Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese.

Introduction: Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. Methods: To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. Results and discussion: The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from ß-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from ß-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for ß-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.

Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures.

The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.

Effect of Fermentation with Streptococcus thermophilus Strains on In Vitro Gastro-Intestinal Digestion of Whey Protein Concentrates.

Three Streptococcus thermophilus strains, namely RBC6, RBC20, and RBN16, were proven to release bioactive peptides during whey protein concentrate (WPC) fermentation, resulting in WPC hydrolysates with biological activities. However, these bioactive peptides can break down during gastro-intestinal digestion (GID), hindering the health-promoting effect of fermented WPC hydrolysates in vivo. In this work, the effect of simulated GID on three WPC hydrolysates fermented with S. thermophilus strains, as well as on unfermented WPC was studied in terms of protein hydrolysis, biological activities, and peptidomics profiles, respectively. In general, WPC fermentation enhanced protein hydrolysis compared to unfermented WPC. After in vitro GID, WPC fermented with S. thermophilus RBC20 showed the highest antioxidant activity, whereas WPC fermented with strain RBC06 displayed the highest angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPP-IV)-inhibitory activities. Peptidomics analysis revealed that all digested WPC samples were highly similar to each other in peptide profiles, and 85% of the 46 identified bioactive peptides were shared among fermented and unfermented samples. However, semi-quantitative analysis linked the observed differences in biological activities among the samples to differences in the amount of bioactive peptides. The anti-hypertensive peptides VPP and IPP, as well as the DPP-IV-inhibitory peptide APFPE, were quantified. In conclusion, WPC fermentation with S. thermophilus positively impacted protein hydrolysis and bioactive peptide release during GID.

Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait.

Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.

Microbial Consortia Involved in Traditional Sicilian Sourdough: Characterization of Lactic Acid Bacteria and Yeast Populations.

Sourdough is one of the oldest starters traditionally used for making baked goods, offering several advantages to the sensory, rheology, and shelf life of final products. The present study investigated, for the first time, the microbiota of spontaneously fermented Maiorca dough samples collected from bakeries located in Sicily (Italy). Four sourdough samples (M1, M2, M3, and M4), were produced using Triticum vulgare Host. var. albidum Koern (Maiorca grain) were subjected to LAB and yeasts isolation and identification at the species level. The in-depth characterization of the lactobacilli population revealed that Lactiplantibacillus plantarum and Levilactobacillus brevis unquestionably dominated the Maiorca sourdough ecosystem. Concerning the yeasts community, high species diversity was found. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In addition, Torulaspora delbrueckii, Pichia kluyveri, Candida boidinii, and Candida diddensiae were also detected. Investigations on both pro-technological and functional traits of the isolated strains could lead to the selection of starters for the production of baked goods.

Metabolomic and Transcriptional Profiling of Oleuropein Bioconversion into Hydroxytyrosol during Table Olive Fermentation by Lactiplantibacillus plantarum.

This study aims to elucidate the mechanisms responsible for the bioconversion of oleuropein into low-molecular-weight phenolic compounds in two selected Lactiplantibacillus plantarum strains, namely, C11C8 and F3.5, under stress brine conditions and at two different temperatures (16°C and 30°C). For this purpose, we adopted an experimental strategy that combined high-resolution mass spectrometry, in silico functional analysis of glycoside hydrolase family 1 (GH1)-encoding candidate genes, and gene expression studies. The oleuropein hydrolysis products and the underlying enzymatic steps were identified, and a novel putative bgl gene was detected, using seven strains belonging to the same species as controls. According to metabolomic analysis, a new intermediate compound (decarboxymethyl dialdehydic form of oleuropein aglycone) was revealed. In addition, strain C11C8 showed a decrease in the oleuropein content greater than that of the F3.5 strain (30% versus 15%) at a temperature of 16°C. The highest increase in hydroxytyrosol was depicted by strain C11C8 at a temperature of 30°C. PCR assays and sequencing analyses revealed that both strains possess bglH1, bglH2, and bglH3 genes. Furthermore, a reverse transcription-PCR (RT-PCR) assay showed that bglH3 is the only gene transcribed under all tested conditions, while bglH2 is switched off in strain C11C8 grown at cold temperatures, and no transcription was detected for the bglH1 gene. The bglH3 gene encodes a 6-phospho-ß-glucosidase, suggesting how phospho-ß-glucosidase activity could belong to the overall metabolic strategy undertaken by L. plantarum to survive in an environment poor in free sugars, like table olives. IMPORTANCE In the present study, a new candidate gene, bglH3, responsible for the ß-glucosidase-positive phenotype in L. plantarum was detected, providing the basis for the future marker-assisted selection of L. plantarum starter strains with a ß-glucosidase-positive phenotype. Furthermore, the ability of selected strains to hydrolyze oleuropein at low temperatures is important for application as starter cultures on an industrial scale.

Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese.

In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and ß-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved ß-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1' subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and ß-casein hydrolysis.

Characterization of Yeasts Isolated from Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization.

Whey is the main byproduct 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 population 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. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. 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 trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency 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.

Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts.

Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.

An Integrated Peptidomics and In Silico Approach to Identify Novel Anti-Diabetic Peptides in Parmigiano-Reggiano Cheese.

Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano-Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure-activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC50 = 49.5 ± 0.5 µmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management.

Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation.

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 Saccharomycescerevisiae ale × Saccharomyceseubayanus 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 × Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae × 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 × S. eubayanus × S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2 L 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 designing de novo lager beer hybrids and may help to diversify aroma profiles in lager beer.

Peptide Profiling and Biological Activities of 12-Month Ripened Parmigiano Reggiano Cheese.

Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced fat/salt content showed the highest antihypertensive and antioxidant activities.

Cultivable non-starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti-hypertensive peptides.

The impact of salt and fat intake on human health drives the consumer's attention towards dairy food with reduced salt and fat contents. How changes in salt and fat content modulate dairy LAB population and the associated proteolytic activities have been poorly studied. Here, non-starter LAB populations from 12 Parmigiano Reggiano (PR) cheeses (12-month ripened), clustered in low salt and fat content (LL-PR) and high salt and fat content (HH-PR) groups, were investigated and identified at specie-level with molecular assays. Lactobacillus rhamnosus was dominant in HH-PR samples, whereas Lactobacillus paracasei in LL-PR samples. (GTG)5 rep-PCR analysis discriminated 11 and 12 biotypes for L. rhamnosus and L. paracasei isolates, respectively. Screening for proteolytic activity identified L. rhamnosus strains more proteolytic than L. paracasei, and, within L. rhamnosus species, HH-PR strains were generally more proteolytic than LL-PR strains. Two L. rhamnosus representatives, namely strain 0503 from LL-PR and strain 2006 from HH-PR, were functionally characterized in cow milk fermentation assay. HH-PR strain 2006 overcame LL-PR strain 0503 in acidification performance, leading to a fermented milk with higher angiotensin I-converting enzyme inhibitory and antioxidant activities. L. rhamnosus 2006 was more prone to release VPP, while L. rhamnosus 0503 released higher amount of IPP. This study provides evidences that salt/fat content affects NSLAB cultivable fraction and the associated proteolytic ability resulting in a complex occurrence of bioactive peptides featuring health-promoting properties.

Valorization of cheese whey using microbial fermentations.

Cheese whey (CW), the liquid resulting from the precipitation and removal of milk casein during cheese-making, and the second cheese whey (SCW) derived from the production of cottage and ricotta cheeses are the main byproducts of dairy industry. The major constituent of CW and SCW is lactose, contributing to the high BOD and COD content. Because of this, CW and SCW are high-polluting agents and their disposal is still a problem for the dairy sector. CW and SCW, however, also consist of lipids, proteins, and minerals, making them useful for production of various compounds. In this paper, microbial processes useful to promote the bioremediation of CW and SCW are discussed, and an overview on the main whey-derived products is provided. Special focus was paid to the production of health-promoting whey drinks, vinegar, and biopolymers, which may be exploited as value-added products in different segments of food and pharmaceutical industries.

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii.

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

Interplay of Chimeric Mating-Type Loci Impairs Fertility Rescue and Accounts for Intra-Strain Variability in Zygosaccharomyces rouxii Interspecies Hybrid ATCC42981.

The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii. Recently, different mating-type-like (MTL) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MATa/MATα expression loci, in contrast to MATα in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MATα expressed locus by gene deletion. The resulting MATa/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HMLα cassette masks the loss of heterozygosity at the MAT locus. We also found that MATα deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast.

Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives.

In the present study, the ß-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.

Draft Genome Sequences of the Highly Halotolerant Strain Zygosaccharomyces rouxii ATCC 42981 and the Novel Allodiploid Strain Zygosaccharomyces sapae ATB301T Obtained Using the MinION Platform.

Here, we report draft genome sequences of the halotolerant and allodiploid strains Zygosaccharomyces rouxii ATCC 42981 and Zygosaccharomyces sapae ABT301T. Illumina and Oxford Nanopore MinION sequencing revealed genome sizes of 20.9 and 24.7 Mb, respectively. This information will be useful for deciphering the genetics of hybrid adaptation to high salt and sugar concentrations in nonconventional yeasts.

Evaluation of fingerprinting techniques to assess genotype variation among Zygosaccharomyces strains.

Molecular typing techniques are key tools in surveillance of food spoilage yeasts, in investigations on intra-species population diversity, and in tracing selected starters during fermentation. Unlike previous works on strain typing of Zygosaccharomyces spoilage species, here Zygosaccharomyces mellis and the Zygosaccharoymces rouxii complex yeasts, which include Z. rouxii, Zygosaccharomyces sapae, and a mosaic lineage (ML) of putatively hybrids, were evaluated by three typing methods for intra- and inter-species resolution. Overall these yeasts are relevant for food fermentation and spoilage, but are quite difficult to discriminate at strain and species level as they evolved by reticulation. A pool of 76 strains from different sources were typed by M13 and (GTG)5 MSP-PCR fingerprinting and PCR-RFLP of ribosomal intergenic spacer region (IGS). We demonstrated that M13 overcame (GTG)5 fingerprinting to group Z. sapae, Z. rouxii, Z. mellis and the ML isolates in congruent distinct clusters. Even if (GTG)5 primer yielded a number of DNA fingerprints comparable with those obtained by M13 primer, it failed to discriminate Z. sapae, Z. mellis and Z. rouxii at species level. Clustering of IGS RFLP patterns obtained with three endonucleases produced groups congruent with species assignment and highlighted intra-species diversity similar to that observed by M13 fingerprinting. However, IGS PCR amplification failed for 14 ML and 6 Z. mellis strains under the experimental conditions tested here, indicating that this marker could be less easy to use in fast typing protocol. Finally, our results posit that the genetic diversity within Z. sapae and Z. mellis could be shaped by isolation source. The information generated in this study would facilitate the monitoring of these yeasts during food processing and storage, and provides preliminary evidences about Z. sapae and Z. mellis intra-species diversity.

Mating-type switching in CBS 732T derived subcultures unveils potential genetic and phenotypic novelties in haploid Zygosaccharomyces rouxii.

In haploid Saccharomyces cerevisiae, a complex recombination system regulates mating-type switching and requires one MAT expression locus, two donor cassettes (HML and HMR) and the HO endonuclease that catalyses gene conversion. Zygosaccharomyces rouxii is the most distant species from S. cerevisiae with a functional HO, but with a poorly understood mating-type switching. Here, we described that two subcultures of the type strain CBS 732T underwent the α to a genotype switching leading to mixed MATα and MATa populations. Remarkably, during this event the donor cassette was copied into the MAT locus, except for its own 3΄ end, resulting in a new MATa2 gene copy different from the silenced HMRa2. Moreover, CBS 732T cells bypassed the cell-cycle control, which oversees HO transcription in S. cerevisiae, and expressed HO at the stationary phase. Despite HO dysregulation, mating-type switching seemed to occur rarely or belatedly during CBS 732T colony formation in most of the tested conditions. When morphology and mating behaviour were analysed, two subcultures displayed distinct outcross fertility responses. Overall, our data support that mating-type switching causes genotype instability and phenotypic novelties in CBS 732T, and open the question whether this mechanism is shared by other Z. rouxii haploid homothallic strains.