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.

Investigating the biotechnological potential of lactic acid bacteria strains isolated from different Algerian dairy and farm sources.

Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

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.

Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp.

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer.IMPORTANCELactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

Glucose- and Lipid-Related Biomarkers Are Affected in Healthy Obese or Hyperglycemic Adults Consuming a Whole-Grain Pasta Enriched in Prebiotics and Probiotics: A 12-Week Randomized Controlled Trial.

BACKGROUND: Synbiotic foods, which combine the action of prebiotics and probiotics along the gastrointestinal tract, can affect inflammatory and glucose-related markers. OBJECTIVE: The aim of this study was to investigate the effects on inflammatory and glycemia-related markers of a whole-grain pasta containing barley ß-glucans and Bacillus coagulans BC30, 6086 in healthy overweight or obese volunteers. METHODS: A single-blind, parallel, randomized, placebo-controlled dietary intervention study was carried out. Forty-one healthy sedentary overweight (body mass index [BMI] 25-29.9 kg/m2) and obese (BMI ≥30) volunteers, aged 30-65 y and low consumers of fruit and vegetables, ate 1 serving/d of whole-grain control (CTR) or innovative (INN) pasta for 12 wk and maintained their habitual diets. Biological samples were collected at baseline and every 4 wk for primary (plasma high-sensitivity C-reactive protein [hs-CRP] and fasting plasma lipid profile) and secondary outcomes (glycemia-related markers, blood pressure, fecal microbiota composition, and body weight). Between (CTR compared with INN) and within (among weeks) group differences were tested for the whole population and for subgroups stratified by baseline values of BMI (≥30) and glycemia (≥100 mg/dL). RESULTS: INN or CTR pasta consumption had no effect on primary and secondary outcomes over time, except for a significant increase in plasma γ-glutamyltransferase (GGT) after 12 wk of CTR pasta consumption. Comparisons between intervention groups revealed differences only at 12 wk: plasma GGT was higher in the CTR group; plasma hs-CRP, plasma LDL/HDL cholesterol ratio, and Bifidobacterium spp. were lower in the INN subgroup of obese volunteers; plasma resistin was lower and Faecalibacterium prausnitzii abundance was higher in the INN subgroup of hyperglycemic volunteers. CONCLUSIONS: A daily serving of a synbiotic whole-grain pasta had limited effects on primary and secondary outcomes in the entire group of volunteers but affected glycemia- and lipid-related markers and resistin in a subgroup of healthy obese or hyperglycemic volunteers. This trial was registered at clinicaltrials.gov as NCT02236533.

Antimicrobial spectrum activity of bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk.

Bacteriocins have attracted great attention as potential alternatives to antibiotics and chemical food additives. In the present study, 243 Staphylococcus isolates from milk samples (n = 110) of goat and sheep herds located in Fars province, Iran, were screened for antimicrobial substance production. Twenty-eight isolates showed an antagonistic activity against the indicator strain Micrococcus luteus ATCC 4698. The susceptibility of all antimicrobial substances to proteolytic enzymes allowed us to consider them as bacteriocin-like inhibitory substances (BLIS). The term BLIS is applied to uncharacterized proteinaceous antimicrobials produced by gram-positive bacteria. Based on molecular identification methods, the isolates belonged to the species Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus pseudintermedius, Staphylococcus aureus, and Staphylococcus agnetis. Pulsed-field gel electrophoresis revealed a high level of genotype diversity among the Staph. chromogenes isolates. All of the isolates harbored nukA or bsaA2 genes, suggesting that their BLIS were related to nukacin or Bsa. The antimicrobial compounds from test strains were not effective against gram-negative pathogens, including Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Klebsiella pneumonia as well as the indicator mold Aspergillus fumigatus. All the gram-positive targets, including Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis Ef37 (a tyramine-producer strain), Lactobacillus saerimneri 30a (a histamine-producer strain), and methicillin-resistant Staph. epidermidis, were inhibited by the Staph. chromogenes isolates. Staphylococcus haemolyticus 4S12 was able to inhibit the majority of gram-positive bacteria. Listeria monocytogenes strains were the only indicators sensitive to the antimicrobial agents produced by Staph. agnetis 4S97B. The other Staphylococcus strains were ineffective on all the organisms tested. Based on their inhibitory capacities, the BLIS produced by the Staph. chromogenes isolates seem to be interesting candidates for developing novel antimicrobial agents.

Safety hazards in bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk.

In this study, 28 bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk were subjected to the PCR detection of enterotoxin genes (sea-see), enterotoxin-like toxin Q gene (selq), toxic shock syndrome toxin gene (tst1), and antibiotic resistance genes. They were also evaluated for phenotypic resistance against 10 antibiotics and hemolytic activity. The tyramine and histamine production was investigated using the agar plate assay and capillary zone electrophoretic analysis (CZE). Twenty-five isolates harbored at least one enterotoxin gene. The gene sec was the most frequent (89%). The gene tst1 was found in 84% of sec-positive isolates. The occurrence of antibiotic resistance genes was in the order of blaZ/tetK (100%), mecA/ermB (86%), ermC (50%), and tetM (18%). The genes ermA, aac(6')Ie-aph(2″)Ia, vanA, and vanB were absent in all the isolates. Nineteen isolates were phenotypically susceptible to all the antibiotics. The only isolate with phenotypic resistance to penicillin G and oxacillin was S. epidermidis 4S93 which had a different SmaI-PFGE profile from those of the other S. epidermidis strains. All the S. haemolyticus and S. pseudintermedius isolates were not susceptible to trimethoprim. Twenty-five isolates showed complete or partial hemolytic activity. None of the isolates was able to decarboxylate tyrosine, while CZE analysis revealed histamine formation activity in S. haemolyticus 4S12. The occurrence of safety risks in the isolates reinforces the need for regular monitoring of food-producing animals to mitigate the risks of multidrug resistant and zoonotic pathogens. Moreover, none of the isolates fulfilled the safety criteria to be used as starter cultures or biopreservatives.

Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay.

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

Variability in gene content and expression of the thioredoxin system in Oenococcus oeni.

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions.

Effect of thyme essential oil and Lactococcus lactis CBM21 on the microbiota composition and quality of minimally processed lamb's lettuce.

The main aim of this work was to evaluate, at pilot scale in an industrial environment, the effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared to chlorine, used in the washing step of fresh-cut lamb's lettuce, on the microbiota and its changes in relation to the time of storage. The modification of the microbial population was studied through pyrosequencing in addition to the traditional plate counts. In addition, the volatile molecule and sensory profiles were evaluated during the storage. The results showed no significant differences in terms of total aerobic mesophilic cell loads in relation to the washing solution adopted. However, the pyrosequencing data permitted to identify the genera and species able to dominate the spoilage associations over storage in relation to the treatment applied. Also, the analyses of the volatile molecule profiles of the samples during storage allowed the identification of specific molecules as markers of the spoilage for each different treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the panelists for samples washed with the combination thyme EO and the biocontrol agent. These samples were preferred for attributes such as flavor, acceptability and overall quality. These results highlighted the effect of the innovative washing solutions on the quality of lettuce through the shift of microbiota towards genera and species with lower potential in decreasing the sensory properties of the product.

Association between intestinal permeability and faecal microbiota composition in Italian children with beta cell autoimmunity at risk for type 1 diabetes.

BACKGROUND: Pancreatic organ-specific autoimmunity in subjects at risk for type 1 diabetes (T1D) is associated with increased intestinal permeability and an aberrant gut microbiota, but these factors have not yet been simultaneously investigated in the same subjects. Thus, the aim of this study was to assess both intestinal permeability and gut microbiota composition in an Italian sample of children at risk for T1D. METHODS: Ten Italian children with beta cell autoimmunity at risk for T1D and 10 healthy children were involved in a case-control study. The lactulose/mannitol test was used to assess intestinal permeability. Analysis of microbiota composition was performed using polymerase chain reaction followed by denaturing gradient gel electrophoresis, based on the 16S rRNA gene. RESULTS: Intestinal permeability was significantly higher in children at risk for T1D than in healthy controls. Moreover, the gut microbiota of the former differed from that of the latter group: Three microorganisms were detected - Dialister invisus, Gemella sanguinis and Bifidobacterium longum - in association with the pre-pathologic state. CONCLUSIONS: The results of this study validated the hypothesis that increased intestinal permeability together with differences in microbiota composition are contemporaneously associated with the pre-pathological condition of T1D in a sample of Italian children. Further studies are necessary to confirm the microbial markers identified in this sample of children as well as to clarify the involvement of microbiota modifications in the mechanisms leading to increased permeability and the autoimmune mechanisms that promote diabetes onset. Copyright © 2016 John Wiley & Sons, Ltd.

Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study.

Probiotics are microorganisms that confer beneficial effects on the host; nevertheless, before being allowed for human consumption, their safety must be verified with accurate protocols. In the genomic era, such procedures should take into account the genomic-based approaches. This study aims at assessing the safety traits of Bacillus coagulans GBI-30, 6086 integrating the most updated genomics-based procedures and conventional phenotypic assays. Special attention was paid to putative virulence factors (VF), antibiotic resistance (AR) genes and genes encoding enzymes responsible for harmful metabolites (i.e. biogenic amines, BAs). This probiotic strain was phenotypically resistant to streptomycin and kanamycin, although the genome analysis suggested that the AR-related genes were not easily transferrable to other bacteria, and no other genes with potential safety risks, such as those related to VF or BA production, were retrieved. Furthermore, no unstable elements that could potentially lead to genomic rearrangements were detected. Moreover, a workflow is proposed to allow the proper taxonomic identification of a microbial strain and the accurate evaluation of risk-related gene traits, combining whole genome sequencing analysis with updated bioinformatics tools and standard phenotypic assays. The workflow presented can be generalized as a guideline for the safety investigation of novel probiotic strains to help stakeholders (from scientists to manufacturers and consumers) to meet regulatory requirements and avoid misleading information.

Biocide and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from the swine meat chain.

In this study nine strains of Enterococcus faecalis and 12 strains of Enterococcus faecium, isolated from different sample types in the swine meat chain and previously characterized for the presence of antibiotic resistance genes, were examined for phenotypic tolerance to seven biocides (chlorexidine, benzalkonium chloride, triclosan, sodium hypochlorite, 2-propanol, formaldehyde and hydrogen peroxide) and resistance to nine antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol). Moreover, the presence of efflux system encoding genes qacA/B, qacC, qacE, qacEΔ1, emeA, and stress response genes, sigV and gsp65, involved in the tolerance to biocides, was analysed. Most strains were not tolerant to the biocides, but showed minimum inhibitory concentrations (MICs) higher than the recommended cut-off values for all the antibiotics tested, except for vancomycin and chloramphenicol. Only weak correlations, if any, were found between biocide and antibiotic resistance data. One E. faecalis strain was tolerant to triclosan and one E. faecium strain, with higher tolerance to chlorexidine than the other strains tested, was found to carry a qacA/B gene. Our results indicated that phenotypic resistance to antibiotics is very frequent in enterococcal isolates from the swine meat chain, but phenotypic tolerance to biocides is not common. On the other hand, the gene qacA/B was found for the first time in the species E. faecium, an indication of the necessity to adopt measures suitable to control the spread of biocide resistance determinants among enterococci.

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains.

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.

Horizontal gene transfer among microorganisms in food: current knowledge and future perspectives.

The possibility of horizontal gene transfer (HGT) among microorganisms in food matrices has been specifically targeted in a few investigations, though most current knowledge has been obtained indirectly or derived from genome sequence analyses. In this review, we have assembled reported examples of the HGT events that probably occurred in food matrices since the bacterial partners involved are commonly found in association in a food matrix or are specifically adapted to it. Exchanged genes include those encoding for substrate utilization, bacteriocin, exopolysaccharide and biogenic amine (BA) production, immunity to bacteriophages and antibiotic resistance (AR). While the acquisition of new traits involved in substrate utilization led to the natural genetic improvement of the microbial cultures for food production, the acquisition of hazardous traits, e.g., AR, virulence or BA production genes, can give rise to health concerns in otherwise innocuous species. Available evidence suggests that it would be opportune to determine what conditions favour HGT among bacteria in food ecosystems in order to naturally obtain improved starter or adjunct cultures, and also to prevent the propagation of hazardous traits.

Selection of Botrytis cinerea and Saccharomyces cerevisiae strains for the improvement and valorization of Italian passito style wines.

In order to improve the quality of Italian passito wine, produced from withered grapes that can be naturally infected by noble rot, in this study, a novel protocol was developed to select suitable cultures of both Botrytis cinerea to infect grapes (as noble rot) and of Saccharomyces cerevisiae to ferment grapes. A total of 16 B. cinerea isolated from withered grapes were typified by RAPD-PCR, and three representative strains were selected for physiological characterization. The strains showed different mycelial growth and enzymatic activities (i.e. polygalacturonase, protease, and laccase). A total of 15 yeasts were isolated from spontaneous fermented wines, these were identified as S. cerevisiae, and typified at strain level. Seven strains were selected according to RAPD-PCR profiles and tested for their fermentation performances. The effects of B. cinerea and S. cerevisiae cultures on the aroma profile of sweet style wine were preliminary evaluated fermenting artificially botrytized grapes induced with B. cinerea infection. The combination of selected fungi affected the aroma profile of wine according to the variation of the content of important molecules (i.e. alcohols, esters, and lactones). This study has provided valuable information to develop new natural cultures destined to induce grape botrytization and manage fermentation in passito winemaking.

Biodiversity and characterization of indigenous coagulase-negative staphylococci isolated from raw milk and cheese of North Italy.

The aim of the current study was to detect coagulase-negative staphylococci (CNS) in raw milk and cheeses produced in North Italy, and to analyze isolates for their biodiversity, safety aspects and technological properties. Molecular identification methods revealed a high biodiversity among isolates and assigned them to 17 species. The most recovered species were Staphylococcus equorum (12%), Staphylococcus lentus (12%), Staphylococcus simulans (12%), Staphylococcus sciuri (10%), and Staphylococcus xylosus (9%). The presence of ten transferable antibiotic resistance (AR) genes was verified by PCR and 19% of isolates were positive, with tet(K) being the most frequent gene (10%); interestingly, no strain carried multiple AR genes. Twenty-four isolates displayed hemolytic activity; tyrosine decarboxylase gene (tdcA) was found in two isolates, while histidine decarboxylase gene (hdcA) and enterotoxin genes (se) were not detected. Isolates were further characterized for the presence of some relevant technological properties; 16% of isolates displayed proteolytic activity and 39% lipolytic activity, while no one of the isolates was found to exhibit antimicrobial activity against Staphylococcus aureus. This study provided evidence of a low occurrence of safety hazards in CNS isolated from dairy products.

Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes.

The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387(T) or Clostridium tyrobutyricum LMG 1285(T). In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria. PCR detection of thermophilin genes proved unreliable in predicting antimicrobial activity. For S. thermophilus PRI36 and PRI45, with relevant inhibitory features, the identity of the bacteriocin genes present in the thermophilin 9 cluster was defined, thus revealing novel variants for this genome region.

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.