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My PhD project was intended, throughout the selection of probiotics from human milk and healthy vaginal environment, for the development of tailored fermented foods. According to this aim, several activities were carried out. The first one, concerning the isolation of Lactobacillus and Bifidobacterium strains from human milk to find new probiotic candidates to be included in food products showed promising results. Probiotics have been also proposed to improve female genital health and microbial strains isolated and connected with healthy vaginal ecosystem could be used to prevent or treat vaginal dysbiosis. In this context vaginal lactobacilli previously characterized for their technological features and antagonistic activity against several female uro-genital pathogens were investigated for their metabolic aptitude and additional probiotic features, showing interesting results hypothesizing their inclusion in foods. In addition, in order to preserve vaginal strains viability during food processing/digestion it was also evaluated the potential of microencapsulation by spray-drying. In this framework the results obtained were highly promising from the perspective of using encapsulated powders in food formulations. Another activity connected with the main idea to develop a food strategy for the administration of these vaginal strains was carried out. Lactobacillus crispatus BC4, was supplemented in a Squacquerone cheese, and its digestive fate was evaluated adopting SHIME® system. The results showed that during colonic fermentation, L. crispatus BC4 was metabolically active. Additionally, although probiotic delivery to humans has traditionally been associated with fermented dairy foods, recently the demand for non-dairy-alternatives as potential probiotics carrier is increasing. In this framework, my latest activity was connected with the development of fermented soy milks with encapsulated and non-encapsulated L. crispatus BC4 and L. gasseri BC9. The same fermented soy milks were also investigated for their nutritional qualities and after in vitro digestion for their specific functionality on post-menopausal fecal microbiota and protein bioaccessibility.

The use of milling by-products as ingredients in food formulations has increased gradually over the past years, due to their well-recognized health properties. Fermentation performed with selected microbial strains or microbial consortia is the most promising way to reduce antinutritional factors of cereals and bran, while increasing their nutritional and functional properties. This work, developed within the BBI project INGREEN, was aimed to study the functional, nutritional and technological features of a pre-fermented ingredient obtained from the fermentation of a mixture of rye bran and wheat germ by a selected microbial consortium composed of yeasts (Kazachstania unispora and Kazachstania servazii) and lactic acid bacteria (Latilactobacillus curvatus) using as reference the unfermented mixture and the same mixture fermented by a baker’s yeast. The selected microbial consortium improved the complexity of the volatile molecules such as acids, alcohols and esters. A better retention of color parameters was maintained compared to the product fermented by a baker’s yeast. In addition, the fermentation by the selected consortium showed a significant increase in short chain fatty acids (more than 5-fold), antioxidant activity (22–24%), total phenol content (53–71%), bioactive peptides (39–52%), a reduction of 20–28% in phytic acid content and an increase in prebiotic activity not only compared to the unfermented product but also compared to the preferment obtained with a baker’s yeast. Overall, the fermentation by the selected microbial consortium can be considered a valuable way to valorize milling by-products and promote their exploitation as food ingredients.

The present research is aimed at investigating the potential of two commercial Saccharomyces cerevisiae strains (EC1118 and AWRI796) to generate wine-specific volatile molecule fingerprinting in relation to the initial must applied. To eliminate the effects of all the process variables and obtain more reliable results, comparative fermentations on interlaboratory scale of five different regional red grape musts were carried out by five different research units (RUs). For this purpose, the two S. cerevisiae strains were inoculated separately at the same level and under the same operating conditions. The wines were analyzed by means of SPME-GC/MS. Quali-quantitative multivariate approaches (two-way joining, MANOVA and PCA) were used to explain the contribution of strain, must, and their interaction to the final wine volatile fingerprinting. Our results showed that the five wines analyzed for volatile compounds, although characterized by a specific aromatic profile, were mainly affected by the grape used, in interaction with the inoculated Saccharomyces strain. In particular, the AWRI796 strain generally exerted a greater influence on the aromatic component resulting in a higher level of alcohols and esters. This study highlighted that the variable strain could have a different weight, with some musts experiencing a different trend depending on the strain (i.e., Negroamaro or Magliocco musts).

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory- scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster Frontiers in Microbiology | www.frontiersin.org 1 February 2022 | Volume 13 | Article 830277 Romano et al. Wine S. cerevisiae Protocol Validation INTRODUCTION Saccharomyces cerevisiae is the predominant yeast species in winemaking. Due to its adaptability to the stressful conditions imposed by grape must fermentation, it easily competes with other yeasts and bacteria, and being the main actor of the transformation of grape must into wine, it is universally known as the “wine yeast.” In the last decades, a wide number of molecular and physiological studies demonstrated the high genotypic and phenotypic diversity of S. cerevisiae wine strains (Romano et al., 2008; Csoma et al., 2010; Mercado et al., 2011; Capece et al., 2013; Tristezza et al., 2013; Legras et al., 2018; Peter et al., 2018). This biodiversity is strictly associated with a significant high technological variability (Pretorius, 2000) and is of great importance for a successful strain selection and the development of new starters able to modulate the organoleptic quality of wine (Romano et al., 2003). Wild strains of S. cerevisiae are genetically and phenotypically distinguished from the selected commercial starter strains that are the result of selection programs (Peter et al., 2018; Pontes et al., 2020). In general, the commercial strains are characterized by high ethanol and low-pH tolerance, and they exhibit scarce production of aromatic compounds and low sporulation activity and biodiversity level (Duan et al., 2018; Kang et al., 2019). On the contrary, the wild strains, possessing high genotypic and phenotypic diversity, produce relatively high amounts of different secondary metabolites, thus offering considerable potential for utilization in industrial applications (Kang et al., 2019). Therefore, wild isolates from flowers and sugar-rich sources can lead to an aromatic profile characterized by specific volatile compounds capable of characterizing wine (Pontes et al., 2020; Alfonzo et al., 2021). As an example, wine fermentations using native wild strains obtained from oaks produce earthy and sulfurous organoleptic characteristics but intense of citrus and floral attributes (Hyma et al., 2011). Thus, although industrial yeast strains represent a fundamental tool for reproducing the final quality of table wines, their massive use is not recommended for traditional wines in which peculiar traits are desired (Spano et al., 2010; Capozzi and Spano, 2011). For these reasons, indigenous yeast starters, which are supposedly well adapted to a specific grape must and reflect the biodiversity of a particular “terroir” are more and more requested by winemakers (Bokulich et al., 2014; Gilbert et al., 2014; Feghali et al., 2020). Indeed, it is hypothesized that in different vitivinicultural regions, specific yeast strains are naturally selected and that they are able to exalt the sensorial and aromatic profile of wine produced in that area. In fact, Knight and Goddard (2015) showed that genetically differentiated population of S. cerevisiae in New Zealand had a different impact on wine quality due to the production of a different complex mix of chemicals. Setati et al. (2012), while studying the spatial distribution of fungal microbial communities within and between vineyards from the same “terroir” found higher yeast heterogeneity on grape samples collected at different points inside individual vineyards than between vineyards with very contrasting farming strategies. Thus, the myriad of microclimates occurring within each vineyard due to differential shading of grapes by leaves, and the aspect of each grape cluster, greatly affects the qualitative/quantitative composition of the vineyard-associated yeast microbiota. Bokulich et al. (2014) used a high-throughput short-amplicon sequencing approach to demonstrate that specific regional and grape-variety factors shape the biodiversity of fungal and bacterial consortia inhabiting wine-grape surfaces. Indeed, the microbial assemblages correlate with specific climatic features, and this suggests a link between vineyard environmental conditions and microbial residence patterns. Taken together, these findings reveal the importance of microbial populations for the regional identity of wine (Bokulich et al., 2016) and underline that the utilization of S. cerevisiae indigenous strain with selected traits is fundamental to modulate the final characteristics of the wine. The first step toward the attainment of indigenous S. cerevisiae wine starters is the clonal selection of the yeast strains associated with the wine-producing area of interest. Clonal selection is based on the evaluation of a number of phenotypic characteristics that are requested to guarantee the production of wines with peculiar sensorial properties. Traditionally, these are distinguished in technological and qualitative characteristics. Technological characteristics, such as fermentation power (ethanol production), analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

The aromatic complexity of a wine is mainly influenced by the interaction between grapes and fermentation agents. This interaction is very complex and affected by numerous factors, such as cultivars, degree of grape ripeness, climate, mashing techniques, must chemical–physical characteristics, yeasts used in the fermentation process and their interactions with the grape endogenous microbiota, process parameters (including new non-thermal technologies), malolactic fermentation (when desired), and phenomena occurring during aging. However, the role of yeasts in the formation of aroma compounds has been universally recognized. In fact, yeasts (as starters or naturally occurring microbiota) can contribute both with the formation of compounds deriving from the primary metabolism, with the synthesis of specific metabolites, and with the modification of molecules present in the must. Among secondary metabolites, key roles are recognized for esters, higher alcohols, volatile phenols, sulfur molecules, and carbonyl compounds. Moreover, some specific enzymatic activities of yeasts, linked above all to non-Saccharomyces species, can contribute to increasing the sensory profile of the wine thanks to the release of volatile terpenes or other molecules. Therefore, this review will highlight the main aroma compounds produced by Saccharomyces cerevisiae and other yeasts of oenological interest in relation to process conditions, new non-thermal technologies, and microbial interactions.

Recently, application of high-pressure homogenization (HPH) treatments has been widely studied to improve shelf life and rheological and functional properties of vegetable and fruit juices. Another approach that has drawn the attention of researchers is the use of biocontrol cultures. Nevertheless, no data on their possible combined effect on fruit juices shelf life and functionality have been published yet. In this work, the microbial, organoleptic, and technological stability of extremely perishable carrot juice and its functionality were monitored for 12 and 7 days (stored at 4 and 10 °C, respectively) upon HPH treatment alone or in combination with a fermentation step using the biocontrol agent L. lactis LBG2. HPH treatment at 150 MPa for three passes followed by fermentation with L. lactis LBG2 extended the microbiological shelf life of the products of at least three and seven days when stored at 10 °C and 4 °C, respectively, compared to untreated or only HPH-treated samples. Moreover, the combined treatments determined a higher stability of pH and color values, and a better retention of β-carotene and lutein throughout the shelf-life period when compared to unfermented samples. Eventually, use of combined HPH and LBG2 resulted in the production of compounds having positive sensory impact on carrot juice.