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The butyric acid content produced by three Lactobacillus plantarum (Lactiplantibacillus plantarum) strains (FP37, FP38 and FP48) deposited as probiotic strains was determined. The experimentation was carried out to evaluate whether the production of butyric acid was due to lipolytic activity for the presence of specific lipases or was related to the fermentation of saccharides. Therefore, the three strains were inoculated in different substrates with and without the addition of fat (tributyrin). The free butyric acid contents were determined by gas chromatography after distilling each culture medium. The results showed that butyric acid production occurred mainly in the substrates supplemented with fat and therefore it was linked to the activity of specific L. plantarum lipase. The L. plantarum FP48 strain produced the highest amount of butyric acid in fat substrates and could be a useful strain in the development of dairy products with functional properties.

In the last several years, the popularity of homebrewed beers has skyrocketed. However, this type of product is extremely vulnerable to microbial deterioration. Twelve homemade beers, some characterized by defects or stuck fermentation, were analysed by using a polyphasic approach encompassing culturomics and culture-independent techniques to better understand mechanisms that drive microbiota evolution throughout production and to highlight determinants responsible for crowning with success. Two sour beers, one apple-flavoured ale, two Italian grape ales, and seven standard ales were sampled. Microbiological characterization was obtained by plating on nine different media coupled with High- throughput sequencing analysis of fungal and bacterial communities by targeting ITS1–2 and the V3–V4 re- gions of the 16S rRNA, respectively. 2 7 TotalmicrofloraonPCAlargelyvariedamongsamples,rangingfrom

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.

To design a rapid, simple, and low-cost procedure for yeast selection with differential adsorption activities toward phenolics and ochratoxin A, 284 yeast strains were screened. This was done by evaluating the type of growth during grape must fermentation, acetic acid production on Chalk agar, H2S production on BiGGY agar, and spore-formation on acetate agar. After that initial step, 84 strains were pre-selected and further studied by Petri plate tests and to determine their wine-making ability in trials and evaluating their differential adsorption activities toward phenolics and ochratoxin A. Three yeast strains were selected based on the above evaluations. After confirming that they belonged to Saccharomyces cerevisiae species and were diploids, a spore clonal selection was performed. The strain Sc1741A_1D was selected and used in winemaking at six Calabrian wineries and found to be suitable as wine starter to improve quality and safety of red wines.

“Grottone” is a pasta filata hard cheese produced in Campania region from cow’s milk and characterized by holes formation due to CO2 development by Propionic Acid Bacteria. The contamination of raw milk with butyric acid-producing spore-forming clostridia represent a major concern for cheese producers since clostridia outgrowth may lead to the cheese late blowing defect during ripening. Detection of clostridial endospores in milk before processing and the use of antimicrobial compounds may represent an important control strategy. The present study is aimed to point out the most suitable procedure for the determination of clostridial spores in dairy samples, and to assess the inhibitory activity of several antimicrobial compounds against Cl. sporo- genes. Based on results, MPN counts on Bryant and Burkey medium and CFU on RCM proved to be the most suitable proto- cols for routine testing. By using these pro- cedures clostridial spores were detected in 10 out 13 milk samples and in all cheeses with late blowing defect. Within antimicro- bial compounds, sodium nitrate is still the best choice for preventing late blowing, nevertheless a protective culture of Lacticaseibacillus casei proved to be a promising alternative. Nevertheless, the use of this protective culture in six Grottone cheese productions carried out at farm level, led to unsatisfactory results. Holes’ devel- opment was hampered likely for an inhibi- tion of the PAB starter and the expected ‘Grouviera-type’ taste was not perceived by panellists. Based on results, the use of pro- tective cultures needs to be contextualized and interactions with starters needs to be evaluated case by case.

The search for sourdough starters for the direct production of baked goods with all the advantages of biological sourdough fermentation is still a crucial issue. In this study, 43 Lactic Acid Bacteria strains isolated from mature sourdoughs were evaluated for features of technological interest and tested for fermentation ability. Three microbial combinations were selected and used to produce bread. Based on GC-MS and sensory analysis, bread made by using the three combinations of strains was characterized by a more complex aroma profile with the prevalence of VOCs typical of sourdough bread. To set up the best way to keep microbial viability upon drying, the three combinations were subject to freeze-drying and wet granulation, with the latter being used for the first time for food starters’ stabilization. Wet granulation ensured optimal strains’ viability. Surprisingly, the height attained by mature sourdoughs when inoculated with wet granulated starters was constantly higher than the height reached by sourdoughs made with the same starters as fresh cells. The microbial combination E75-B72 exhibited the best performances and may represent a starter able to ensure sourdough bread production in 16 h of fermentation at 28 °C.