Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses.

In the present study 177 Lactobacillus spp. strains, isolated from Ragusano and Pecorino Siciliano cheeses, were in vitro screened for probiotic traits, and their characteristics were compared to those of Lactobacillus rhamnosus GG, commercial strain. Based on acidic and bile salt resistance, thirteen Lactobacillus strains were selected. The multiplex-PCR application revealed that nine strains belonged to L. rhamnosus species and four to Lactobacillus paracasei species. All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal tract (GI), for adhesion capacity to human intestinal cell lines, for hydrophobicity, for co-aggregation and auto-aggregation and for antimicrobial activities. Moreover, antibiotic resistance, hemolytic and bile salt hydrolase activities were investigated for safety assessment. Viable counts after simulated gastric and duodenal transit revealed that overall the selected lactobacilli tolerated better pancreatic juice and bile salts than acidic juice. In particular, three L. rhamnosus strains (FS10, FS2, and PS11) and one L. paracasei strain (PM8) increased their cell density after the simulated GI transit. The same strains showed also high percentage of auto-aggregation and co-aggregation with Escherichia coli. All strains were effective against both Staphylococcus aureus and E. coli and variability was achieved versus Listeria monocytogenes and Enterococcus faecalis used as pathogenic indicator strains. Different behavior was revealed by strains for adhesion ability and hydrophobicity, which are not always linked each other and are strongly strain-dependent. From the safety point of view, no isolate showed hemolytic and bile salt hydrolase activities, except one, and most of the strains were sensitive to a broad range of clinical antibiotics. This work showed that the L. rhamnosus FS10 and the L. paracasei PM8 are good promising probiotic candidates for further in vivo investigations.

Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding.

AIMS: The present work proposes a two-step molecular strategy to select inter- and intra-species Saccharomyces hybrids obtained by spore-to-spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts. METHODS AND RESULTS: As low spore viability and haplo-selfing are the main causes of failed mating, at first, we used colony screening PCR (csPCR) of discriminative gene markers to select hybrids directly on dissection plate and discard homozygous diploid colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted to recursive streaking and conventional PCR in order to discriminate between the hybrids with stable genomic background and the false-positive admixtures of progenitor cells both undergone haplo-selfing. csPCRs of internal transcribed spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI, respectively, were efficient to select six new Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses. Intragenic minisatellite regions in PIR3, HSP150, and DAN4 genes showed high inter-strain size variation detectable by cost-effective agarose gel electrophoresis and were successful to validate six new intra-species S. cerevisiae hybrids from 34 crosses. CONCLUSIONS: Both protocols reduce significantly the number of massive DNA extractions, prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be easily implemented in yeast labs without any specific instrumentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a method for the marker-assisted selection of several inter- and intra-species yeast hybrids in a cost-effective, rapid and reproducible manner.

Probiotic supplementation in systemic nickel allergy syndrome patients: study of its effects on lactic acid bacteria population and on clinical symptoms.

AIMS: The study aimed to evaluate the effects of probiotic Lactobacillus reuteri DSM 17938 strain supplementation in patients suffering from systemic nickel allergy syndrome, in terms of modulation of faecal LAB population linked to a reduction of GI and cutaneous symptoms and to an increase of patient's quality of life. METHODS AND RESULTS: A preliminary double-blind randomized placebo-controlled study was planned and a culture-independent method based on denaturing gradient gel electrophoresis (DGGE) analysis coupled to the 16S rRNA gene sequencing was applied to investigate on the dynamics of faecal LAB communities before and during a low-Ni diet, supplemented with the probiotic strain. Moreover, the severity and the frequency of GI and cutaneous reactions as well as patient's clinical condition perception (VAS scores) were estimated by statistical analysis. PCR-DGGE fingerprinting obtained using LAB-specific primers revealed significant shift in faecal community with an increase in biodiversity in patients supplemented with probiotic Lact. reuteri strain. In addition, GI reactions such as symptoms related to meals and type of stools significantly improved only in patients treated with Lact. reuteri, while severity and frequency of cutaneous symptoms (urticaria, itch and eczema) and recurrent abdominal pain (RAP) as well as VAS scores statistically decreased in both groups. CONCLUSIONS: Our preliminary findings suggest that probiotic Lact. reuteri could be a useful supplementation during a low-Ni diet of patients with SNAS, to increase LAB population diversity, which could contribute to restore the intestinal homoeostasis conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: To date, no information is available on probiotics application and on their effects, in terms of intestinal microbiota modulation, on patients suffering from SNAS. Therefore, the identification of dominant LAB community and the study of its shift during the probiotic supplementation could enhance the knowledge of the SNAS syndrome.

Pecorino Crotonese cheese: study of bacterial population and flavour compounds.

The diversity and dynamics of the dominant bacterial population during the manufacture and the ripening of two artisanal Pecorino Crotonese cheeses, provided by different farms, were investigated by the combination of culture-dependent and -independent approaches. Three hundred and thirty-three strains were isolated from selective culture media, clustered using Restriction Fragment Length Polymorphism and were identified by 16S rRNA gene sequencing. The results indicate a decrease in biodiversity during ripening, revealing the presence of Lactococcus lactis and Streptococcus thermophilus species in the curd and in aged cheese samples and the occurrence of several lactobacilli throughout cheese ripening, with the dominance of Lactobacillus rhamnosus species. Bacterial dynamics determined by Denaturant Gradient Gel Electrophoresis provided a more precise description of the distribution of bacteria, highlighting differences in the bacterial community among cheese samples, and allowed to detect Lactobacillus plantarum, Lactobacillus buchneri and Leuconostoc mesenteroides species, which were not isolated. Moreover, the concentration of flavour compounds produced throughout cheese ripening was investigated and related to lactic acid bacteria presence. Fifty-seven compounds were identified in the volatile fraction of Pecorino Crotonese cheeses by Gas Chromatography-Mass Spectrometry. Esters, alcohols and free fatty acids were the most abundant compounds, while aldehydes and hydrocarbons were present at low levels.

Application of molecular approaches to study lactic acid bacteria in artisanal cheeses.

The present review focuses on the application of the most common molecular approaches for the investigation of the diversity of LAB in the artisanal cheese environment, and for their tracking within this complex ecosystem. Historically the detection and identification of lactic acid bacteria (LAB) has largely been hampered by the incomplete knowledge of their optimal in vitro culture conditions. Recent years have seen an explosion in the application of molecular tools for elucidating both qualitatively and quantitatively on the abundance of LAB species and how their presence interacts with the environment. Many of these approaches, predominantly rooted in the use of ribosomal RNA (rRNA) and its encoding genes, are successfully applied to describe the relationship between LAB and their individual identity. In the present work emphasis is given to the methodologies to determine the complex communities as well as to the detection of individual groups of LAB of several artisanal cheeses. Moreover, analyses of viability of LAB during the manufacture and ripening, through culture-independent approaches is also discussed. The potential of novel advances in molecular technologies such as metagenomic for analysis of the cheese ecosystem is also provided.

Effect of wild strains used as starter cultures and adjunct cultures on the volatile compounds of the Pecorino Siciliano cheese.

The effect of six wild strains on the volatile profile of the PS cheese was investigated and compared to that generated from industrial starters generally used to produce PS cheese. All cheeses were subjected to microbiological, physicochemical, and volatile compounds analyses. The DGGE of the 16S rDNA analysis was also applied. The volatile compounds generated during ripening were studied through the SPME and the GC-MS methods. No difference was detected between the experimental and control cheeses throughout chemical and microbiological analyses, while the DGGE results showed the presence of Streptococcus thermophilus in all cheeses, and the dominance of Enterococcus durans, Lactobacillus rhamnosus, and Lactobacillus casei in most of the experimental cheeses. Moreover, the presence of Lactococcus lactis species as in the control and in the experimental P2 and P4 cheeses was also revealed. The SPME results showed more pronounced volatile compounds in the experimental cheese samples than in the control ones.

Preliminary characterization of wild lactic acid bacteria and their abilities to produce flavour compounds in ripened model cheese system.

AIMS: The aim of this work was to preliminary characterize wild lactic acid bacteria (LAB), previously isolated during artisanal Pecorino Siciliano (PS) cheese-making for technological and flavour formation abilities in a model cheese system. METHODS AND RESULTS: Twelve LAB were studied for the ability to grow at 10 and 45 degrees C, to coagulate and acidify both reconstituted skim milk and ewe's milk. Moreover, the capacity of the strains to generate aroma compounds was evaluated in a model cheese system at 30- and 60-day ripening. Flavour compounds were screened by sensory analysis and throughout gas chromatography (GC)-mass spectrometry (MS). Most of the strains were able to grow both at 10 and 45 degrees C and exhibited high ability to acidify and coagulate ewes' milk. Sensory evaluation revealed that the wild strains produced more significant flavour attributes than commercial strains in the 60-day-old model cheese system. GC-MS data confirmed the results of sensory evaluations and showed the ability of wild lactobacilli to generate key volatile compounds. Particularly, three wild lactobacilli strains, belonging to Lactobacillus casei, Lb. rhamnosus and Lb. plantarum species, generated both in 60- and 30-day-old model cheeses system, the 3-methyl butan(al)(ol) compound, which is associated with fruity taste. CONCLUSIONS: The present work preliminarily demonstrated that the technological and flavour formation abilities of the wild strains are strain-specific and that wild lactobacilli, which produced key flavour compounds during ripening, could be used as tailor-made starters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the technological characterization and flavour formation ability of wild LAB strains isolated from artisanal Pecorino cheese and highlights that the catabolic activities were highly strain dependent. Hence, wild lactobacilli could be selected as tailor-made starter cultures for the PS cheese manufacture.

Bacterial population in traditional sourdough evaluated by molecular methods.

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.

Identification of Pichia anomala isolated from yoghurt by RFLP of the ITS region.

Several packs of swollen retailed plain and flavoured yoghurt were examined. The most commonly found species was Pichia anomala, identified both by physiological tests and RFLP analysis of the rDNA internal transcribed spacer (ITS) region. The isolated strains did not ferment lactose and were positive for galactose fermentation, confirming the hypothesis that galactose-fermenting yeast could be the cause of spoilage in yoghurt.

Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto.

Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rDNA, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group.

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains.

The chromosomal DNAs of cryotolerant Saccharomyces bayanus, non-cryotolerant Saccharomyces cerevisiae strains and their intra and interspecific hybrids were separated by pulsed field electrophoresis (PFGE). The cryotolerant and non-cryotolerant strains gave distinctly different electrophoretic profiles. The hybrids cryotolerant x cryotolerant and non-cryotolerant x non-cryotolerant were fertile and they gave the same electrophoretic karyotype as the respective parents. The cryotolerant x non-cryotolerant hybrids were sterile and gave electrophoretic karyotypes which showed both the bands the parents have in common and those they do not share.

Karyotyping of Saccharomyces strains with different temperature profiles.

This study examined the karyotype, the fermentation performance and the optimum growth temperature (Topt) of 28 yeast strains all identified as species belonging to Saccharomyces sensu stricto. The strains were isolated from fermented musts, which had not been inoculated, at two temperature ranges: 20-40 degrees C and approximately 0-6 degrees C. The results demonstrated a correlation between the Topt and the chromosome organization. In particular, strains with Topt of less than 30 degrees C showed only two bands in the region between 365 and 225 kb, while those with a Topt greater than 30 degrees C had three bands in this size range. From a taxonomic viewpoint, the Topt is a better indicator for the Saccharomyces sp. than the ceiling temperature of 37 degrees C currently used to differentiate cryotolerant Saccharomyces bayanus and S. pastorianus from non-cryotolerant S. cerevisiae and S. paradoxus strains.