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months of life, reaching the state of mature microbiota when the child is around two years old. Even so, diet, lifestyle, antibiotic consumption or the aging process will determine changes in its composition and, as a consequence, the possibility of suffering chronic non-communicable diseases. Currently, there is sufficient scientific evidence to strengthen the importance of diet for the establishment, structure and functional activity of the gut microbiota, as well as many studies that demonstrate the role of diet in certain diseases through its effects on microbial communities of the gut. Modulation of the gut microbiota through dietary intervention is an emerging therapeutic and preventive strategy for many conditions. Probiotics and prebiotics may also be effective for restoration of beneficial bacteria and microbiota diversity capable to shift from disease to health promoting states.

Epidemiological studies have found a higher prevalence of allergic symptoms and positive prick tests in patients with Meniere's disease (MD); however the effect of allergenic extracts in MD has not been established. Thus, this study aims to determine the effect of Aspergillus and Penicillium stimulation in cytokine release and gene expression profile in MD. Patients with MD showed higher basal levels of IL-1β, IL-1RA, IL-6 and TNF-α when compared to healthy controls. We observed that IL-1β levels had a bimodal distribution suggesting two different subgroups of patients, with low and high basal levels of cytokines. Gene expression profile in peripheral blood mononuclear cells (PBMC) showed significant differences in patients with high and low basal levels of IL-1β. We found that both mold extracts triggered a significant release of TNF-α in MD patients, which were not found in controls. Moreover, after mold stimulation, MD patients showed a different gene expression profile in PBMC, according to the basal levels of IL-1β. The results indicate that a subset of MD patients have higher basal levels of proinflammatory cytokines and the exposure to Aspergillus and Penicillium extracts may trigger additional TNF-α release and contribute to exacerbate inflammation.

Background : Cochlear and vestibular epithelial non-hair cells (ENHCs) are the supporting elements of the cellular architecture in the organ of Corti and the vestibular neuroepithelium in the inner ear. Intercellular and cell-extracellular matrix interactions are essential to prevent an abnormal ion redistribution leading to hearing and vestibular loss. The aim of this study is to define the main pathways and molecular networks in the mouse ENHCs. Methods : We retrieved microarray and RNA-seq datasets from mouse epithelial sensory and non-sensory cells from gEAR portal (http://umgear.org/index.html) and obtained gene expression fold-change between ENHCs and non-epithelial cells (NECs) against HCs for each gene. Differentially expressed genes (DEG) with a log2 fold change between 1 and -1 were discarded. The remaining genes were selected to search for interactions using Ingenuity Pathway Analysis and STRING platform. Specific molecular networks for ENHCs in the cochlea and the vestibular organs were generated and significant pathways were identified. Results : Between 1723 and 1559 DEG were found in the mouse cochlear and vestibular tissues, respectively. Six main pathways showed enrichment in the supporting cells in both tissues: (1) "Inhibition of Matrix Metalloproteases"; (2) "Calcium Transport I"; (3) "Calcium Signaling"; (4) "Leukocyte Extravasation Signaling"; (5) "Signaling by Rho Family GTPases"; and (6) "Axonal Guidance Si". In the mouse cochlea, ENHCs showed a significant enrichment in 18 pathways highlighting "axonal guidance signaling (AGS)" ( p = 4.37 × 10 -8 ) and "RhoGDI Signaling" ( p = 3.31 × 10 -8 ). In the vestibular dataset, there were 20 enriched pathways in ENHCs, the most significant being "Leukocyte Extravasation Signaling" ( p = 8.71 × 10 -6 ), "Signaling by Rho Family GTPases" ( p = 1.20 × 10 -5 ) and "Calcium Signaling" ( p = 1.20 × 10 -5 ). Among the top ranked networks, the most biologically significant network contained the "auditory and vestibular system development and function" terms. We also found 108 genes showing tonotopic gene expression in the cochlear ENHCs. Conclusions : We have predicted the main pathways and molecular networks for ENHCs in the organ of Corti and vestibular neuroepithelium. These pathways will facilitate the design of molecular maps to select novel candidate genes for hearing or vestibular loss to conduct functional studies.

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) is a rare disorder with an unknown etiology. We present a British family with presumed autosomal dominant CANVAS with incomplete penetrance and variable expressivity. Exome sequencing identified a rare missense variant in the ELF2 gene at chr4:g.140058846 C > T, c.10G > A, p.A4T which segregated in all affected patients. By using transduced BE (2)-M17 cells, we found that the mutated ELF2 (mt-ELF2) gene increased ATXN2 and reduced ELOVL5 gene expression, the causal genes of type 2 and type 38 spinocerebellar ataxias. Both, western blot and confocal microscopy confirmed an increase of ataxin-2 in BE(2)-M17 cells transduced with lentivirus expressing mt-ELF2 (CEE-mt- ELF2 ), which was not observed in cells transduced with lentivirus expressing wt-ELF2 (CEE-wt-ELF2). Moreover, we observed a significant decrease in the number and size of lipid droplets in the CEE-mt- ELF2 -transduced BE (2)-M17 cells, but not in the CEE-wt-ELF2-transduced BE (2)-M17. Furthermore, changes in the expression of ELOVL5 could be related with the reduction of lipid droplets in BE (2)-M17 cells. This work supports that ELF2 gene regulates the expression of ATXN2 and ELOVL5 genes, and defines new molecular links in the pathophysiology of cerebellar ataxias.

Tinnitus is a common medical condition which interfaces many different disciplines, yet it is not a priority for any individual discipline. A change in its scientific understanding and clinical management requires a shift toward multidisciplinary cooperation, not only in research but also in training. The European School for Interdisciplinary Tinnitus research (ESIT) brings together a unique multidisciplinary consortium of clinical practitioners, academic researchers, commercial partners, patient organizations, and public health experts to conduct innovative research and train the next generation of tinnitus researchers. ESIT supports fundamental science and clinical research projects in order to: (1) advancing new treatment solutions for tinnitus, (2) improving existing treatment paradigms, (3) developing innovative research methods, (4) performing genetic studies on, (5) collecting epidemiological data to create new knowledge about prevalence and risk factors, (6) establishing a pan-European data resource. All research projects involve inter-sectoral partnerships through practical training, quite unlike anything that can be offered by any single university alone. Likewise, the postgraduate training curriculum fosters a deep knowledge about tinnitus whilst nurturing transferable competencies in personal qualities and approaches needed to be an effective researcher, knowledge of the standards, requirements and professionalism to do research, and skills to work with others and to ensure the wider impact of research. ESIT is the seed for future generations of creative, entrepreneurial, and innovative researchers, trained to master the upcoming challenges in the tinnitus field, to implement sustained changes in prevention and clinical management of tinnitus, and to shape doctoral education in tinnitus for the future.

Meniere's disease (MD) is a rare disorder characterized by episodic vertigo, sensorineural hearing loss, tinnitus, and aural fullness. It is associated with a fluid imbalance between the secretion of endolymph in the cochlear duct and its reabsorption into the subarachnoid space, leading to an accumulation of endolymph in the inner ear. Epidemiological evidence, including familial aggregation, indicates a genetic contribution and a consistent association with autoimmune diseases (AD). We conducted a case-control study in two phases using an immune genotyping array in a total of 420 patients with bilateral MD and 1,630 controls. We have identified the first locus, at 6p21.33, suggesting an association with bilateral MD [meta-analysis leading signal rs4947296, OR = 2.089 (1.661-2.627); p  = 1.39 × 10 -09 ]. Gene expression profiles of homozygous genotype-selected peripheral blood mononuclear cells (PBMCs) demonstrated that this region is a trans -expression quantitative trait locus (eQTL) in PBMCs. Signaling analysis predicted several tumor necrosis factor-related pathways, the TWEAK/Fn14 pathway being the top candidate ( p  = 2.42 × 10 -11 ). This pathway is involved in the modulation of inflammation in several human AD, including multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In vitro studies with genotype-selected lymphoblastoid cells from patients with MD suggest that this trans-eQTL may regulate cellular proliferation in lymphoid cells through the TWEAK/Fn14 pathway by increasing the translation of NF-κB. Taken together; these findings suggest that the carriers of the risk genotype may develop an NF-κB-mediated inflammatory response in MD.

Purpose: Genetic contributions to tinnitus have been difficult to determine due to the heterogeneity of the condition and its broad etiology. Here, we evaluated the genetic and nongenetic influences on self-reported tinnitus from the Swedish Twin Registry (STR)., Methods: Cross-sectional data from the STR was obtained. Casewise concordance rates (the risk of one twin being affected given that his/her twin partner has tinnitus) were compared for monozygotic (MZ) and dizygotic (DZ) twin pairs (N = 10,464 concordant and discordant twin pairs) and heritability coefficients (the proportion of the total variance attributable to genetic factors) were calculated using biometrical model fitting procedures., Results: Stratification of tinnitus cases into subtypes according to laterality (unilateral versus bilateral) revealed that heritability of bilateral tinnitus was 0.56; however, it was 0.27 for unilateral tinnitus. Heritability was greater in men (0.68) than in women (0.41). However, when female pairs younger than 40 years of age were selected, heritability of 0.62 was achieved with negligible effects of shared environment., Conclusion: Unlike unilateral tinnitus, bilateral tinnitus is influenced by genetic factors and might constitute a genetic subtype. Overall, our study provides the initial evidence for a tinnitus phenotype with a genetic influence.Genet Med advance online publication 23 March 2017.

The aim of the study was to evaluate the potential use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for fast and reliable detection of strains producing the lantibiotics nisin, lacticin 481 and lacticin 3147 in a large collection of lactococci. A total of one hundred lactococcal isolates from traditional ewe's and goat's raw milk cheeses were identified to the species level as Lactococcuslactis by MALDI-TOF MS based on comparison with lactococcal entries in the BioTyper database. Mass spectra in the range 2000-4000Da of the identified isolates were compared to reference spectra of three lactococcal strains producing lacticin 481 (IFPL 330), lacticin 3147 (IFPL 105) and nisin (IFPL 503). Only eight isolates had mass spectra with peaks that could be unequivocally identified as lacticin 481 (2900.47Da) or nisin (3330.31Da). None of the assayed isolates matched the mass spectra corresponding to the two-peptide lacticin 3147 (2847.97 and 3306.29Da). The results obtained by MALDI-TOF MS were genetically validated by amplification of the corresponding structural gene coding for lacticin 481, nisin and lacticin 3147. MALDI-TOF MS can be used as a fast and reliable technique to screen a large number of lactococcal isolates for the ability to produce the lantibiotics nisin, lacticin 481 and lacticin 3147., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The identification of disease-causing variants in autosomal dominant diseases using exome-sequencing data remains a difficult task in small pedigrees. We combined several strategies to improve filtering and prioritizing of heterozygous variants using exome-sequencing datasets in familial Meniere disease: an in-house Pathogenic Variant (PAVAR) score, the Variant Annotation Analysis and Search Tool (VAAST-Phevor), Exomiser-v2, CADD, and FATHMM. We also validated the method by a benchmarking procedure including causal mutations in synthetic exome datasets., Results: PAVAR and VAAST were able to select the same sets of candidate variants independently of the studied disease. In contrast, Exomiser V2 and VAAST-Phevor had a variable correlation depending on the phenotypic information available for the disease on each family. Nevertheless, all the selected diseases ranked a limited number of concordant variants in the top 10 ranking, using the three systems or other combined algorithm such as CADD or FATHMM. Benchmarking analyses confirmed that the combination of systems with different approaches improves the prediction of candidate variants compared with the use of a single method. The overall efficiency of combined tools ranges between 68 and 71% in the top 10 ranked variants., Conclusions: Our pipeline prioritizes a short list of heterozygous variants in exome datasets based on the top 10 concordant variants combining multiple systems.

Scope: There is growing interest in understanding how human colonic microbiota can be modified by dietary habits. We examined the influence of moderate red wine intake on the colonic microbiota of 15 healthy volunteers, related to the high concentration of polyphenols present in this beverage. The volunteers were classified into high, moderate, and low polyphenol metabolizers (metabotypes) due to their ability to metabolize polyphenols and the results were compared with that of five control (no wine intake) subjects., Methods and Results: We analyzed the composition, diversity, and dynamics of their fecal microbiota before and after 1 month of wine consumption. The 16S rDNA sequencing allowed detection of 2324 phylotypes, of which only 30 were found over the 0.5% of mean relative frequency, representing 84.6% of the total taxonomical assignments. The samples clustered more strongly by individuals than by wine intake or metabotypes, however an increase in diversity, after the wine intake, was observed., Conclusion: The results of this study suggest an increase in the global fecal microbial diversity associated to the consumption of red wine, confirm the high variability of the microbiota from different individuals, and show the stability of their singular microbiota composition to small and short-term dietary changes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autosomal dominant (AD) familial Meniere's disease (FMD) is a rare disorder involving the inner ear defined by sensorineural hearing loss, tinnitus and episodic vertigo. Here, we have identified two novel and rare heterozygous variants in the SEMA3D and DPT genes segregating with the complete phenotype that have variable expressivity in two pedigrees with AD-FMD. A detailed characterization of the phenotype within each family illustrates the clinical heterogeneity in the onset and progression of the disease. We also showed the expression of both genes in the human cochlea and performed in silico analyses of these variants. Three-dimensional protein modelling showed changes in the structure of the protein indicating potential physical interactions. These results confirm a genetic heterogeneity in FMD with incomplete penetrance and variable expressivity.

There is limited information that relates the intake of food-derived bioactive peptides and the gut microbiota. We have previously described a pepsin hydrolysate of egg white (EWH) that ameliorates fat accumulation and dyslipidemia, while reducing oxidative stress and inflammation markers in obese Zucker rats. The aim of this study was to associate the beneficial effects of EWH with gut microbiota changes in these animals. Obese Zucker rats received daily 750 mg kg -1 EWH in drinking water for 12 weeks and faeces were analysed for microbial composition and metabolic compounds in comparison with Zucker lean rats and obese controls. EWH supplementation modulated the microbiological characteristics of the obese rats to values similar to those of the lean rats. Specifically, counts of total bacteria, Lactobacillus/Enterococcus and Clostridium leptum in EWH fed obese Zucker rats were more similar to the lean rats than to the obese controls. Besides, feeding the obese Zucker rats with EWH reduced (P < 0.05) the faecal concentration of lactic acid. The physiological benefits of EWH in the improvement of obesity associated complications of Zucker rats could be associated with a more lean-like gut microbiota and a tendency to diminish total short-chain fatty acids (SCFA) production and associated obesity complications. The results warrant the use of pepsin egg white hydrolysate as a bioactive food ingredient.

The microbiological and metabolic changes of an overweight-associated colonic microbiota after reducing in vitro the carbohydrate supply and its supplementation with oligosaccharides derived from lactulose (OsLu) were evaluated using a dynamic simulator of the gastrointestinal tract. The differentiation and stability of the microbial communities within each colon compartment were reached after two weeks of feeding the system with a high energy (HE) medium based on fructose and readily fermentable starches. The effect of reducing the energy content (low-energy medium, LE) and the supplementation with OsLu caused minor variations in bacterial counts, except for Enterobacteriaceae. The LE medium caused an effect on the microbial metabolic activity that was characterized by an absence of net butyrate production and an increase in ammonium content. This shift from fermentative to proteolytic metabolism was not observed when the LE medium was supplemented with OsLu. This oligosaccharide mixture was mainly metabolized in the proximal colonic compartment. The results obtained in this study indicate that the substitution in the diet of easily digestible carbohydrates by OsLu maintains the fermentative functionality of the intestinal microbiota, allowing the net production of butyric acid with potential beneficial effects on health, and avoiding a full transition to proteolytic metabolism profiles., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Meniere's Disease (MD) is a complex disorder associated with an accumulation of endolymph in the membranous labyrinth in the inner ear. It is characterized by recurrent attacks of spontaneous vertigo associated with sensorineural hearing loss (SNHL) and tinnitus. The SNHL usually starts at low and medium frequencies with a variable progression to high frequencies. We identified a novel missense variant in the PRKCB gene in a Spanish family with MD segregating low-to-middle frequency SNHL. Confocal imaging showed strong PKCB II protein labelling in non-sensory cells, the tectal cells and inner border cells of the rat organ of Corti with a tonotopic expression gradient. The PKCB II signal was more pronounced in the apical turn of the cochlea when compared with the middle and basal turns. It was also much higher in cochlear tissue than in vestibular tissue. Taken together, our findings identify PRKCB gene as a novel candidate gene for familial MD and its expression gradient in supporting cells of the organ of Corti deserves attention, given the role of supporting cells in K +  recycling within the endolymph, and its apical turn location may explain the onset of hearing loss at low frequencies in MD., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: This study has evaluated the effect of regular and moderate red wine consumption on the diversity and occurrence of different groups of bacteria that are representative in human saliva., Methods: Saliva from twenty-two healthy volunteers (age range 20-48 years) was analyzed in this study. Fourteen individuals consumed red wine (250mL/day) during 4 weeks, whereas 8 volunteers were included in the control group. The evolution and composition of the microbial community in saliva was evaluated by PCR-DGGE and quantitative PCR., Results: The microbial inter-individual variability observed in the PCR-DGGE band patterns was higher than the differences observed after the 4-weeks period of red wine intake. Bifidobacterium dentium, Bifidobacterium spp. and Alloscardovia omnicolens were the most representative bifidobacterial species, whereas the Streptococcus mitis-Streptococcus oralis group predominated within Streptococcus. This genus was the most numerous of the bacterial groups assayed, reaching average counts above 8 log copy numbers/mL. On the other hand, the lowest counts were recorded for Actinomyces, Fusobacterium, Haemophilus, Neisseria and Veillonella, which showed average values of 5 log copy numbers/mL. The results showed no significant differences (P>0.5) in bacterial counts after the period of red wine intake., Conclusion: The overall diversity and stability of representative bacterial groups of the human saliva is not disturbed due to regular-moderate red wine consumption., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, we have assessed the phenolic metabolism of a cranberry extract by microbiota obtained from the ascending colon and descending colon compartments of a dynamic gastrointestinal simulator (SHIME). For comparison, parallel fermentations with a grape seed extract were carried out. Extracts were used directly without previous intestinal digestion. Among the 60 phenolic compounds targeted, our results confirmed the formation of phenylacetic, phenylpropionic and benzoic acids as well as phenols such as catechol and its derivatives from the action of colonic microbiota on cranberry polyphenols. Benzoic acid (38.4μg/ml), 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid (26.2μg/ml) and phenylacetic acid (19.5μg/ml) reached the highest concentrations. Under the same conditions, microbial degradation of grape seed polyphenols took place to a lesser extent compared to cranberry polyphenols, which was consistent with the more pronounced antimicrobial effect observed for the grape seed polyphenols, particularly against Bacteroides, Prevotella and Blautia coccoides-Eubacterium rectale., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Meniere's disease (MD) is a chronic disorder of the inner ear defined by sensorineural hearing loss, tinnitus and episodic vertigo, and familial MD is observed in 5-15% of sporadic cases. Although its pathophysiology is largely unknown, studies in human temporal bones have found an accumulation of endolymph in the scala media of the cochlea. By whole-exome sequencing, we have identified two novel heterozygous single-nucleotide variants in FAM136A and DTNA genes, both in a Spanish family with three affected cases in consecutive generations, highly suggestive of autosomal-dominant inheritance. The nonsense mutation in the FAM136A gene leads to a stop codon that disrupts the FAM136A protein product. Sequencing revealed two mRNA transcripts of FAM136A in lymphoblasts from patients, which were confirmed by immunoblotting. Carriers of the FAM136A mutation showed a significant decrease in the expression level of both transcripts in lymphoblastoid cell lines. The missense mutation in the DTNA gene produces a novel splice site which skips exon 21 and leads to a shorter alternative transcript. We also demonstrated that FAM136A and DTNA proteins are expressed in the neurosensorial epithelium of the crista ampullaris of the rat by immunohistochemistry. While FAM136A encodes a mitochondrial protein with unknown function, DTNA encodes a cytoskeleton-interacting membrane protein involved in the formation and stability of synapses with a crucial role in the permeability of the blood-brain barrier. Neither of these genes has been described in patients with hearing loss, FAM136A and DTNA being candidate gene for familiar MD., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Diet is one of the main factors that could affect quantitatively and qualitatively the stability of the gut microbiota. Polyphenols are abundantly present in the human diet and have an antimicrobial effect inducing selective changes in the microbiota composition, with potential beneficial effects for the human health. Our aim was to determine the human gut microbiota susceptibility toward wine polyphenols. Susceptibility to two commercial wine phenolic extracts (Vitaflavan(®) and Provinols™) was determined in isolates from fecal samples from 36 gastrointestinal healthy volunteers. To select the polyphenol-resistant isolates, feces were seeded in plates containing 1 mg/ml of phenolic extract. The minimal inhibitory concentration to polyphenols in the collected isolates was assessed by the agar dilution method. Overall results showed that Gram-negative isolates are most tolerant to the presence of both grape seed and red wine extracts. Furthermore, we purified to homogeneity the phenolic fractions by high-performance liquid chromatography (HPLC) to determine their antimicrobial effect and their influence on bacterial growth in four selected ATCC strains using the BioScreen apparatus. Results showed that the antimicrobial activity of the wine polyphenols is the result of the interaction of both the flavan-3-ol type and the bacteria. Bacterial Intraspecies differences in the phenolic susceptibility suggest the existence of polyphenol-resistant mechanisms that are uncharacterized as yet.

Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

Probiotic properties are highly strain-dependent but rarely studied in enological lactic acid bacteria (LAB). In this study, the probiotic features of 11 strains of Lactobacillus spp., Pediococcus spp., and Oenococcus oeni, including saliva and acid resistance, bile tolerance and exopolysaccharides' production, were investigated. The assays included two probiotic reference strains (Lactobacillus plantarum CLC 17 and Lactobacillus fermentum CECT5716). The Lactobacillus and Pediococcus strains showed high resistance to lysozyme (>80% resistance to 100 mg/L of lysozyme under conditions simulating the in vivo dilution by saliva) and were capable of surviving at low pH values (pH 1.8) and bile salts, suggesting good adaptation of the wine strains to gastrointestinal conditions. The ability of the strains to adhere to the intestinal mucosa and the inhibition of the adhesion of Escherichia coli to human intestinal cells were also evaluated. Adhesion levels of enological LAB to Caco-2 cells varied from 0.37% to 12.2%, depending on the strain. In particular, Pediococcus pentosaceus CIAL-86 showed a high percentage of adhesion to intestinal cells (>12%), even higher than that shown by the probiotic reference strains, and a high anti-adhesion activity against E. coli CIAL-153 (>30%), all of which support this wine LAB strain as a potential probiotic., (Published by Elsevier Ltd.)

Meniere's disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10(-8)), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.

Purpose of Review: Recent advances in next generation sequencing techniques (NGS) are increasing the number of novel genes associated with cerebellar and vestibular disorders. We have summarized clinical and molecular genetics findings in neuro-otolology during the last 2 years., Recent Findings: Whole-exome and targeted sequencing have defined the genetic basis of dizziness including new genes causing ataxia: GBA2, TGM6, ANO10 and SYT14. Novel mutations in KCNA1 and CACNA1A genes are associated with episodic ataxia type 1 and type 2, respectively. Moreover, new variants in genes such as COCH, MYO7A and POU4F3 are associated with nonsyndromic deafness and vestibular dysfunction. Several susceptibility loci have been linked to familial vestibular migraine, suggesting genetic heterogeneity, but no specific gene has been identified. Finally, loci for complex and heterogeneous diseases such as bilateral vestibular hypofunction or familial Ménière disease have not been identified yet, despite their strong familial aggregation., Summary: Cerebellar and vestibular disorders leading to dizziness or episodic vertigo may show overlapping clinical features. A deep phenotyping including a complete familial history is a key step in performing a reliable molecular genetic diagnosis using NGS. Personalized molecular medicine will be essential to understand disease mechanisms as well as to improve their diagnosis and treatment.

Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160-170 and 210-230 μg mL(-1), resp.) and, also, in reconstituted skim milk (75-95 and 210-244 μg mL(-1), resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90-105 μg mL(-1)) and CLNA (290-320 μg mL(-1)) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk.

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This work aimed to unravel the role of Lactobacillus plantarum IFPL935 strain in the colonic metabolism of a polyphenolic red wine extract, when added to a complex human colonic microbiota from the dynamic simulator of the human intestinal microbial ecosystem (SHIME). The concentration of microbial-derived phenolic metabolites and microbial community changes along with fermentative and proteolytic activities were monitored. The results showed that L. plantarum IFPL935 significantly increased the concentration of the initial microbial ring-fission catabolite of catechins and procyanidins, diphenylpropanol, and, similarly, 4-hydroxy-5-(3'-hydroxyphenyl)valeric acid production. Overall, the addition of L. plantarum IFPL935 did not have an impact on the total concentration of phenolic metabolites, except for batches inoculated with colonic microbiota from the effluent compartment (EC), where the figures were significantly higher when L. plantarum IFPL935 was added (24 h). In summary, the data highlighted that L. plantarum IFPL935 may have an impact on the bioavailability of these dietary polyphenols. Some of the microbial-derived metabolites may play a key role in the protective effects that have been linked to a polyphenol-rich diet.

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the α-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953ΔpanE was an efficient strain producing volatile compounds related to cheese aroma.

For economical reasons and to accommodate current market trends, cheese manufacturers and product developers are increasingly interested in controlling cheese flavor formation and developing new flavors. Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to their typical flavors. Thus, the control of VSCs formation offers considerable potential for industrial applications. This paper gives an overview of the main VSCs found in cheese, along with the major pathways and key enzymes leading to the formation of methanethiol from methionine, which is subsequently converted into other sulfur-bearing compounds. As these compounds arise primarily from methionine, the metabolism of this amino acid and its regulation is presented. Attention is focused in the enzymatic potential of lactic acid bacteria (LAB) that are widely used as starter and adjunct cultures in cheese-making. In view of industrial applications, different strategies such as the enhancement of the abilities of LAB to produce high amounts and diversity of VSCs are highlighted as the principal future research trend.

Formation of cheese aroma compounds by Lactococcus lactis from amino acid catabolism depends on a complex network of reactions, which involve enzymes such as aminotransferases, dehydrogenases, lyases, and decarboxylases, among others. Based on the ability of some L. lactis strains to grow with low requirements of amino acids, we have studied in L. lactis IFPL730 the effect of the branched chain amino acid (BCAA) content on the expression of functional genes related to amino acid catabolism and aroma compound formation (araT, bcaT, kivD, ytjE and panE). L. lactis IFPL730 growth rate decreased under leucine, valine or isoleucine starvation but the strain reached similar viable counts at the stationary phase in all culture conditions studied. The level of expression of some genes encoding enzymes involved in amino acid catabolism changed significantly (P<0.05) when those conditions were compared. Specially, α-ketoisovalerate decarboxylase (kivD), BCAA-specific aminotransferase (bcaT) and C-S lyase (yjtE) gene expressions increased markedly by both isoleucine and valine starvation. In addition to gene expression, formation of volatile compounds was determined in all growth conditions. The results showed that BCAA starvation conditions caused a significant increase (P<0.05) in the formation of metabolic end products related to cheese aroma, such as 3-methylbutanal and 3-methylbutanol., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.

The effect of dietary flavan-3-ols on the adhesion of potential probiotic lactobacilli strains to intestinal cells was unraveled. The inhibitory activity of these compounds on intestinal cells was highlighted. The cytotoxic effect was shown to depend on both the compound's chemical structure (galloylation and polymerization) and degree of differentiation of intestinal cells. The effect of flavan-3-ols on bacteria adhesion differed greatly between compounds, strains, and intestinal cells. All flavan-3-ols inhibited significantly Lactobacillus acidophilus LA-5 and Lactobacillus plantarum IFPL379 adhesion except epigallocatechin gallate, which enhanced L. acidophilus LA-5 adhesion to Caco-2. Procyanidins B1 and B2 increased remarkably the adhesion of Lactobacillus casei LC115 to HT-29 cells, whereas epigallocatechin increased L. casei LC115 adhesion to Caco-2. These data showed the potential of flavan-3-ols to alter gut microecology by modifying adhesion of lactobacilli strains to intestinal cells.

Lactobacillus plantarum IFPL935 was incubated with individual monomeric flavan-3-ols and dimeric A- and B-type procyanidins to identify new metabolites and to determine the effect of compound structural features on bacterial growth and catabolism. Complex extracts rich in A-type proanthocyanidins and phenolic acids from cranberry were also tested. The results showed that L. plantarum IFPL935 exhibited higher resistance to nongalloylated monomeric flavan-3-ols, A-type dimeric procyanidins, and cranberry extract than to (-)-epicatechin-3-O-gallate and B-type dimeric procyanidins. Despite these findings, the strain was capable of rapidly degrading (-)-epicatechin-3-O-gallate, but not A- or B-type dimeric procyanidins. However, it was able to produce large changes in the phenolic profile of the cranberry extract mainly due to the catabolism of hydroxycinnamic and hydroxybenzoic acids. Of most relevance was the fact that L. plantarum IFPL935 cleaved the heterocyclic ring of monomeric flavan-3-ols, giving rise to 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)propan-2-ol, activity exhibited by only a few human intestinal bacteria.

Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.

In this paper, β-lactoglobulin (β-Lg) and sodium caseinate (SC) have been glycated via Maillard reaction with galactose and lactose and, subsequently, the effect of glycoconjugates hydrolyzed under simulated gastrointestinal digestion on the growth of pure culture of Lactobacillus, Streptococcus and Bifidobacterium has been investigated. Glycopeptides were added to the growth media as the sole carbon source. None of the bacterial strains was able to grow in hydrolysates of native and control heated β-Lg and SC. However, glycopeptides were fermented, in different degree, by Lactobacillus and Bifidobacterium and hardly any effect was detected on the growth of Streptococcus. Digested β-Lg glycoconjugates showed a strain-dependent effect whereas growth profiles of bacteria when hydrolysates of SC glycoconjugates were used as substrates were very similar, regardless of the strain. A general preference towards peptides from β-Lg/SC glycated with galactose, particularly at the state of the reaction in which the highest content in the Amadori compound tagatosyl-lysine is present, was observed. SC glycoconjugates were quickly fermented by some strains, promoting their growth in a greater extent than β-Lg complexes or even glucose. Therefore, from the results obtained in this work it can be concluded that conjugation of both milk proteins with galactose and lactose via the Maillard reaction could be an efficient method to obtain novel food ingredients with a potential prebiotic character., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Food polyphenols are able to selectively modify the growth of susceptible micro-organisms. This study describes the effect of a flavan-3-ol enriched grape seed extract (GSE) on the growth of several lactic acid bacteria (LAB) and bifidobacteria and the ability of the resistant strains to metabolize these compounds. Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus vaginalis strains showed a remarkable sensitivity to the phenolic extracts assayed, including a GSE fraction consisting mainly in (+)-catechin and (-)-epicatechin (GSE-M). On the other hand, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus strains reached maximal growth with the GSE fractions, including a rich-oligomeric (GSE-O) fraction. Within bifidobacteria, Bifidobacterium lactis BB12 showed the highest sensitivity to the phenolic extracts assayed, whereas Bifidobacterium breve 26M2 and Bifidobacterium bifidum HDD541 reached maximum growth in presence of GSE-O and GSE-M fractions. Metabolism of flavan-3-ols by LAB and bifidobacteria resistant strains was investigated in vitro. The results revealed that only L. plantarum IFPL935 was able to metabolize the polyphenols studied by means of galloyl-esterase, decarboxylase and benzyl alcohol dehydrogenase activities that led to the formation of gallic acid, pyrogallol and catechol, respectively. An unknown metabolite that does not exhibit a phenolic-acid-type structure was also detected, which suggests a new enzyme activity in L. plantarum IFPL935 able to degrade flavan-3-ol monomers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of structure on the fermentative properties of potential prebiotic trisaccharides derived from lactulose like 6'-galactosyl-lactulose (β-d-galactopyranosyl-(1→6)-β-d-galactopyranosyl-(1→4)-β-d-fructopyranose), 4'-galactosyl-lactulose (β-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→4)-β-d-fructopyranose), and 1-galactosyl-lactulose (β-d-galactopyranosyl-(1→4)-β-d-fructopyranosyl-(1→1)-β-d-galactopyranose); and from lactose like 4'-galactosyl-lactose (β-d-galactopyranosyl-(1→4)-β-d-galactopyranosyl-(1→4)-β-d-glucopyranose) and 6'-galactosyl-lactose (β-d-galactopyranosyl-(1→6)-β-d-galactopyranosyl-(1→4)-β-d-glucopyranose), has been assessed in vitro. Fermentations with twelve pure strains of Lactobacillus, Streptococcus and Bifidobacterium were carried out using the purified trisaccharides as the sole carbon source, and bacteria growth was evaluated at 600nm by means of a microplate reader during 48h. Maximum growth rates (μ(max)) and lag phase were calculated. In general, all the strains tested were able to utilize lactulose and pure trisaccharides derived from lactulose and lactose when they were used as sole carbon source. Nonetheless, glycosidic linkage and/or the monosaccharide composition of the trisaccharides affected the individual strains lag phase, cell densities and growth rates. A general preference towards β-galactosyl residues β(1-6) and β(1-1) linked over those β(1-4) linked was observed, and some strains showed higher cell densities and speed of growth on 6'-galactosyl-lactulose than on 6'-galactosyl-lactose. This is the first study of the effect of lactulose-derived oligosaccharides on pure culture growth which shows that transglycosylation of lactulose allows for obtaining galactooligosaccharides with new glycosidic structures and would open new routes to the synthesis of compounds with potential prebiotic effects., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Volatile sulphur compounds (VSCs) are of prime importance in the overall aroma of cheese and make a significant contribution to their typical flavours. Thus, the control of VSCs formation offers considerable potential for industrial applications. Here, lactic acid bacteria (LAB) from different ecological origins were screened for their abilities to produce VSCs from L-methionine. From the data presented, VSC-forming abilities were shown to be strain-specific and were correlated with the C-S lyase enzymatic activities determined using different approaches. High VSCs formation were detected for those strains that were also shown to possess high thiol-producing abilities (determined either by agar plate or spectrophotometry assays). Moreover, differences in C-S lyase activities were shown to correspond with the enzymatic potential of the strains as determined by in situ gel visualization. Therefore, the assessment of the C-S lyase enzymatic potential, by means of either of these techniques, could be used as a valuable approach for the selection of LAB strains with high VSC-producing abilities thus, representing an effective way to enhance cheese sulphur aroma compounds synthesis. In this regard, this study highlights the flavour forming potential of the Streptococcus thermophilus STY-31, that therefore could be used as a starter culture in cheese manufacture. Furthermore, although C-S lyases are involved in both biosynthetic and catabolic pathways, an association between methionine and cysteine auxotrophy of the selected strains and their VSCs-producing abilities could not be found., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This report shows the level of scientific consensus on definition, characteristics and health benefits of probiotics. The content of the report has derived from the scientific meeting: Workshop on Probiotics and Health. Scientific evidence, that congregated several Spanish experts, including gastroenterologists, microbiologists, nutritionists, immunologists and food technologists, among others, who have agreed with the statements shown in this document. Each statement has been sustained with the most relevant scientific aspects that were discussed during the Workshop and the following evaluation of the report by all experts who approved and signed it.

Phenolic acids (benzoic, phenylacetic and phenylpropionic acids) are the most abundant phenolic structures found in fecal water. As an approach towards the exploration of their action in the gut, this paper reports the antimicrobial activity of thirteen phenolic acids towards Escherichia coli, Lactobacillus spp., Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The growth of E. coli ATCC 25922 was inhibited by only four of the phenolic acids tested at a concentration of 1000 microg/mL, whereas pathogenic E. coli O157:H7 (CECT 5947) was susceptible to ten of them. The genetically manipulated E. coli lpxC/tolC strain was highly susceptible to phenolic acids. The growth of lactobacilli (Lactobacillus paraplantarum LCH7, Lactobacillus plantarum LCH17, Lactobacillus fermentum LPH1, L. fermentum CECT 5716, Lactobacillus brevis LCH23, and Lactobacillus coryniformis CECT 5711) and pathogens (S. aureus EP167 and C. albicans MY1055) was also inhibited by phenolic acids, but to varying extents. Only P. aeruginosa PAO1 was not susceptible to any of the phenolic compounds tested. Structure-activity relationships of phenolic acids and some of their diet precursors [(+)-catechin and (-)-epicatechin] were established, based on multivariate analysis of microbial activities. The antimicrobial properties of phenolic acids reported in this paper might be relevant in vivo., (2010 Elsevier Masson SAS. All rights reserved.)

A total of 96 lactic acid bacteria (LAB) were isolated from African indigenous fermented products and cow's intestines to study their inhibitory capability against multi-drug-resistant uropathogens. Escherichia coli accounted for approximately 45% of isolated uropathogens, followed by Staphylococcus spp. (20%). The Gram negative uropathogens were highly resistant to quinolones, co-trimoxazole, teicoplanin and some beta-lactams, while the Staphylococcus spp. showed high resistance to aminoglycosides, beta-lactams and macrolides. Twenty-four LAB isolates were selected based on their antimicrobial activity against two uropathogenic Staphylococcus aureus strains and bacteriocin production. LAB strains showing antimicrobial activity were grouped into smaller groups through amplified ribosomal DNA restriction analysis (ARDRA). Representative strains were identified as Weissella spp., Enterococcus faecium, Lactococcus lactis and Lactobacillus brevis through sequencing of 16S rDNA. The Weissella spp. and L. brevis strains demonstrated remarkable inhibitory activity against seven strains of Gram negative uropathogens. Two strains of L. lactis produced a bacteriocin-like inhibitory substance active against Lactobacillus sakei. In this study, an unusual high rate of co-trimoxazole, quinolones and macrolides resistance among uropathogens from south west Nigeria was discovered. Based on their sensitivity to Weissella spp., there is a potential for using these LAB as a natural approach for the protection against the uropathogens assayed.

This work is believed to be the first report on the physiological and biochemical characterization of alpha-l-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for alpha-l-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of alpha-l-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by l-rhamnose. The expression of the two alpha-l-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2P1B1, was repressed by glucose and induced by l-rhamnose, showing regulation at the transcriptional level. The two alpha-l-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 degrees C and at neutral pH and RhaB2 maximal activity was detected at 60 degrees C and at pH 5, with high residual activity at 70 degrees C. Both enzymes showed a preference for the alpha-1,6 linkage of l-rhamnose to beta-d-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the alpha-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two alpha-l-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.

Lactobacillus acidophilus La-5 is a probiotic strain used in dairy products. Production of bacteriocin by L. acidophilus La-5 was achieved when it was grown in co-cultures with the yogurt starter species Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. However, bacteriocin induction was not observed when heat-killed cells were used as inducers. This study demonstrates that L. acidophilus La-5 produces lactacin B and that the bacteriocin expression is controlled by an auto-induction mechanism involving the secreted peptide IP&#95;1800. The transcript level of the lactacin B gene cluster expression was investigated in co-cultures between L. acidophilus La-5 and S. thermophilus STY-31 and a remarkable increase of the bacteriocin structural gene (lbaB) transcription was observed. However, lbaB was transcribed constitutively in uninduced L. acidophilus La-5 cells, but the levels of the secreted bacteriocin were not enough to be detected by the agar diffusion assay. A new method for bacteriocin detection was formulated based on the monitoring on real time of Lactobacillus sakei subsp. sakei growth in presence of the supernatant and the cell wall extracts of pure and induced L. acidophilus La-5. These results showed that part of lactacin B secreted remains adhered to cell envelope.

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-gamma-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

The alpha-ketoisovalerate decarboxylase (Kivd) is a unique lactococcal key enzyme in the decarboxylation of branched-chain alpha-keto acids derived from branched-chain amino acids transamination into aldehydes. These products are important aroma compounds produced during cheese ripening. In this study, the Kivd expression of Lactococcus lactis IFPL730 growing either in nitrogen-rich media or in chemically defined media supplemented with different concentrations of branched-chain amino acids and casitone was analyzed. Isoleucine starvation increased 4-fold the Kivd activity of L. lactis IFPL730 compared to cells grown in chemically defined medium with casitone as nitrogen source. Regulation of expression was at the transcription level, probably mediated by CodY, for which a consensus CodY-box sequence was identified in the kivd promoter region.

Eight dairies, located in two distant geographic regions of Portugal, were screened along the production cycle in order to evaluate the presence and distribution of Listeria spp. in their environment. Three dairies in each region were positive for the presence of listeriae and 213 isolates were obtained. Based on an integrated analysis of RAPD fingerprints with three primers, molecular identification and genomic typing of isolates was performed followed by spatial and temporal mapping on dairy plants. The occurrence of Listeria species by region was noticeable different. Listeria monocytogenes prevailed in South Portugal dairies and L. innocua presented the highest occurrence in Azores, whereas L. seeligeri and L. ivanovii were detected in distinct regions. Dairies were at risk of contamination, from more than one source, whatever the stage in the production cycle and the surface materials used. For the three prevalent species, most of the genomic types were dairy and sampling time specific. Nonetheless, more than one type could be found in each dairy at a particular site and, in a few cases, even for different species. Some dairies also shared types, mainly for L. innocua and usually at the same stage of the production cycle. For L. monocytogenes, PCR serotyping was applied and 52% of genomic types were serotype 4b. An equal frequency of genomic types (24%) was found for serotypes 1/2b or 3b and 1/2a or 3a. The global pattern of types within a dairy is not constant, suggesting cycles of elimination and recontamination along the production cycle.

Aims: The use of randomly amplified polymorphic DNA (RAPD)-PCR fingerprinting and plasmid profiles to determine at the strain level, the similarity of Lactococcus lactis isolates obtained during sampling of traditional cheeses and to verify its correspondence to the selected phenotypic characteristics., Methods and Results: A total of 45 L. lactis isolates were genotypically analysed by RAPD-PCR fingerprinting and plasmid patterns. Phenotypic traits used to compare strains were proteolytic, acidifying, aminotransferase (aromatic and branched chain aminotransferase) and alpha-ketoisovalerate decarboxylase (Kivd) activities. The results show that 23 isolates could be grouped in clusters that exhibited 100% identity in both their RAPD and plasmid patterns, indicating the probable isolation of dominant strains during the cheese sampling process. However, there were phenotypic differences between isolates within the same cluster that included the loss of relevant technological properties such as proteinase activity and acidifying capacity or high variation in their amino acid converting enzyme activities. Likewise, the analysis of a specific attribute, Kivd activity, indicated that 7 of 15 isolates showed no detectable activity despite the presence of the encoding (kivd) gene., Conclusion: Phenotypic differences found between genotypically similar strains of L. lactis strains could be linked to differences in enzymatic expression., Significance and Impact of the Study: Phenotypic analysis of L. lactis isolates should be considered when selecting strains with new cheese flavour forming capabilities.

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of alpha-keto acids. In addition, a novel alpha-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of alpha-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and alpha-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of alpha-keto acids. However, alpha-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of alpha-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in alpha-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide alpha s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.