Metataxonomic insights in the distribution of Lactobacillaceae in foods and food environments.

Members of the family Lactobacillaceae, which now includes species formerly belonging to the genera Lactobacillus and Pediococcus, but also Leuconostocaceae, are of foremost importance in food fermentations and spoilage, but also as components of animal and human microbiota and as potentially pathogenic microorganisms. Knowledge of the ecological distribution of a given species and genus is important, among other things, for the inclusion in lists of microorganisms with a Qualified Presumption of Safety or with beneficial use. The objective of this work is to use the data in FoodMicrobionet database to obtain quantitative insights (in terms of both abundance and prevalence) on the distribution of these bacteria in foods and food environments. We first explored the reliability of taxonomic assignments using the SILVA v138.1 reference database with full length and partial sequences of the 16S rRNA gene for type strain sequences. Full length 16S rRNA gene sequences allow a reasonably good classification at the genus and species level in phylogenetic trees but shorter sequences (V1-V3, V3-V4, V4) perform much worse, with type strains of many species sharing identical V4 and V3-V4 sequences. Taxonomic assignment at the genus level of 16S rRNA genes sequences and the SILVA v138.1 reference database can be done for almost all genera of the family Lactobacillaceae with a high degree of confidence for full length sequences, and with a satisfactory level of accuracy for the V1-V3 regions. Results for the V3-V4 and V4 region are still acceptable but significantly worse. Taxonomic assignment at the species level for sequences for the V1-V3, V3-V4, V4 regions of the 16S rRNA gene of members of the family Lactobacillaceae is hardly possible and, even for full length sequences, and only 49.9 % of the type strain sequences can be unambiguously assigned to species. We then used the FoodMicrobionet database to evaluate the prevalence and abundance of Lactobacillaceae in food samples and in food related environments. Generalist and specialist genera were clearly evident. The ecological distribution of several genera was confirmed and insights on the distribution and potential origin of rare genera (Dellaglioa, Holzapfelia, Schleiferilactobacillus) were obtained. We also found that combining Amplicon Sequence Variants from different studies is indeed possible, but provides little additional information, even when strict criteria are used for the filtering of sequences.

Effect of oil-born yeasts on the quality of extra-virgin olive oils of Basilicata region.

The olive oil microbiota mainly consists of yeasts, which may positively or negatively affect the physicochemical and sensory features of product. In this study, 17 yeast strains belonging to Candida boidinii, Lachancea fermentati, Nakazawaea molendinolei, N. wickerhamii and Schwanniomyces polymorphus species were collected during olive oil production, identified and tested for the ability to ferment sugars, to grow at low temperatures, for the occurrence of different enzymatic activities, for the tolerance and degradation of phenolic compounds, radical scavenging activities, biofilm formation, survival to simulated gastro-intestinal (GIT) tract. Yeasts were also inoculated in extra virgin olive oils (EVOO; from Leccino and Coratina cultivar) to evaluate their survival and their effect on EVOO quality (changes in analytical indices) during 6-months of storage. Most of strains were able to grow at 15°C, while the ability to ferment different sugars was strain-specific. All strains had ß-glucosidase activity, while none exhibited lipolytic activity; peroxidase was widespread among the strains, while protease activity was strain-dependent. Esterase and the ability to hydrolyse oleuropein and form hydroxytyrosol was present only in N. wickerhamii strains. All strains were able to survive in olive mill wastewater, used as a model of phenolic compounds-rich matrix. A potential biofilm formation was observed only in N. wickerhamii, while the ability to scavenge radical and to cope with GIT-associated stresses were strain-dependent. High levels of survival were observed for almost strains (except S. polymorphus), in both Leccino and Coratina samples. Yeasts limited the acidity rise in olive oils, but overtime they contributed to increase the parameters related to oxidative phenomena (i.e. peroxides, K232, K270), resulting in a declassification of EVOOs. The total phenolic content (TPC) was correlated to the presence of yeasts and, at the end of storage period (6 months) inoculated samples had significantly lower concentrations compared to the control oils. This study confirms that yeasts are able to survive in olive oils and, therefore, the control of their occurrence during extraction process and storage conditions is needed to obtain high-quality products and to maintain the standards of EVOO classification.

The Production of γ-Aminobutyric Acid from Free and Immobilized Cells of Levilactobacillus brevis Cultivated in Anaerobic and Aerobic Conditions.

γ-aminobutyric acid (GABA) has several beneficial effects on human health. GABA may be produced via chemical synthesis or through microbial metabolism, and Levilactobacillus brevis is recognized as a GABA-producing species. In this study, 11 Lvb. brevis strains were screened for GABA production, and the best producers were selected to verify the effect of aerobic (AE) and respiratory (RS) cultivations on growth parameters, biomass, and GABA accumulation. Lvb. brevis LB12 was then used to evaluate the combined effect of the incubation atmosphere (anaerobiosis vs. aerobiosis), cell protection (free vs. immobilized cells), and cell recycling (fresh vs. starved cells) on GABA production. Glutamate (GLU) consumption and GABA accumulation were detected by Thin-layer Chromatography (TLC) and RP-HPLC analyses. The ability to produce GABA was widespread among the strains. AE and RS growth improved biomass production, but oxygen availability impaired GLU to GABA conversion, and the anaerobically growing cells had the highest GABA productivity. Immobilized strains had lower efficiency in both GLU uptake and conversion compared to free cells, probably due to the poor diffusion in alginate beads. The use of resting cells allowed further GABA production without the cultivation step, but cell activity was exhausted after three cycles of reutilization. Lvb. brevis LB12 is an excellent GABA producer, and AE cultivation can be exploited to improve the final cell density; however, the conditions for boosting GLU to GABA conversion and cell regeneration need to be further investigated.

Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains.

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods.

FoodMicrobionet v4: A large, integrated, open and transparent database for food bacterial communities.

With the availability of high-throughput sequencing techniques our knowledge of the structure and dynamics of food microbial communities has made a quantum leap. However, this knowledge is dispersed in a large number of papers and hard data are only partly available through powerful on-line databases and tools such as QIITA, MGnify and the Integrated Microbial Next Generation Sequencing platform, whose annotation is not optimized for foods. Here, we present the 4th iteration of FoodMicrobionet, a database of the composition of bacterial microbial communities of foods and food environments. With 180 studies and 10,151 samples belonging to 8 major food groups FoodMicrobionet 4.1.2 is arguably the largest and best annotated database on food bacterial communities. This version includes 1684 environmental samples and 8467 food samples, belonging to 16 L1 categories and 196 L6 categories of the EFSA FoodEx2 classification and is approximately 4 times larger than previous version (3.1, https://doi.org/10.1016/j.ijfoodmicro.2019.108249). As a representative case study among the many potential applications of FoodMicrobionet, we confirm that taxonomic assignment at the genus level can be performed confidently for the majority of amplicon sequence variants using the most commonly used 16S RNA gene target regions (V1-V3, V3-V4, V4), with best results with higher quality sequences and longer fragment lengths, but that care should be exercised in confirming the assignment at species level. Both FoodMicrobionet and related data and software conform to FAIR (findable, accessible, interoperable, reusable/reproducible) criteria for scientific data and software and are freely available on public repositories (GitHub, Mendeley data). Even if FoodMicrobionet does not have the sophistication of QIITA, IMNGS and MGnify, we feel that this iteration, due to its size and diversity, provides a valuable asset for both the scientific community and industrial and regulatory stakeholders.

A review of methods for the inference and experimental confirmation of microbial association networks in cheese.

Interactions among microorganisms deeply affect the dynamics of cheese microbial communities and, as a consequence, multiple aspects of cheese quality, from the production of metabolites affecting the taste, aroma and flavour, to body, texture and colour. Understanding and exploiting interactions among beneficial or detrimental microorganisms is therefore key to managing cheese quality. This is true for the simplest systems (fresh cheeses produced from pasteurized milk using defined starters) and the more so for complex, dynamic systems, like surface ripened cheese produced from raw milk, in which a dynamic succession of diverse microorganisms is essential for obtained the desired combination of sensory properties while guaranteeing safety. Positive (commensalism, protocooperation) and negative (competition, amensalism, predation and parasitism) interactions among members of the cheese biota have been reviewed multiple times. However, even if the complex, multidimensional datasets generated by multi-omic approaches to cheese microbiology and biochemistry are ideally suited for the representation of biotic and metabolic interactions as networks, network science concepts and approaches are rarely applied to cheese microbiology. In this review we illustrate concepts relevant to the description of microbial interactions using a network science framework. Then, we briefly review methods used for the inference and analysis of microbial association networks (MAN) and their potential use in the interpretation of the cheese interactome. Finally, since these methods can only be used for mining microbial associations, we review the experimental methods used to confirm the nature of microbial interactions among cheese microbes.

Selection of Lactiplantibacillus Strains for the Production of Fermented Table Olives.

Lactiplantibacillus strains (n. 77) were screened for technological properties (e.g., xylose fermentation, EPS production, antimicrobial activity, tolerance to NaCl and phenolic compounds, oleuropein degradation and hydroxytyrosol formation) relevant for the production of fermented table olives. Survival to olive mill wastewater (OMW) and to simulated gastro-intestinal tract (GIT), the capability to grow at different combinations of NaCl and pH values, radical scavenging activities and biofilm formation were further investigated in 15 selected strains. The screening step revealed high diversity among Lactiplantibacillus strains. Most of the strains were able to ferment xylose, while only a few strains produced EPS and had inhibitory activity against Y. lipolytica. Resistance to phenolic compounds (gallic, protocatechuic, hydroxybenzoic and syringic acids), as well as the ability to release hydroxytyrosol from oleuropein, was strain-specific. OMWs impaired the survival of selected strains, while combinations of NaCl ≤ 6% and pH ≥ 4.0 were well tolerated. DPPH and hydroxyl radical degradation were strain-dependent, while the capability to form biofilm was affected by incubation time. Strains were very tolerant to the GIT. The genome of Lpb. pentosus O17 was sequenced and analysed to verify the presence of genes involved in the degradation and metabolism of phenolic compounds. O17 lacks carboxylesterase and gallate decarboxylase (subunits B and D) sequences, and its gene profile differs from that of other publicly available Lpb. pentosus genomes.

The Effect of Respiration, pH, and Citrate Co-Metabolism on the Growth, Metabolite Production and Enzymatic Activities of Leuconostoc mesenteroides subsp. cremoris E30.

Leuconostoc mesenteroides includes strains used as starter and/or adjunct cultures for the production of several fermented foods. In this study, the effect of anaerobic and respiratory cultivations, as well as of citrate supplementation and different pH values, was evaluated on growth, biomass, metabolite, and enzymatic activities (pyruvate oxidase, POX; NADH-dependent oxidase, NOX; NADH-dependent peroxidase, NPR) of Leuconostoc mesenteroides subsp. cremoris E30. We compared the respiration-increased growth rate and biomass production of Leuc. mesenteroides E30 to anaerobic cultivation. A supplementation of citrate impaired the growth rate of the respiratory cells. As expected, anaerobic cultures did not consume oxygen, and a similar trend in oxygen uptake was observed in respiratory cultures. The aerobic incubation caused changes in the metabolic pattern, reducing the production of ethanol in favour of acetic acid. Citrate was already exhausted in the exponential phase and did not affect the yields in acetic acid and ethanol. NOX activity increased in the presence of oxygen, while catalase was also detected in the absence of hemin. The absence of H2O2 suggested its degradation by NPR and catalase. Respiratory cultivation provided benefits (increase in growth rate, biomass, and activity in antioxidant enzymes) for Leuc. mesenteroides E30. Therefore, the exploitation of respiratory phenotypes may be useful for the formulation of competitive starter or adjunct cultures.

Metataxonomic and metagenomic approaches for the study of undefined strain starters for cheese manufacture.

Undefined strain starters are used for the production of many traditional and artisanal cheeses. Composition of undefined starters depends on several factors, and the diversity in strains and species significantly affects cheese quality and features. Culture-dependent approaches have long been used for the microbial profiling and functionalities of undefined cultures but underestimate their diversity due to culturability biases. Recently, culture-independent methods, based on high-throughput sequencing (HTS), have been preferred, with a significant boost in resolution power and sensitivity level. Amplicon targeted (AT) metagenomics, based on 16S rRNA sequencing, returned a larger microbiota diversity at genus and, sometimes, at species levels for artisanal starters of several PDO cheeses, but was inappropriate for populations with high strain diversity, and other gene targets were tested in AT approaches. Shotgun metagenomics (total DNA) and metatranscriptomics (total RNA), although are more powerful in depicting diversity and functionality of undefined cultures, have been rarely applied because of some limitations (e.g., high costs and laboriousness, need for bioinformatics skills). The advantages of HTS technologies are undoubted, but some hurdles need to be still overcame (e.g., resolution power, discrepancy between active and inactive cells, robust analytic pipelines, cost and time reduction for integrated approaches) so that HTS become routinary and convenient for defining complexity, microbial interactions (including host-phage relationships) and evolution in cheeses of undefined starters.

Microbiological Stability and Overall Quality of Ready-To-Heat Meals Based on Traditional Recipes of the Basilicata Region.

The quality of ready meals is affected by several factors that may impair stability and nutritional value. In this work, we evaluated the overall quality of four traditional meals (Basilicata region) prepared according to the cook&chill method, packaged in air or modified atmosphere packaging (MAP; 70% N2 and 30% CO2), and stored at 4 °C for seven days. The shelf-life was determined by Listeria monocytogenes challenge testing and inactivation by microwave (MW) heating was assessed. The counts at the production day were excellent in three meals out of four, whereas one had high levels of spoilage and pathogens both as soon as the preparation and after seven days. MAP was partially effective only against the growth of the aerobic mesophilic species, whereas sensory analysis revealed that MAP may preserve many of sensory attributes. The average shelf-life of the meals ranged from 11 to 13 days, however, the potential shelf-life was undetectable in one out four meals, as L. monocytogenes growth was inhibited two days after the inoculum. In the inoculated meals, MW heating provided a partial inactivation (25%) of the pathogen. The overall quality of type the meals was partially satisfactory; post-cooking contaminations may affect the microbial load and reduce the palatability over the storage period and, above all, may involve biological hazards which consumers' habits may not be able to eliminate.

Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

Advancing integration of data on food microbiome studies: FoodMicrobionet 3.1, a major upgrade of the FoodMicrobionet database.

We present a new version of FoodMicrobionet, a database for the exploration of food bacterial communities. The database, available as an app built with the Shiny package of R, includes data from 44 studies and 2234 samples (food or food environment), covering dairy, meat, fruit and vegetables, cereal based and ready-to-eat foods. The interactive interface allows exploration of data, access to external resources (on line versions of the studies, sequence data on SRA, taxonomic databases), filtering samples on the basis of a number of criteria, aggregation of samples and bacterial taxa and export of data in a variety of formats. FoodMicrobionet is the largest collection of data on food bacterial communities and, due to the structure of sample metadata, largely derived from the European Food Safety Agency FoodEx2 classification, makes comparison and re-analysis of data from published and unpublished studies easy. Data exported from FoodMicrobionet can be readily used for graphical and statistical meta-analyses using open-source software (Gephi, Cytoscape, CoNet, and R packages and apps, such as phyloseq and Shiny-Phyloseq) thus providing scientists, risk assessors and industry with a wealth of information on the structure of food biomes.

Effect of Respiratory Growth on the Metabolite Production and Stress Robustness of Lactobacillus casei N87 Cultivated in Cheese Whey Permeate Medium.

Cheese whey permeate (WP) is a low-cost feedstock used for the production of biomass and metabolites from several lactic acid bacteria (LAB) strains. In this study, Lactobacillus casei N87 was cultivated in an optimized WP medium (WPM) to evaluate the effect of anaerobic and respiratory conditions on the growth performances (kinetics, biomass yield), consumption of sugars (lactose, galactose, glucose) and citrate, metabolite production [organic acids, volatile organic compounds (VOCs)] and stress survival (oxidative, heat, freezing, freeze-drying). The transcription of genes involved in the main pathways for pyruvate conversion was quantified through Real Time-PCR to elucidate the metabolic shifts due to respiratory state. Cultivation in WPM induced a diauxic growth in both anaerobic and respiratory conditions, and L. casei N87 effectively consumed the lactose and galactose present in WPM. Genomic information suggested that membrane PTS system and tagatose-6-P pathway mediated the metabolism of lactose and galactose in L. casei N87. Respiration did not affect specific growth rate and biomass production, but significantly altered the pyruvate conversion pathways, reducing lactate accumulation and promoting the formation of acetate, acetoin and diacetyl to ensure the redox balance. Ethanol was not produced under either cultivation. Pyruvate oxidase (pox), acetate kinase (ack), α-acetolactate decarboxylase (ald), acetolactate synthase (als) and oxaloacetate decarboxylase (oad) genes were up-regulated under respiration, while L-lactate dehydrogenase (ldh), pyruvate formate lyase (pfl), pyruvate carboxylase (pyc), and phosphate acetyltransferase (pta) were down regulated by oxygen. Transcription analysis was consistent with metabolite production, confirming that POX-ACK and ALS-ALD were the alternative pathways activated under aerobic cultivation. Respiratory growth affected the production of volatile compounds useful for the development of aroma profile in several fermented foods, and promoted the survival of L. casei N87 to oxidative stresses and long-term storage. This study confirmed that the respiration-based technology coupled with cultivation on low-cost medium may be effectively exploited to produce competitive and functional starter and/or adjunct cultures. Our results, additionally, provided further information on the activation and regulation of metabolic pathways in homofermentative LAB grown under respiratory promoting conditions.

Impact of aerobic and respirative life-style on Lactobacillus casei N87 proteome.

Lactic acid bacteria (LAB) are used as starter, adjunct and/or probiotic cultures in fermented foods. Several species are recognized as oxygen-tolerant anaerobes, and aerobic and respiratory cultivations may provide them with physiological and technological benefits. In this light, mechanisms involved in the adaptation to aerobic and respiratory (supplementation with heme and menaquinone) growth conditions of the O2-tolerant strain Lactobacillus casei N87 were investigated by proteomics. In fact, in this bacterial strain, respiration induced an increase in biomass yield and robustness to oxidative, long-term starvation and freeze-drying stresses, while high concentrations of dissolved O2 (dO2 60%) negatively affected its growth and cell survival. Proteomic results well paralleled with physiological and metabolic features and clearly showed that aerobic life-style led to a higher abundance of several proteins involved in carbohydrate metabolism and stress response mechanisms and, concurrently, impaired the biosynthesis of proteins involved in nucleic acid formation and translation processes, thus providing evidence at molecular level of the significant damage to L.casei N87 fitness. On the contrary, the activation of respiratory pathways due to heme and menaquinone supplementation, led to a decreased amount of chaperones and other stress related proteins. These findings confirmed that respiration reduced oxidative stress condition, allowing to positively modulate the central carbohydrate and energy metabolism and improve growth and stress tolerance features. Results of this study could be potentially functional to develop competitive adjunct and probiotic cultures effectively focused on the improvement of quality of fermented foods and the promotion of human health.

Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air.

Thawed hake (Merluccius capensis and M. paradoxus) and plaice (Pleuronectes platessa) fillets were used as a model to evaluate the effect of storage temperature (0 or 10 °C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on non-selective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 107 cfu/g within 2-3 days at 10 °C and 7-8 days at 0 °C. Plate counts on three media (Plate Count Agar +0.5% NaCl, Iron Agar Lyngby and Pseudomonas Selective medium) and 16S rRNA gene counts estimated by qPCR were highly correlated. Growth was modelled using the D-model and specific growth rate ranged between 0.97 and 1.24 d-1 and 3.54 and 5.90 d-1 at 0 and 10 °C, respectively. The initial composition of the microbiota showed lot-to-lot variation, but significant differences between the two fish species were detected. Alpha diversity significantly decreased during storage. When bacterial counts exceeded 107 cfu/g, the microbiota was dominated by members of the genera Pseudomonas, Psychrobacter, Acinetobacter, Serratia, Flavobacterium, Acinetobacter, Carnobacterium, Brochothrix and Vagococcus. However, Photobacterium and Shewanella, two genera frequently associated with fish spoilage, were either absent or minor components of the microbiota. As expected, storage temperature significantly affected the abundance of several species. The inference of microbial association networks with three different approaches (an ensemble approach using the CoNet app, Sparse Correlations for Compositional data, and SParse InversE Covariance Estimation for Ecological Association Inference) allowed the detection of both a core microbiota, which was present throughout storage, and a number of taxa, which became dominant at the end of spoilage and were characterized by a disproportionate amount of negative interactions.

Aerobic and respirative growth of heterofermentative lactic acid bacteria: A screening study.

Heterofermentative lactic acid bacteria (76 strains) belonging to Lactobacillus, Leuconostoc and Weissella species which are important in fermentation, spoilage or as probiotics were screened in a factorial experiment for their ability to grow, produce catalase and consume oxygen in aerobiosis or in anaerobiosis, with or without supplementation with hemin and/or menaquinone in a medium containing glucose as a carbohydrate source. Aerobiosis improved growth with a few exceptions. The effect of supplementation with heme and/or menaquinone was strain specific and clear evidence of heme-boosted respiration was found in some cases. Heme-catalase was produced by strains of L. brevis, W. minor and Leuc. mesenteroides; some strains of the latter species produced non-heme catalase. Shaken flasks experiments showed that aerobic growth resulted in increased maximum growth rate and in a limited increase in biomass. Heme supplementation during aerobic growth resulted in a further increase in growth rate and final biomass only for a few strains; this was often related to catalase, which was also responsible for increased tolerance of H2O2. In both experiments we found evidence of heme toxicity, especially in anaerobiosis and in absence of menaquinone. Dose response curves for aerobic growth in the presence of combinations of hemin and menaquinone were non-monotonic, with growth stimulation at low doses of heme (<2.5 mg/l) and toxicity at higher doses. Menaquinone at 0.25-8 mg/l increased growth stimulation and partially reduced toxicity.

Factors affecting gene expression and activity of heme- and manganese-dependent catalases in Lactobacillus casei strains.

Catalases reduce oxidative stress by degrading hydrogen peroxide to molecular oxygen and water. The presence of heme-dependent or manganese-dependent catalases was observed for a long time in lactic acid bacteria (LAB) but, to date, knowledge on the factors affecting gene expression and enzymatic functionality are limited to a very few strains. In this study, the effect of atmosphere of incubation (not aerated static growth vs aerated shaken growth) and supplementation with Fe2+, hemin, Mn2+ or their combinations on the catalase production of respiration-competent strain Lactobacillus casei N87 was evaluated using a 24 factorial design. Kinetics of growth, enzymatic activity, tolerance of oxidative stress and expression of heme- and Mn-catalase genes were assessed. A phylogenetic analysis of heme- and Mn-catalase sequences retrieved for all published LAB genomes was performed. The presence of cofactors, especially when combined, improved biomass production in L. casei N87 in both aerated and not aerated conditions. The genome of L. casei N87 harboured sequences for both catalases and hemin and Mn supplementation was crucial for gene expression and enzyme functionality. Iron and oxygen had an additive stimulatory effect. Tolerance of oxidative stress was higher in aerated cultures supplemented with hemin and/or Mn, because of high catalase activities. The presence of both enzymes was confirmed in other respirative strains of L. casei. Clustering of catalase sequences reflected in most of cases the phylogenetic distance between LAB genomes, but in other cases significant differences were found within the same genus, indicating a different evolutionary history. The occurrence of both genes is rare in LAB genomes. The exploitation of LAB with both heme- and Mn-catalases may ensure protection from oxidative stress in different conditions and may be relevant for several food (reduction of oxidative processes on food components) and health (prevention of human diseases) related applications.

Investigation of Factors Affecting Aerobic and Respiratory Growth in the Oxygen-Tolerant Strain Lactobacillus casei N87.

Aerobic and respiratory cultivations provide benefits for some lactic acid bacteria (LAB). Growth, metabolites, enzymatic activities (lactate dehydrogenase; pyruvate and NADH oxidases, NADH peroxidase; catalase), antioxidant capability and stress tolerance of Lactobacillus casei N87 were evaluated in anaerobic, aerobic and respiratory (aerobiosis with heme and menaquinone supplementation) batch cultivations with different dissolved oxygen (DO) concentrations. The expression of pox (pyruvate oxidase) and cydABCD operon (cytochrome bd oxidase complex) was quantified by quantitative Real Time polymerase chain reaction. Respiration increased biomass production compared to anaerobiosis and unsupplemented aerobiosis, and altered the central metabolism rerouting pyruvate away from lactate accumulation. All enzymatic activities, except lactate dehydrogenase, were higher in respiratory cultures, while unsupplemented aerobiosis with 60% of DO promoted H2O2 and free radical accumulation. Respiration improved the survival to oxidative and freeze-drying stresses, while significant numbers of dead, damaged and viable but not cultivable cells were found in unsupplemented aerobic cultures (60% DO). Analysis of gene expression suggested that the activation of aerobic and respiratory pathways occurred during the exponential growth phase, and that O2 and hemin induced, respectively, the transcription of pox and cydABCD genes. Respiratory cultivation might be a natural strategy to improve functional and technological properties of L. casei.

Polymorphism of the phosphoserine phosphatase gene in Streptococcus thermophilus and its potential use for typing and monitoring of population diversity.

The phosphoserine phosphatase gene (serB) of Streptococcus thermophilus is the most polymorphic gene among those used in Multilocus Sequence Typing schemes for this species and has been used for both genotyping of isolates and for evaluation of population diversity. However, the information on the potential of this gene as a marker for diversity in S. thermophilus species is still fragmentary. In this study, we evaluated serB nucleotide polymorphism and its potential impact on protein structure using data from traditional sequencing. In addition we evaluated the ability of serB targeted high-throughput sequencing in studying the diversity of S. thermophilus populations in cheese and starter cultures. Data based on traditional cultivation based techniques and sequencing provided evidence that the distribution of serB alleles varies significantly in some environments (commercial starter cultures, traditional starter cultures, cheese). Mutations had relatively little impact on predicted protein structure and were not found in domains that are predicted to be important for its functionality. Cultivation independent, serB targeted high-throughput sequencing provided evidence for significantly different alleles distribution in different cheese types and detected fluctuations in alleles abundance in a mixed strain starter reproduced by backslopping. Notwithstanding some shortcomings of this method that are discussed here, the cultivation independent approach appears to be more sensitive than cultivation based approaches based on isolation and traditional sequencing.

Draft Genome Sequence of the Respiration-Competent Strain Lactobacillus casei N87.

Lactobacillus casei is used as a starter, adjunct, and/or probiotic culture in the production of fermented and functional foods. Here, we report the draft genome sequence of the respiration-competent strain L. casei N87, isolated from infant feces. This genome information may be useful for the study of respiratory metabolism in lactic acid bacteria.