Technological and Safety Characterization of Coagulase-Negative Staphylococci Isolated from Sardinian Fermented Sausage Made by Ovine Meat.

Ripened sheep sausages are widely consumed in Italy, particularly in Sardinia. Despite their driving role in flavor and color development, coagulase-negative staphylococci in these products have been rarely investigated. A total of 70 CoNS cultures isolated from Sardinian sheep sausages were characterized by rep-PCR and M13-RAPD typing and identified by 16S rDNA sequencing. S. xylosus and S. equorum accounted for more than 70% of the total isolates, whilst S. pasteuri (8.5%), S. succinus (2.8%), and S. haemolyticus (2.8%) were less represented. The genes encoding the synthesis of putrescine, tyramine, cadaverine, and histamine were evaluated by PCR. None of the strains hosted genes for decarboxylases, except one S. pasteuri strain that was potentially a tyramine-producer. Antibiotic resistance was evaluated, along with nitrate reductase, lipolytic, and proteolytic activity, in a pool of selected cultures. Resistance to the primary antibiotics was rather widespread. S. xylosus, S. equorum, and S. pasteuri strains were all resistant to amoxicillin and kanamycin. S. equorum strains were sensitive to all tested antibiotics. S. xylosus strains were all resistant to penicillin B. Conversely, all S. pasteuri strains were resistant to both ampicillin and penicillin B, and four out of five strains exhibited tetracycline resistance. The high variability in the production of sheep sausages makes the search for adjunct cultures of crucial relevance. According to this perspective, the characterization of the autochthonous CSN population represents the first step to approach a starter selection.

Addition of Organic Acids to Base Wines: Impacts on the Technological Characteristics and the Foam Quality of Sparkling Wines.

Climate change is causing a significant decrease in the total acidity of grapes and related wines. This represents a serious issue for sparkling wine production. Consequently, before the second fermentation, the acidification of base wines is often necessary. However, the impacts of the most important organic acids on the foam properties of sparkling wines are not yet well known. The impacts of the addition of tartaric, malic, citric, and lactic acid on the quality of Falanghina and Bombino sparkling wines were evaluated. Analyses were performed soon after the second fermentation and one year after aging sur lees. The addition of each different organic acid to the two base wines resulted in significant changes in the sparkling wines not only in terms of pH, titratable acidity, and buffering capacity but also in the content of total amino acids and, in some cases, in the height of the foam and its stability over time. For both grape varieties, acidified wines showed a lower content of total amino acids in comparison with the control wines. The addition of lactic acid determined a higher persistency of the foam even after one year of aging sur lees only in Falanghina wines. The results obtained herein highlight the importance of organic acids and the pH of the base wines for the content of amino acids in sparkling wines. No strict correlation between organic acid addition and the foamability of wines was observed.

Genetic diversity and signature of divergence in the genome of grapevine clones of Southern Italy varieties.

Sexual reproduction has contributed to a significant degree of variability in cultivated grapevine populations. However, the additional influence of spontaneous somatic mutations has played a pivotal role in shaping the diverse landscape of grapevine agrobiodiversity. These naturally occurring selections, termed 'clones,' represent a vast reservoir of potentially valuable traits and alleles that hold promise for enhancing grape quality and bolstering plant resilience against environmental and biotic challenges. Despite their potential, many of these clones remain largely untapped.In light of this context, this study aims to delve into the population structure, genetic diversity, and distinctive genetic loci within a collection of 138 clones derived from six Campanian and Apulian grapevine varieties, known for their desirable attributes in viticulture and winemaking. Employing two reduced representation sequencing methods, we extracted Single-Nucleotide Polymorphism (SNP) markers. Population structure analysis and fixation index (FST) calculations were conducted both between populations and at individual loci. Notably, varieties originating from the same geographical region exhibited pronounced genetic similarity.The resulting SNP dataset facilitated the identification of approximately two hundred loci featuring divergent markers (FST ≥ 0.80) within annotated exons. Several of these loci exhibited associations with essential traits like phenotypic adaptability and environmental responsiveness, offering compelling opportunities for grapevine breeding initiatives. By shedding light on the genetic variability inherent in these treasured traditional grapevines, our study contributes to the broader understanding of their potential. Importantly, it underscores the urgency of preserving and characterizing these valuable genetic resources to safeguard their intra-varietal diversity and foster future advancements in grapevine cultivation.

Probiotic bacilli incorporation in foods: is really so easy?

Foods containing probiotic bacilli spores are becoming more and more popular because of their resistance to acidic pH, low water activity, and, most importantly, heat processes. Even though spores may engage in some functions, such as immunomodulation, the majority of the beneficial characteristics are unique to vegetative cells. As matter of fact, the development of foods fortified with spores ought to even ensure the germination of the spores along the gastrointestinal tract. In this perspective, vegetative cells derived from spore germination were separately counted on a minimal medium because the traditional approach is based on the use of complex media that allows the spores to germinate independently by stimuli arising from processing or digestion. In more detail, three Bacillus spp. cultures with claimed probiotic properties were added to two entirely unrelated foods (pasta and croissants), and tolerance, as well as germination, was monitored before and after exposure to simulated GIT, as well as at the beginning and end of the products shelf life. For the first time, potential probiotic bacilli were included in a frozen ready-to-bake product. Germination appears to be prevented in this instance, and the impact of baking, matrix and cold storage on spores was examined independently. All of the parameters appeared to contribute, although further research is needed due to the unpredictable behavior exhibited by spores during freezing.

Isolation and Characterization of Cryotolerant Yeasts from Fiano di Avellino Grapes Fermented at Low Temperatures.

A fermentation of Fiano di Avellino grape must was carried out at 9°C with the aim of selecting cryotolerant yeast strains and testing their fermentative performances and volatile production following molecular characterization. A total of 20 yeast cultures were isolated at different fermentation stages. Based on molecular identification and characterization, Metschnikowia (M.) pulcherrima, Hanseniaspora (H.) uvarum, Staremerella (St.) bacillaris, Saccharomyces (S.) cerevisiae, S. kudriavzevii, and S. paradoxus were found to be the yeast species dominating the fermentation. S. paradoxus has been rarely isolated in vineyards and never in the cellar environment. Moreover, in this study, S. kudriavzevii is detected for the first time in vine-wine environments. Both S. kudriavzevii and S. paradoxus co-occurred with S. cerevisiae when grapes were micro-fermented at low temperatures. The growth kinetics of the three species were greatly affected by the fermentation temperature. As a consequence, Fiano wines obtained with S. kudriavzevii and S. paradoxus significantly differed from those made by S. cerevisiae in terms of chemical and volatile composition.

Stuck or sluggish fermentations in home-made beers: Beyond the surface.

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 regions of the 16S rRNA, respectively. Total microflora on PCA largely varied among samples, ranging from <102 CFU/mL up to around 107 CFU/mL often reflecting yeast counts on WL and LM. LAB population's levels on MRS and SDBm did not overlap, with the counts on the latter being even 5 Log CFU/mL greater. Acetic Acid bacteria were retrieved in Sour beers, as well as in one IGA, even though acetic acid was not detectable by HPLC in this last sample. Brettanomyces spp. were only found in sour beers, as expected, whereas Enterobacteriaceae were never counted. A total of 63 yeasts were randomly isolated from countable plates. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In many cases, Interdelta analysis biotyping of S. cerevisiae isolates consistently allowed the detection of the starter strain. By HST S. cerevisiae dominated the mycobiota in four samples, even if in one of them residual maltose and ethanol contents suggested a stuck fermentation. W. anomalus was found to be the dominant species in two beers. Fifty-five LAB cultures were isolated and identified. Pediococcus damnosus was the only species retrieved in sour beers and two Ales, while Levilactobacillus brevis was found in two Ale samples. HTS did not confirm this result in one Ale sample since the genus Panotea spp. accounted for over 90 % of the microbiota. Enterobacteriaceae which were never counted dominated the microbiome of two Ale beers. Biogenic amines content largely varied with three Ale samples greatly contaminated. Based on chemical and microbiological outcomes only one beer ASAle out of 12 could be considered acceptable. Furthermore, the widespread presence of LAB by culturomics and Enterobacteriaceae by HTS raises concerns about the final products' safety.

Late blowing defect in Grottone cheese: detection of clostridia and control strategies.

"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. sporogenes. Based on results, MPN counts on Bryant and Burkey medium and CFU on RCM proved to be the most suitable protocols for routine testing. By using these procedures clostridial spores were detected in 10 out 13 milk samples and in all cheeses with late blowing defect. Within antimicrobial 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' development was hampered likely for an inhibition of the PAB starter and the expected 'Grouviera-type' taste was not perceived by panellists. Based on results, the use of protective cultures needs to be contextualized and interactions with starters needs to be evaluated case by case.

Development of a Wet-Granulated Sourdough Multiple Starter for Direct Use.

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.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

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 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.

Technological Parameters, Anti-Listeria Activity, Biogenic Amines Formation and Degradation Ability of L. plantarum Strains Isolated from Sheep-Fermented Sausage.

The aim of this work was to identify and characterize, from a technological and safety point of view, the lactic acid bacteria (LAB) isolated from traditional sheep-fermented sausage. First, LABs were identified then were screened for some technological parameters such as acidifying and growth ability, proteolytic and lipolytic activity and for antimicrobial activity. Finally, biogenic amine production and degradation abilities were also evaluated. This research reveals the predominance of Lactiplantibacillus (L.) plantarum on LAB community. Almost all L. plantarum strains were active against Listeria monocytogenes strains (inhibition zone diameters > 1 cm). None of the tested strains were positive in histidine (hdcA), lysine (ldc) and tyrosine (tyrdc) decarboxylase genes and only one (L. plantarum PT9-2) was positive to the agmatine deiminase (agdi) gene. Furthermore, given the positive results of the sufl (multi-copper oxidase) gene detection, all strains showed a potential degradation ability of biogenic amines.

Characterization of a new type of mead fermented with Cannabis sativa L. (hemp).

Mead, one of the oldest existing drinks, is a fermented product based on honey, water, and the possible addition of spices and selected yeasts. In this work, various parts (inflorescences, leaves, and steams) of Cannabis sativa L. at different concentrations and Saccharomyces cerevisiae biotype M3/5 were added during mead fermentation. The physicochemical parameters (pH, alcoholic content, sugar content, titratable acidity, and organic acids) of the mead were assessed at the beginning and end of fermentation. Moreover, polyphenols, cannabidiol and volatile organic compounds were identified at the end of fermentation and compared with the control sample prepared without hemp and with only indigenous yeasts. The mead fermented with hemp showed the highest quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg. The volatile organic compounds found were mainly alcohols, esters and terpenes, which were present at higher concentrations in the mead prepared with C. sativa L. than in the control mead and conferred freshness and "hemp aroma" characteristics. PRACTICAL APPLICATION: Inflorescences, leaves, and steams of Cannabis sativa L. were added at different concentrations during mead fermentation. This type of mead showed high quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg which have anxiolytic and neuro-protective properties. Moreover the volatile organic compounds found (mainly alcohols, esters, and terpenes) conferred freshness and "hemp aroma" characteristics.

Dominance of S. cerevisiae Commercial Starter Strains during Greco di Tufo and Aglianico Wine Fermentations and Evaluation of Oenological Performances of Some Indigenous/Residential Strains.

In order to evaluate dominance/implantation of starter cultures for wine fermentation, both commercial starters and wild strains were monitored during the fermentation of Greco di Tufo (GR) and Aglianico of Taurasi (AGL) musts. Preliminary characterization of commercial strains was carried out by several molecular markers. Five fermentations-four starter-inoculated and one spontaneous-were carried out in duplicates by using grapes from GR and AGL. Trials were monitored, and yeast cultures were isolated within the dominant microflora. Comparison of Interdelta patterns allowed to assess the real occurrence of both starters and indigenous strains. A high genetic diversity within S. cerevisiae strains was detected. In starter-led fermentations (except for few cases), in addition to the starter strains, indigenous S. cerevisiae biotypes were found, as well. Native strains isolated from replicates of the same fermentation showed different genetic profiles. Spontaneous fermentations were conducted, during the first 5 days, by non-Saccharomyces yeasts and, afterwards, by a high number (16 in the AGL and 20 in the GR) of S. cerevisiae biotypes. Indigenous biotypes isolated by GR revealed a high variability in oenological features and, in several cases, showed better performances than those recorded for commercial strains. The study further highlighted the low dominance of some commercial starter cultures. Moreover, autochthonous yeast strains proved to be sometimes more aggressive in terms of fermentation vigor in GR must, likely because better adapted to ecological and technological conditions occurring during winemaking. Finally, the use of such strains for production of autochthonous "pied de cuve" may be a useful strategy for lowering production cost of winemaking.

New insights into the formation of precipitates of quercetin in Sangiovese wines.

Wines produced from Sangiovese (sg) grapes, the most cultivated red grape variety in Italy and widely grown across the world, is often subjected to loss of clarity due to the formation of a deposit constituted by fine needle-shaped crystals. In this work, a qualitative study by 1H-NMR and 13C-NMR analysis of the deposit obtained by filtering cloudy sg wines showed that it was constituted by crystals of quercetin (Q). The analysis of hydro-alcoholic solutions (12% ethanol and pH 3.2.) and red wines added with increasing amounts of Q showed that, above 3 mgL-1 of Q, a deposit can be detected and, the time necessary for its formation depends on the medium. The comparison among sg and other 11 monovarietal wines showed that sg was the richest in Q and quercetin glycosides (GQ). Both Q and GQ decreased in the analyzed solutions over time and the decrease was faster for Q than for GQ. The controlled exposure to oxygen determined a decrease of Q higher than the 50% of the initial values. Data obtained in this study suggested that practices as micro-oxygenation and wood aging could help to decrease the amount of Q in sg wines.

Microbiological characterization of Gioddu, an Italian fermented milk.

Gioddu, also known as "Miciuratu", "Mezzoraddu" or "Latte ischidu" (literally meaning acidulous milk), is the sole variety of traditional Italian fermented milk. The aim of the present study was to elucidate the microbiota and the mycobiota occurring in artisan Gioddu sampled from three Sardinian producers by combining the results of viable counting on selective culture media and high-throughput sequencing. Physico-chemical parameters were also measured. The overall low pH values (3.80-4.22) recorded in the analyzed Gioddu samples attested the strong acidifying activity carried out by lactic acid bacteria during fermentation. Viable counts revealed the presence of presumptive lactococci, presumptive lactobacilli and non-Saccharomyces yeasts. A complex (kefir-like) microbiota of bacteria and yeasts was unveiled through sequencing. In more detail, Lactobacillus delbrueckii was found to dominate in Gioddu together with Streptococcus thermophilus, thus suggesting the establishment of a yogurt-like protocooperation. Unexpectedly, in all the three analyzed batches from two out of the three producers Lactobacillus kefiri was also detected, thus representing an absolute novelty, which suggests the presence of bioactive compounds (e.g. exopolysaccharides) similar to those characterizing milk kefir beverage. Mycobiota population, studied for the very first time in Gioddu, revealed a more complex composition, with Kluyveromyces marxianus, Galactomyces candidum and Geotrichum galactomyces constituting the core species. Further research is needed to disclose the eventual occurence in Gioddu of probiotic cultures and bioactive compounds (e.g. exopolysaccharides, angiotensin-converting enzyme inhibitory peptides and antimicrobial compounds) with potential health-benefits for the consumers.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk.

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S-23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated. The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6. Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers' health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Impact of a selected Debaryomyces hansenii strain's inoculation on the quality of Sardinian fermented sausages.

Dominant yeast species in Salsiccia Sarda, a traditional fermented sausage produced in Sardinia (Italy), were evaluated through the monitoring three typical production processes. Six different species were identified by molecular techniques, but Debaryomyces (D.) hansenii proved to be dominant. A D. hansenii strain was selected according to its technological features and used in three experimental sausage productions at farm scale with the aim to evaluate its antifungal effect. In all cases, two batches were inoculated with a previously selected autochthonous starter cultures (Lactobacillus plantarum and Staphylococcus xylosus), whereas two batches were left to spontaneous fermentation. D. hansenii was inoculated on the sausages surface by brushing after the sausages drying, by immersion in a yeast suspension after the stuffing, or, alternatively, casings were dipped in a yeast suspension before the dough stuffing. Microbial counts in the sausages core did not appear to be affected by D. hansenii application, while outcomes obtained for casings appeared soundly diversified. Brushing on the sausages surface at the onset of fermentation proved to be the best approach to treat sausages. Yeast inoculation exerted a noteworthy anti-mould effect, independently of the mode of application and, on the other hand, did not affect the overall quality and typical features of the product.

Influence of microbial communities on the chemical and sensory features of Falanghina sweet passito wines.

Natural (N) as well as starter inoculated (S, inoculated with Saccharomyces cerevisiae M3-5; CZS, Candida zemplinina T13, Zygosaccharomyces bailii NS113 and Saccharomyces cerevisiae M3-5) fermentations of Falanghina must from dehydrated grape were monitored. Culture dependent analyses and amplicon-based high-throughput sequencing targeting 18S rRNA and 16S rRNA genes were used to monitor the fungal and bacterial communities (8 sampling points during 65 days). The resulting wines were subject to both sensory evaluation and volatile organic compounds analysis. Fungal community of un-inoculated musts (N) at beginning of the fermentation was mainly represented by Aureobasidium, Cladosporium, Sclerotinia, while Candida, Debaryomyces, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Zygosaccharomyces showed a very low occurrence. The dominance of Hanseniaspora vineae and/or Hanseniaspora uvarum was clear up to 29th days of fermentation. S. cerevisiae occurred in all the phases but become dominant only at the end of the process. The odour profiles as evaluate by Quantitative Descriptive Analysis (QDA) highlighted a significant impact of the fungal populations on the olfactory profiles of the wines. Raisins, dried fruits, Sherry and liqueur were stronger in both S and CZS, while N was mostly discriminated by solvent/chemical and floral features. Outcomes underpin the impact of microbiota on the chemical and odour traits of Falanghina passito wines.

Alternative Methods to SO2 for Microbiological Stabilization of Wine.

The use of sulfur dioxide (SO2 ) as wine additive is able to ensure both antioxidant protection and microbiological stability. In spite of these undeniable advantages, in the last two decades the presence of SO2 in wine has raised concerns about potential adverse clinical effects in sensitive individuals. The winemaking industry has followed the general trend towards the reduction of SO2 concentrations in food, by expressing at the same time the need for alternative control methods allowing reduction or even elimination of SO2. In the light of this, research has been strongly oriented toward the study of alternatives to the use of SO2 in wine. Most of the studies have focused on methods able to replace the antimicrobial activity of SO2 . This review article gives a comprehensive overview of the current state-of-the-art about the chemical additives and the innovative physical techniques that have been proposed for this purpose. After a focus on the chemistry and properties of SO2 in wine, as well as on wine spoilage and on the conventional methods used for the microbiological stabilization of wine, recent advances on alternative methods proposed to replace the antimicrobial activity of SO2 in winemaking are presented and discussed. Even though many of the alternatives to SO2 showed good efficacy, nowadays no other physical technique or additive can deliver the efficacy and broad spectrum of action as SO2 (both antioxidant and antimicrobial), therefore the alternative methods should be considered a complement to SO2 in low-sulfite winemaking, rather than being seen as its substitutes.

Brettanomyces bruxellensis population survey reveals a diploid-triploid complex structured according to substrate of isolation and geographical distribution.

Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO2). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

Improving in vivo conversion of oleuropein into hydroxytyrosol by oral granules containing probiotic Lactobacillus plantarum 299v and an Olea europaea standardized extract.

This study reports novel food-grade granules for co-delivery of L. plantarum 299v and a standardized extract of Olea europaea leaves (Phenolea®) as oral carrier of probiotics and hydroxytyrosol. Different granule formulations containing either L. plantarum 299v (Lac), or the olive leave extract (Phe) or their combination (Lac-Phe) have been successfully produced through wet granulation employing excipients generally regarded as safe as granulating/binding agents. L. plantarum cells withstood the manufacturing process and were stable upon storage at 4 °C for more than 6 months. In vitro dissolution studies in simulated gastro-intestinal fluids showed the capability of the granules to rapidly dissolve and deliver both olive leave phenols and living L. plantarum cells. In simulated digestion conditions, Lac and Lac-Phe granules protected L. plantarum against the harsh environment of the gastro-intestinal tract. Co-administration of Lac and Phe oral granules to healthy mice provided for higher amounts of hydroxytyrosol in urines as compared to Phe granules alone, suggesting that L. plantarum 299v boosted in vivo conversion of oleuropein to hydroxytyrosol. On the other hand, PCR-assisted profiling of the Lactobacillus population in faeces obtained from mice treated with Lac or Lac plus Phe confirmed that the probiotic arrived alive to colon and was there able to exert a sort of perturbing effect on the climax colonic microflora. Overall, these results pave the way towards the development of a nutraceutical useful for combined delivery of bioactive hydroxytyrosol and probiotics to colon site.