The genomic basis of Lactobacilli as health-promoting organisms

Lactobacilli occupy a unique position in human culture and scientific history. Like brewer's and baker's yeast, lactobacilli have been associated with food production and preservation for thousands of years. Lactobacillus species are used in mixed microbial cultures, such as the classical Lactobacillus bulgaricus/Streptococcus thermophilus inoculum for yogurt fermentation, or combinations of diverse lactobacilli/yeasts in kefir grains. The association of lactobacilli consumption with greater longevity and improved health formed the basis for developing lactobacilli as probiotics, whose market has exploded worldwide in the past 10 years. The decade that followed the determination of the first genome sequence of a food-associated species, Lactobacillus plantarum, saw the application to lactobacilli of a full range of functional genomics methods to identify the genes and gene products that govern their distinctive phenotypes and health associations. In this review, we will briefly remind the reader of the range of beneficial effects attributed to lactobacilli, and then explain the phylogenomic basis for the distribution of these traits across the genus. Recognizing the strain specificity of probiotic effects, we review studies of intraspecific genomic variation and their contributions to identifying probiotic traits. Finally we offer a perspective on classification of lactobacilli into new genera in a scheme that will make attributing probiotic properties to clades, taxa, and species more logical and more robust

When regulation challenges innovation: the case of the genus Lactobacillus

The majority of probiotic bacteria belong to the genus Lactobacillus which includes a large number of safe species integral to fermented food production. In the European Union the conversion of ensuing data into successful claims that are compliant with regulatory requirements has proved difficult. Furthermore, the study of lactobacilli has been challenging because of their phenotypic and genomic diversity. Here issues pertaining to the marketing authorization of novel foods and probiotics are outlined, taking Lactobacillus genus as reference. We highlight the drawbacks regarding the taxonomic characterization and the safety assessment of these bacteria and the validation of their beneficial mechanisms

Three-dimensional ISAR imaging: a review

Three-dimensional (3D) inverse synthetic aperture radar (ISAR) imaging has been proven feasible by combining traditional ISAR imaging and interferometry. Such technique, namely inteferometric ISAR (In-ISAR), allows for the main target scattering centres to be mapped into a 3D spatial domain as point clouds. Specifically, the use of an In-ISAR system can overcome the main geometrical interpretation issues imposed by the monostatic acquisition geometry as the problem of cross-range scaling and unknown image projection plane (IPP). However, some issues remain such as scatterer scintillation, shadowing effects, poor SNR etc., which limit the effectiveness of 3D imaging. A solution to such unsolved issues can be found in the use of multiple 3D views, which can be obtained exploiting either multi-temporal or multi-perspective configurations or a combination of both. This study aims to review the main concepts to produce multi-view 3D ISAR images by using In-ISAR systems also presenting real data collected with a multi-static In-ISAR system

Exploring Antibiotic Resistance Diversity in Leuconostoc spp. by a Genome-Based Approach: Focus on the {lsaA} Gene

Leuconostoc spp. are environmental microorganisms commonly associated with fermented foods. Absence of antibiotic resistance (AR) in bacteria is a critical issue for global food safety. Herein, we updated the occurrence of AR genes in the Leuconostoc genus through in silico analyses of the genomes of 17 type strains. A total of 131 putative AR traits associated with the main clinically relevant antibiotics were detected. We found, for the first time, the lsaA gene in L. fallax ATCC 700006T and L. pseudomesenteroides NCDO 768T. Their amino acid sequences displayed high similarities (59.07% and 52.21%) with LsaA of Enterococcusfaecalis V583, involved in clindamycin (CLI) and quinupristin-dalfopristin (QUD) resistance. This trait has different distribution patterns in Leuconostoc nontype strains-i.e., L. pseudomesenteroides, L. lactis and L. falkenbergense isolates from fermented vegetables, cheeses, and starters. To better explore the role of lsaA, MIC for CLI and QUD were assessed in ATCC 700006T and NCDO 768T; both strains were resistant towards CLI, potentially linking lsaA to their resistant phenotype. Contrarily, NCDO 768T was sensitive towards QUD; however, expression of lsaA increased in presence of this antibiotic, indicating an active involvement of this trait and thus suggesting a revision of the QUD thresholds for this species

Suitability of the Nisin Z-producer Lactococcus lactis subsp. lactis CBM 21 to be Used as an Adjunct Culture for Squacquerone Cheese Production

This research investigated the technological and safety effects of the nisin Z producer Lactococcus lactis subsp. lactis CBM 21, tested as an adjunct culture for the making of Squacquerone cheese in a pilot-scale plant. The biocontrol agent remained at a high level throughout the cheese refrigerated storage, without having a negative influence on the viability of the conventional Streptococcus thermophilus starter. The inclusion of CBM 21 in Squacquerone cheesemaking proved to be more effective compared to the traditional one, to reduce total coliforms and Pseudomonas spp. Moreover, the novel/innovative adjunct culture tested did not negatively modify the proteolytic patterns of Squacquerone cheese, but it gave rise to products with specific volatile and texture profiles. The cheese produced with CBM 21 was more appreciated by the panelists with respect to the traditional one

Transcriptional and Metabolic Response of Wine-Related Lactiplantibacillus plantarum to Different Conditions of Aeration and Nitrogen Availability

Lactic acid bacteria (LAB) perform the process of malolactic fermentation (MLF) in wine. Availability of oxygen and nitrogen nutrients could influence LAB growth, malolactic activity, and other metabolic pathways, impacting the subsequent wine quality. The impact of these two factors has received limited investigation within LAB, especially on a transcriptome level. The aim of this study was to evaluate metabolic changes in the strain Lactiplantibacillus plantarum IWBT B063, growing in synthetic grape juice medium (GJM) under different oxygen exposure conditions, and with low availability of nitrogen-based nutrients. Next-generation sequencing was used to analyze expression across the transcriptome (RNA-seq), in combination with conventional microbiological and chemical analysis. L. plantarum consumed the malic acid present in all the conditions evaluated, with a slight delay and impaired growth for nitrogen limitation and for anaerobiosis. Comparison of L. plantarum transcriptome during growth in GJM with and without O-2 revealed differential expression of 148 functionally annotated genes, which were mostly involved in carbohydrate metabolism, genetic information processing, and signaling and cellular processes. In particular, genes with a protective role against oxidative stress and genes related to amino acid metabolism were differentially expressed. This study confirms the suitability of L. plantarum IWBT B063 to carry out MLF in different environmental conditions due to its potential adaption to the stress conditions tested and provides a better understanding of the genetic background of an industrially relevant strain

Treatment of food processing wastes for the production of medium chain fatty acids via chain elongation

The production of medium chain fatty acids (MCFAs) through reverse β-oxidation was investigated both on synthetic and real substrates. From preliminary batch tests emerged that caproic acid was maximized under an acetate/ethanol molar ratio of 5:1 at neutral pH. This ratio was then adopted in different semi-continuous tests operating with different amounts of the two reactants. It emerged that the MCFAs yield reached the maximum level of 6.7% when the total molar substrate amount was around 40–45 mmol/d, while the process was inhibited for values higher than 400 mmol/d. Semi-continuous tests using real waste as substrates, namely food waste condensate, cheese whey, and winery wastewater, confirmed the results obtained with the synthetic substrates. Better performances were obtained when an adequate molar ratio of the acetate and the electron-donor compound was naturally present. Therefore, a MCFAs yield of 25% and 10.5% was obtained for condensate of food waste and acidic cheese whey, respectively. Regarding MCFAs composition, caproic acid was the dominant form but small concentrations of octanoic acid were also found in the tests where ethanol was the electron donor (synthetic substrates and food waste condensate). Octanoic acid was not produced in test where lactic acid represented the electron donor molecules (cheese whey). Condensate and synthetic samples were dominated by Pseudoclavibacter caeni with an abundance of 38.19% and 33.38% respectively, while Thomasclavelia (24.13%) and Caproiciproducens (11.68%) was the most representative genus in acidic cheese whey sample

Whole genome analysis as a tool for the safety assessment of antibiotic resistance in food-processing bacteria

Trabajo presentado en la 2nd EFSA Scientific Conference, celebrado en Milán, Italia, del 14 al 16 de octubre de 2015Acquisition of antibiotic resistances (AR) by pathogens leads ultimately to a failure of antibiotic therapy. The food chain is considered a key player in the transmission of AR determinants to pathogens from reservoirs in commensal and beneficial bacteria. Therefore, the absence of transmissible AR genes in bacteria used as starter and adjunct cultures for food and feed processing is considered to be critical (EFSA, 2012; EFSA Journal, 10:2740). Genome sequencing allows the inspection of the whole genetic makeup of bacteria in the search for the basis of desirable and undesirable traits, including that of AR. Thus, in silico sequence analysis and comparison against databases can be used as a tool for the safety assessment of microorganisms intended to be used in food systems. This communication reports on the genome analysis of three Leuconostoc mesenteroides strains of dairy origin showing atypical resistances to tetracycline (LbT16), erythromycin and clindamycin (LbE15), and kanamycin, streptomycin, tetracycline and virginiamycin (LbE16). Genes encoding for erythromycin [erm(B)] and tetracycline [tet(S)] resistance had already been detected by PCR. Genome analysis confirmed the presence of these genes and identified others which encode uncommon AR in lactic acid bacteria. Analysis of the genes and their flanking regions revealed a potential of some to be horizontally transferred to other bacteria. This study demonstrates the effectiveness of combining genome sequencing and bioinformatics analysis as an affordable tool for the safety assessment of food bacteria. This innovative approach could become a novel paradigm in the selection programs of starters for the food industry.Peer Reviewe

Comparative genomics of the genus Lactobacillus reveals robust phylogroups that provide the basis for reclassification

The genus Lactobacillus includes over 200 species that are widely used in fermented food preservation and biotechnology or that are explored for beneficial effects on health. Naming, classifying, and comparing lactobacilli have been challenging due to the high level of phenotypic and genotypic diversity that they display and because of the uncertain degree of relatedness between them and associated genera. The aim of this study was to investigate the feasibility of dividing the genus Lactobacillus into more homogeneous genera/clusters, exploiting genome-based data. The relatedness of 269 species belonging primarily to the families Lactobacillaceae and Leuconostocaceae was investigated through phylogenetic analysis (by the use of ribosomal proteins and housekeeping genes) and the assessment of the average amino acid identity (AAI) and the percentage of conserved proteins (POCP). For each subgeneric group that emerged, conserved signature genes were identified. Both distance-based and sequence-based metrics showed that the Lactobacillus genus was paraphyletic and revealed the presence of 10 methodologically consistent subclades, which were also characterized by a distinct distribution of conserved signature orthologues. We present two ways to reclassify lactobacilli: a conservative division into two subgeneric groups based on the presence/absence of a key carbohydrate utilization gene or a more radical subdivision into 10 groups that satisfy more stringent criteria for genomic relatedness. Importance: Lactobacilli have significant scientific and economic value, but their extraordinary diversity means that they are not robustly classified. The 10 homogeneous genera/subgeneric entities that we identify here are characterized by uniform patterns of the presence/absence of specific sets of genes which offer potential as discovery tools for understanding differential biological features. Reclassification/subdivision of the genus Lactobacillus into more uniform taxonomic nuclei will also provide accurate molecular markers that will be enabling for regulatory approval applications. Reclassification will facilitate scientific communication related to lactobacilli and prevent misidentification issues, which are still the major cause of mislabeling of probiotic and food products reported worldwide

Reconstruction of Simplified Microbial Consortia to Modulate Sensory Quality of Kombucha Tea

Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation-acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)-is mainly found embedded in an extracellular cellulosic matrix located at the liquid-air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains' metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance