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.

Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

After the storm-Perspectives on the taxonomy of Lactobacillaceae.

In 2020, a taxonomic reorganization of the lactic acid bacteria reclassified over 300 species in 7 genera and 2 families into one family, the Lactobacillaceae, with 31 genera including 23 new genera to include organisms formerly classified as Lactobacillus species. This communication aims to provide a debrief on the taxonomic reorganization of lactobacilli to identify shortcomings in the proposed taxonomic framework, and to outline perspectives and opportunities provided by the current taxonomy of the Lactobacillaceae. The current taxonomy of lactobacilli not only necessitates becoming familiar with 23 new genus names but also provides substantial new opportunities in scientific discovery and regulatory approval of these organisms. First, description of new species in the Lactobacillaceae is facilitated and a solid framework for description of novel genera is provided. Second, the current taxonomy greatly enhances the resolution of genus-level sequencing approaches (e.g., 16S rRNA-based metagenomics) when identifying the composition and function of microbial communities. Third, the current taxonomy greatly facilitates the formulation of hypotheses linking phylogeny to metabolism and ecology of lactobacilli.

Assessing Gut Microbiota in an Infant with Congenital Propionic Acidemia before and after Probiotic Supplementation.

Propionic Acidemia (PA) is a rare inherited metabolic disorder caused by the enzymatic block of propionyl-CoA carboxylase with the consequent accumulation of propionic acid, which is toxic for the brain and cardiac cells. Since a considerable amount of propionate is produced by intestinal bacteria, interest arose in the attempt to reduce propionate-producing bacteria through a monthly antibiotic treatment of metronidazole. In the present study, we investigated the gut microbiota structure of an infant diagnosed at 4 days of life through Expanded Newborn Screening (NBS) and treated the child following international guidelines with a special low-protein diet, specific medications and strict biochemical monitoring. Microbiota composition was assessed during the first month of life, and the presence of Bacteroides fragilis, known to be associated with propionate production, was effectively decreased by metronidazole treatment. After five antibiotic therapy cycles, at 4 months of age, the infant was supplemented with a daily mixture of three bifidobacterial strains, known not to be propionate producers. The supplementation increased the population of bifidobacteria, with Bifidobacterium breve as the dominating species; Ruminococcus gnavus, an acetate and formate producer, was also identified. Metabarcoding analysis, compared with low coverage whole metagenome sequencing, proved to capture all the microbial biodiversity and could be the elected tool for fast and cost-effective monitoring protocols to be implemented in the follow up of rare metabolic disorders such as PA. Data obtained could be a possible starting point to set up tailored microbiota modification treatment studies in the attempt to improve the quality of life of people affected by propionic acidemia.

Non-conventional yeasts for food and additives production in a circular economy perspective.

Yeast species have been spontaneously participating in food production for millennia, but the scope of applications was greatly expanded since their key role in beer and wine fermentations was clearly acknowledged. The workhorse for industry and scientific research has always been Saccharomyces cerevisiae. It occupies the largest share of the dynamic yeast market, that could further increase thanks to the better exploitation of other yeast species. Food-related 'non-conventional' yeasts (NCY) represent a treasure trove for bioprospecting, with their huge untapped potential related to a great diversity of metabolic capabilities linked to niche adaptations. They are at the crossroad of bioprocesses and biorefineries, characterized by low biosafety risk and produce food and additives, being also able to contribute to production of building blocks and energy recovered from the generated waste and by-products. Considering that the usual pattern for bioprocess development focuses on single strains or species, in this review we suggest that bioprospecting at the genus level could be very promising. Candida, Starmerella, Kluyveromyces and Lachancea were briefly reviewed as case studies, showing that a taxonomy- and genome-based rationale could open multiple possibilities to unlock the biotechnological potential of NCY bioresources.

Pangenome analyses of LuxS-coding genes and enzymatic repertoires in cocoa-related lactic acid bacteria.

Lactobacillaceae presents potential for interspecific Quorum Sensing (QS) in spontaneous cocoa fermentation, correlated with high abundance of luxS. Three Brazilian isolates from cocoa fermentation were characterized by Whole Genome Sequencing and luxS gene was surveyed in their genomes, in comparison with public databases. They were classified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus acidilactici. LuxS genes were conserved in core genomes of the novel isolates, but in some non-cocoa related Lactic Acid Bacteria (LAB) it was accessory and plasmid-borne. The conservation and horizontal acquisition of luxS reinforces that QS is determinant for bacterial adaptation in several environments, especially taking into account the luxS has been correlated with modulation of bacteriocin production, stress tolerance and biofilm formation. Therefore, in this paper, new clade and species-specific primers were designed for future application for screening of luxS gene in LAB to evaluate the adaptive potential to diverse food fermentations.

A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.

The genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological and genotypic levels. This study evaluated the taxonomy of Lactobacillaceae and Leuconostocaceae on the basis of whole genome sequences. Parameters that were evaluated included core genome phylogeny, (conserved) pairwise average amino acid identity, clade-specific signature genes, physiological criteria and the ecology of the organisms. Based on this polyphasic approach, we propose reclassification of the genus Lactobacillus into 25 genera including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the Lactobacillus delbrueckii group, Paralactobacillus and 23 novel genera for which the names Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus are proposed. We also propose to emend the description of the family Lactobacillaceae to include all genera that were previously included in families Lactobacillaceae and Leuconostocaceae. The generic term 'lactobacilli' will remain useful to designate all organisms that were classified as Lactobacillaceae until 2020. This reclassification reflects the phylogenetic position of the micro-organisms, and groups lactobacilli into robust clades with shared ecological and metabolic properties, as exemplified for the emended genus Lactobacillus encompassing species adapted to vertebrates (such as Lactobacillus delbrueckii, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensensii, Lactobacillus johnsonii and Lactobacillus acidophilus) or invertebrates (such as Lactobacillus apis and Lactobacillus bombicola).

Fate of Grape-Derived Terpenoids in Model Systems Containing Active Yeast Cells.

Terpenes are important contributors to wine aroma. Free and glycosidically bound terpenes are primarily formed in grapes. During fermentation, they undergo important transformation catalyzed by yeast, so that the terpene profile of grape is substantially different from that of the corresponding wine. The present paper assessed the ability of a Saccharomyces cerevisiae strain to transform 17 different terpenes. Biotransformation was performed by placing target compounds in incubation with resting cells. Volatile compounds produced were extracted by solid-phase extraction and analyzed by gas chromatography-mass spectrometry. Geranyl acetate, neryl acetate, citronellyl acetate, and menthyl acetate were formed from the corresponding terpene alcohols. ß-Citronellol was the main product of geraniol transformation; geranial, an intermediate of this pathway, has also been detected. Limonene was hydroxylated by yeast to form carveol, trans-2,8-menthadien-1-ol, and cis-2,8-menthadien-1-ol. Moreover, yeast cells were found to be able to adsorb a significant portion of the terpenes present in the reaction batches, with the extent of this phenomenon being linked to terpene hydrophobicity.

Volatile organic compounds from Starmerella bacillaris to control gray mold on apples and modulate cider aroma profile.

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.

Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

Alloscardovia theropitheci sp. nov., isolated from the faeces of gelada baboon, the 'bleeding heart' monkey (Theropithecus gelada).

A novel irregularly shaped and slightly curved rod bacterial strain, GLDI4/2T, showing activity of fructose 6-phosphate phosphoketolase was isolated from a faecal sample of an adult gelada baboon (Theropithecus gelada). Phylogenetic analyses based on 16S rRNA genes as well as multilocus sequences (representing fusA, gyrB and xfp genes) and the core genome revealed that GLDI4/2T exhibited phylogenetic relatedness to Alloscardovia omnicolens DSM 21503T and to Alloscardovia macacae DSM 24762T. Comparative analysis of 16S rRNA gene sequences confirmed the phylogenetic results showing the highest gene sequence identity with strain A. omnicolens DSM 21503T (96.0 %). Activities of α- and ß-gluco(galacto)sidases were detected in strain GLDI4/2T, which is characteristic for almost all members of the family Bifidobacteriaceae. Compared to other Alloscardovia species its DNA G+C content (43.8 mol%) was very low. Phylogenetic studies and the evaluation of phenotypic characteristics, including the results of biochemical, physiological and chemotaxonomic analyses, confirmed the novel species status for strain GLDI4/2T, for which the name Alloscardoviatheropitheci sp. nov. is proposed. The type strain is GLDI4/2T (=DSM 106019T=JCM 32430T).

Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking.

The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.

Effects of Probiotics on Cognitive Reactivity, Mood, and Sleep Quality.

Recent demonstration that probiotics administration has positive effects on mood state in healthy populations suggests its possible role as an adjunctive therapy for depression in clinical populations and as a non-invasive strategy to prevent depressive mood state in healthy individuals. The present study extends current knowledge on the beneficial effects of probiotics on psychological well-being, as measured by changes in mood (e.g., cognitive reactivity to sad mood, depression, and anxiety), personality dimensions, and quality of sleep, which have been considered as related to mood. For this double-blind, placebo-controlled study 38 healthy volunteers assigned to an experimental or control group assumed a daily dose of a probiotic mixture (containing Lactobacillus fermentum LF16, L. rhamnosus LR06, L. plantarum LP01, and Bifidobacterium longum BL04) or placebo, respectively, for 6 weeks. Mood, personality dimensions, and sleep quality were assessed four times (before the beginning of the study, at 3 and 6 weeks, and at 3 weeks of washout). A significant improvement in mood was observed in the experimental group, with a reduction in depressive mood state, anger, and fatigue, and an improvement in sleep quality. No between-groups differences were found. These findings corroborate the positive effect of probiotics on mood state and suggest that probiotics administration may improve psychological well-being by ameliorating aspects of mood and sleep quality.

International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms. Minutes of closed and open meetings, 3 September 2018, Berlin, Germany.

The meeting of International Committee on Systematics of Prokaryotes, Subcommittee on the taxonomy of Bifidobacterium,Lactobacillus and related organisms was held within the frame of the FoodMicro 2018 Congress (FoodMicro 2018, 3-6 September 2018, Berlin, Germany). The meeting comprised an open session with a workshop entitled 'Modern approaches of LAB identification and conservation' and a closed session on issues related to ICSP Subcommittee activities.

International Committee on Systematics of Prokaryotes, Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms. Minutes of open and closed meetings, 19 July 2016, Dublin, Ireland.

The meeting of International Committee on Systematics of Prokaryotes, Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms was held within the frame of the FoodMicro 2016 Congress in Dublin (FoodMicro 2016, 19-22 July 2016, Dublin, Ireland). The meeting comprised an open session with a workshop entitled 'Research and networking taxonomy in food with an emphasis on LAB' and a closed session on issues related to ICSP Subcommittee activities.