities of food on demand with potentially high yields and productivities. In this paper we explore the potential of the proposed process to provide food on demand in space, but it should be noted that this novel approach to food production has many valuable terrestrial applications too. For example, enabling food production in climatically challenged environments including turning deserts into food bowls, or utilizing surplus electricity generated from large-scale renewable power sources.

Consumer demands for new sensory experiences have driven the research of unconventional yeasts in beer. While much research exists on the use of various common Saccharomyces cerevisiae strains as well as non-Saccharomyces yeasts, there exists a gap in knowledge regarding other non-cerevisiae Saccharomyces species in the fermentation of beer, outside that of S. pastorianus. Here, five distinct species of Saccharomyces from the UC Davis Phaff Yeast Culture Collection, as well as one interspecies hybrid from Fermentis, were chosen to ferment 40 L pilot scale beers. S. kudriavzevii, S. mikatae, S. paradoxus, S. bayanus, and S. uvarum yeasts were fermented in duplicate, with one fermenter in each pair receiving 10 g/L dry-hop during fermentation. Analytical measurements were made each day of fermentation and compared to controls of SafAle US-05 and SafLager W 34/70 for commercial brewing parameters of interest. Finished beers were also analyzed for aroma, taste, and mouthfeel to determine the flavor of each yeast as it pertains to brewing potential. All beers exhibited spicy characteristics, likely from the presence of phenols; dry-hopping increased fruit notes while also increasing perceived bitterness and astringency. All of the species in this study displayed great brewing potential, and might be an ideal addition to beer depending on a brewery’s desire to experiment with flavor and willingness to bring a new yeast into their production environment

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in food industry and cosmetics: glucose oxidase (GOx), cellobiose dehydrogenase (CBDH), bioremediation: horseradish peroxidase (HRP), laccase (LAC), and medicine for biosensors and miniature biofuel cells (GOx, CBDH, LAC, HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with heterologous expression of oxidoreductase enzymes, since there is requirement not only for multiple OXR genes integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition, and fermentation conditions during expression due to need for adding transition metals (copper, iron), and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find optimal formula using above mentioned approaches, and most recently also using protein engineering and directed evolution for additional increase in the yield of recombinant enzyme production. In this review article we will cover all the current state of the art technologies and most recent advances in the field that yielded high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increase in fermentation yield by the cutting-edge technologies such are directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry and microfluidics.

Biocontainment techniques for genetically modified yeasts (GMYs) are pivotal due to the importance of these organisms for biotechnological processes and also due to the design of new yeast strains by using synthetic biology tools and technologies. Due to the large genetic modifications that many yeast strains display, it is highly desirable to avoid the leakage of GMY cells into natural environments and, consequently, the spread of synthetic genes and circuits by horizontal or vertical gene transfer mechanisms within the microorganisms. Moreover, it is also desirable to avoid that patented yeast gene technologies spread outside the production facility. In this review, it was evaluated the different biocontainment technologies currently available for GMYs. Interestingly, uniplex-type biocontainment approaches (UTBAs), which relies on nutrient auxotrophies induced by gene mutation or deletion, or the expression of simple kill switches apparatus, are still the major biocontainment approaches still in use with GMY. While bacteria like Escherichia coli account for advanced biocontainment technologies based on synthetic biology and multiplex-type biocontainment approaches (MTBAs), GMYs are distant from this scenario due to many reasons. Thus, a comparison of different UTBAs and MTBAs applied for GMY and genetically engineered microorganisms (GEMs) was made, indicating the major advances of biocontainment techniques for GMYs.

The promising antimicrobial activity of essential oils (EOs) led researchers to use them in combination with antimicrobial drugs in order to reduce drug toxicity, side effects, and resistance with single agents. In Pancalieri (Turin, Italy), there is a local production of Mentha x piperita worldwide known as “Mentha of Pancalieri”. The EO from this Mentha is considered as one of the best peppermint EO in the world. In our research, we assessed the antifungal activity of “Mentha of Pancalieri” EO either alone or in combination with azole drugs (fluconazole, itraconazole, ketoconazole) against a wide panel of yeast and dermatophyte clinical isolates. The EO was analyzed by GC-MS and its antifungal properties were evaluated by MIC/MFC parameters, according to the CLSI guidelines, with some modifications. The interaction of peppermint EO with azoles was evaluated through the chequerboard and isobologram methods. Results suggest this EO exerts a fungicidal activity against yeasts, and a fungistatic activity against dermatophytes. Interaction studies with azoles indicate mainly synergistic profiles between itraconazole and peppermint EO vs. Candida spp., Cryptococcus neoformans and Trichophyton mentagrophytes. Peppermint of Pancalieri EO may act as a potential antifungal agent and may serve as a natural adjuvant for fungal infection treatment.

To evaluate the behaviour of the relevant microbial populations during stretched cheese production, the quantitative microbiological analysis was performed during the critical steps of the preparation. The obtained data distributions proved statistically significant increases in all indicators, on average by 4.55 ± 0.64 log CFU/g of presumptive lactococci counts, 4.06 ±0.61 of lactobacilli, 1.53 ± 0.57 log CFU/g of coliforms, 2.42 ± 0.67 log CFU/g of Escherichia coli, 1.53 ± 0.75 log CFU/g of yeasts and moulds, and 0.99 ± 0.27 log CFU/g of presumptive Staphylococcus aureus, from the early stage of milk coagulation until curd ripening (0–24 h). The following steaming/stretching process caused reductions in viable counts with the most significant inactivation effect on coliform bacteria, including E. coli (-4.0 ± 1.0 log CFU/g). Total viable counts and yeasts and moulds showed 2 and almost 3 log reduction (-2.2 ± 1.1 log CFU/g and -2.6 ± 0.9 log CFU/g), respectively. The lowest decreases in presumptive S. aureus counts were estimated at the level of -1.50 ± 0.64 log CFU/g. The counts of yeasts and moulds showed the best indicatory function during the entire storage period of vacuum-packaged cheeses at 6 °C.

Whey is the main by-product of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast fraction inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. We found that PR NWS yeast population consists of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii) in addition to the well-documented Kluyveromyces marxianus, with multiple biotypes scored within each species. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation assay, one thermotolerant strain was suitable to release ethanol with yield of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

Beer is a fermented beverage with a history as old as human civilization and its productive process has been spread all around the world becoming unique in every country and iconic of entire populations. Ales and lagers are by far the most common beers; however, the combination of raw materials, manufacture techniques and aroma profiles are almost infinite, so it is not surprising to notice that there is a large amount of different beer styles, each of them with unique characteristics. Nowadays, diversification is becoming increasingly important in the brewing market and the brewers are continuously interested in improving and extending the already wide range of products, especially in craft brewery. One of the major components that can have a deep impact on the final product is yeast, since it is able to convert carbohydrates in wort, especially maltose and maltotriose, into ethanol, carbon dioxide and other minor aroma-active compounds. Saccharomyces cerevisiae (top‐fermenting yeasts used to produce ales) and Saccharomyces pastorianus (cryotolerant bottom‐fermenting hybrids between S. cerevisiae and Saccharomyces eubayanus responsible for the fermentation of lagers) are most used in breweries. However, an increasing number of different yeast starter cultures are commercially available, to improve the production efficiency also at relative low temperatures and to obtain desirable and diversified aroma profiles avoiding undesired compounds. Four main genetic engineering-free trends are becoming popular in craft brewing yeast development: 1) the research for novel reservoirs as source of new performant S. cerevisiae yeasts; 2) the creation of synthetic hybrids between S. cerevisiae and Saccharomyces non-cerevisiae in order to mimic lager yeasts by expanding their genetic background; 3) the exploitation of evolutionary engineering approaches; 4) the usage of non-Saccharomyces yeasts either in co-coculture or in sequential fermentation with S. cerevisiae. In the present work we summarized pro and contra of these approaches and provided an overview on the most recent advances on how brewing yeast genome evolved and domestication took place. Finally, we delineated how the correlations maps between genotypes and relevant brewing phenotypes can assist and further improve the search for novel craft beer starter yeasts.