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Background: Fermented foods are attracting increasing interest due to their nutritional and health benefits, including a positive impact on gut microbiota exerted by their associated microbes. However, information relative to traditional fermented dairy products, along with their autochthonous microbiota, is still fragmented and poorly standardized. Therefore, our aim was to collect and aggregate data useful for obtaining a comprehensive overview translated in a classical database interface that can be easily handled by users. Methods: a preliminary inventory was built up by systematically collecting data from publicly available resources for the creation of a list of traditional dairy foods produced worldwide, including additional metadata useful for stratifying, and collapsing subgroups. Results: we developed the Fermented Dairy Food Database (FDF-DB), a feasible resource comprising 1852 traditional dairy foods (cheeses, fermented milks, and yogurt) for which microbial content and other associated metadata such as geographical indication label, country/region of origin, technological aspects were gathered. Conclusions: FDF-DB is a useful and user-friendly resource where taxonomic information and processing production details converge. This resource will be of great aid for researchers, food industries, stakeholders and any user interested in the identification of technological and microbiological features characterizing traditional fermented dairy products.

Members of the familyLactobacillaceae, which now includes species formerly belonging to the generaLactobacillusandPediococcus, but alsoLeuconostocaceae, 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 familyLactobacillaceaewith 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 familyLactobacillaceaeis 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 ofLactobacillaceaein 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.

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 1,684 environmental samples and 8,467 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).Using data in 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.

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 β-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, K

Interactions among starter and non-starter microorganisms (starter bacteria, naturally occurring or intentionally added non-starter bacteria, yeasts and filamentous fungi, spoilage and pathogenic microorganisms and, finally bacteriophages and even arthropods) deeply affect the dynamics of cheese microbial communities and, as a consequence, multiple aspects of cheese quality, from metabolites affecting the taste, aroma and flavour, to body, texture and colour. Understanding and exploiting microbial interactions is therefore key to managing cheese quality. This is true for the simplest systems (fresh cheeses produced from pasteurized milk using defined starters composed solely of Lactic Acid Bacteria) 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) among members of the cheese biota have been reviewed multiple times. Although 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 first illustrate concepts relevant to the description of microbial interaction networks using network science concepts. Then, we briefly review methods used for the inference and analysis of microbial association networks and their potential use in the interpretation of the cheese interactome. Since these methods can only be used for mining microbial associations, a review of the experimental methods used to confirm the nature of microbial interactions among cheese microbes. Finally, we demonstrate the potential of microbial association network inference by mining metataxonomic data stored in the public database DairyFMBN, a specialized version of FoodMicrobionet which collates data on 74 metataxonomic studies on dairy products. Microbial association networks were inferred from 34 studies on cheese with up to 4 different methods and the results discussed to evaluate several aspects (choice of method, level of taxonomic resolution for the analysis, network, node and edge properties) which provide insight on the usefulness of this approach as explorative tool in the detection of microbial interactions in cheese.HighlightsApproaches for inference of association networks from metataxonomic data were reviewedA metastudy on association networks in cheese was carried out using 34 recent studiesInference method and taxonomic resolution should be chosen carefullySPIEC-EASI may be used as a conservative method for microbial association inferenceEdge and node properties support the formulation of testable hypotheses for microbial interactions

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 degrees C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on nonselective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 10(7) cfu/g within 2-3 days at 10 degrees C and 7-8 days at 0 degrees 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 degrees 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 10(7) 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.

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 O

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.

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.

Enterocin 1146, a bacteriocin produced by Enterococcus faecium . DPC1146, has a rapid bactericidal effect on Listeria in buffer systems, broth, and milk. In trypticase soy broth, increasing the bacteriocin dose and/or decreasing the pH extended the lag phase of Listeria innocua . A logarithmic relationship was found between response (as proportion of survivors or growth compared to a control) and dose. Increasing the inoculum level of the indicator reduced the effectiveness of enterocin 1146. Log-phase cells of L. innocua were more resistant than stationary-phase cells in both broth and buffer systems. In milk treated with 250 arbitrary units of enterocin 1146/ml and inoculated with 103 or 105 CFU/ml of Listeria monocytogenes Scott A, populations reached only 5-14% of the control after 24 h at 30°C, with numbers exceeding 107, while at 6°C a slow decrease in population was found.

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), ltering 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 classi cation, makes comparison and re-analysis of data from published and unpublished studies easy. Data exported from FoodMicrobionet can be readily used for graphical and sta- tistical 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.

Natural milk cultures are undefined starters produced by a selective treatment (milk pasteurization, incubation at high temperature and backslopping), and used for the production of Mozzarella cheese. The objective of this study was to monitor the microbial composition (at or below the species level) and the variability of two model laboratory cultures (LC) and an artisanal culture (NMC) produced at a dairy plant over 13 reproduction cycles using culture independent and dependent methods. PCR-DGGE of V3 region of 16S RNA gene and of a fragment of the lacSZ operon of Streptococcus thermophilus proved that the cultures were dominated by S. thermophilus but other species ( Lactobacillus delbrueckii , L. helveticus , Lactococcus lactis , enterococci) were present. These results were partially confirmed by culture dependent analysis. Molecular identification and typing (RAPD-PCR and lacSZ PCR-DGGE and partial sequencing of serB in S. thermophilus ) confirmed NMC had the highest diversity, while the two replicate LCs diverged at some early stage. Variability in acid production activity and in aroma production was also observed, and was highest in NMC. S. thermophilus bacteriophages were detected by multiplex PCR.

The number of studies using high throughput sequencing (HTS) methods for the characterisation of microbial communities of milk and dairy products has increased markedly in the last decade. Besides confirming what was previously known from low sensitivity and throughput cultivation based and cultivation independent techniques, HTS studies have provided deeper insights into the structure and function of microbial communities. While the comparison of raw data from different studies is still difficult due the lack of standard operating procedures, the availability of well-structured databases with raw and processed sequences has boosted our ability to get quantitative insights into the factors that shape the microbial communities of milk and dairy products. Here we critically review metataxonomic and metagenomic studies on milk from cows and other dairy species, and discuss potential sources and dynamics of microbiota during storage, transport and processing in liquid pasteurised milk, using the FoodMicrobionet database to carry out meta-analyses.

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.

We developed and validated a tool assessing the adherence to the Mediterranean diet based on current nutritional recommendations for health promotion (MEDI-Quest). Four-hundred-and-eleven clinically healthy subjects completed both the MEDI-Quest-score and the validated MedDietScore (MDS). A good accordance between MEDI-Quest and MDS was observed (R = 0.638), as well as for the percentage of individuals having similar scores with both methods for each food group. Regarding socio-cultural-factors, higher adherence was observed in people with older-age (OR = 1.030, p

Sequencing technologies have deeply changed our approach to the study of food microbial communities. This review describes recent exploitations of high-throughput sequencing applications to improve our knowledge of food microbial consortia. In the past 10 years, target amplicon sequencing has become routinely used in many food microbiology laboratories, providing a detailed picture of food-associated microbiota. Metagenomics and metatranscriptomics approaches are still underexploited in food microbial ecology, despite their potential to uncover the functionality of complex communities. In a near future, sequencing technologies will surely advance our understanding of how to effectively use the invaluable microbial resources to improve food quality and safety. Expected final online publication date for the Annual Review of Food Science and Technology Volume 9 is March 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The microbiological quality, pH, colour, proteolysis and head space composition (using an electronic nose) of several commercial brands of high-moisture Mozzarella cheese produced in Italy were evaluated at the beginning and at the end (5 days) of refrigerated storage in order to evaluate the effect of the acidification system (direct acid addition or use of starter cultures) and storage on the quality of the cheese. A high variability was found for most parameters. At the end of storage all parameters were affected by the mode of acidification and cheese produced by direct acid addition had a significantly lower microbiological quality; counts of psychrotrophs exceeded 10 7 cfu/g for most samples and microbial counts showed a significant correlation with the residual shelf life. Multivariate analysis confirmed that samples at the beginning and at the end of storage were clearly separated but no grouping based on the mode of acidification was found. The electronic nose was only partially successful (80% correct classification) in classifying the cheeses on the basis of storage time or of microbial counts. This is likely to be due to the variety of brands used in the analysis and to differences in the starter systems or acidification mode used.

Aims: Members of the Lactobacillus casei and Lactobacillus plantarum groups are capable of aerobic and respiratory growth. However, they grow poorly in aerobiosis in the currently available chemically defined media, suggesting that aerobic and respiratory growth require further supplementation. Methods and Results: The effect of Tween 80, l-alanine, l-asparagine, l-aspartate, l-proline and l-serine on anaerobic and respiratory growth of Lact. casei N87 was investigated using a 25 factorial design. The effectiveness of modified CDM (mCDM) was validated on 21 strains of Lact. casei and Lact. plantarum groups. Tween 80 supplementation did not affect anaerobic growth, but improved respiratory growth. l-asparagine, l-proline and l-serine were stimulatory for respiring cells, while the presence of l-aspartate, generally, impaired biomass production. mCDM promoted the growth of Lact. casei and Lact. plantarum, with best results for strains showing a respiratory phenotype. Conclusions: The nutritional requirements of anaerobic and respiratory cultures of members of the Lact. casei and Lact. plantarum groups differ. Tween 80 and selected amino acids derived from pathways related to TCA cycle, pyruvate conversion and NADH recycling are required for respiration. Significance and Impact of the Study: The availability of mCDM will facilitate the study of aerobic metabolism of lactobacilli under controlled conditions.

Cultivable NSLAB in traditional Pasta filata and ewes' milk cheeses were studied by both PCR-DGGE of cells from Rogosa agar and by isolation and molecular identification after a simulated gastric juice (SGJ) treatment of the cheese. Two to six species were retrieved from each sample. The majority of isolates were identified as Lactobacillus paracasei , Lactobacillus fermentum , Lactobacillus rhamnosus , Enterococcus or Pediococcus . Bile tolerance and bile salt hydrolase (BSH) activity were tested on 88 strains: 64% were able to grow with ≥0.15% bile and 40% were BSH positive. The effect of simulated digestion was tested on 15 strains. Inactivation ranged from 0.15 to 2.93 log cycles; most of the lethality was associated with pancreatic juice treatment. Although SGJ treatment alone may not provide a correct estimate of tolerance to gastrointestinal transit, it allowed selection of strains with a high tolerance to gastric juice, which may be tested as probiotic candidates.

The strains Lactobacillus casei N87, N811, N2014 (isolated from infant faeces) and CI4368 (from cheese) were used to evaluate the effect of aeration and supplementation with heme and menaquinone (promoting respiration) on the antioxidant capability and on the survival to simulated gastrointestinal transit and oxidative stresses. The probiotic Lactobacillus rhamnosus GG and L. casei Shirota were used for comparative purposes. L. casei N87 and N811 showed potential respiratory traits (increases in biomass and pH, oxygen tolerance and consumption, and production of antioxidant enzymes), while L. rhamnosus GG and L. casei Shirota were unable to activate aerobic pathways. Contrarily to probiotic strains, the four L. casei have the capability to produce catalase in both anaerobic and aerobic conditions, contributing to the expression of highly H2O2-tolerant phenotypes. Oxygen and heme/menaquinone supplementation, generally, improved tolerance of oxidative stresses and degradation of free-radicals, but significantly impaired the survival to simulated gastric juice. This study highlighted the greater robustness of respiration-competent L. casei strains, compared to L. rhamnosus GG and L. casei Shirota. The capability to reduce H2O2 and free-radicals may be exploited in food (prevention of oxidation in fermented foods) and human health (removal of toxic compounds; potential implication in the reduction of gastrointestinal and degenerative diseases) applications.

Scamorza is a semi-hard, pasta filata cheese resembling low-moisture Mozzarella cheese, with a short ripening time (

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.

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 (

The structure of microbial association networks was investigated for seventeen studies on food bacterial communities using the CoNet app. The results were compared with those for host and environmental microbiomes. Microbial association networks of food bacterial communities shared several properties with those of host microbiomes, although they were less complex and lacked a scale-free, small world structure that is characteristic of environmental microbial communities. This may depend on both the initial contamination pattern, whose main source is the raw material microbiome, and on the copiotrophic nature of food environments, with lack of well defined, specific niches. The selective factors which are characteristic of fermentation and spoilage drastically simplified microbial association networks and showed the emergence of negative hubs. Co-presence and mutual exclusion networks had a radically different structure, with high clustering coefficient in the first and high heterogeneity in the latter. Node properties (degree, positive degree, betweenness centrality, abundance) can be combined in plots, which allow a rapid identification of hub species. The combined use of three network inference tools (CoNet, SparCC, and SPIEC-EASI) confirmed that microbial association network detection is method specific, but several coherent copresence or mutual exclusion relationships were detected by at least two different methods. ispartof: Food Microbiology vol:73 pages:49-60 ispartof: location:England status: published

The different pipelines that may be used in 16S rRNA gene profiling of bacterial communities are known to have a significant impact on alpha and beta diversity measures and this may prevent direct comparison of results obtained in studies using different bioinformatic approaches to analyse raw sequences. To evaluate the feasibility of meta-studies on food bacterial communities, we compared four analysis procedures, varying in OTU picking and taxonomy assignment strategies. A closed reference OTU picking resulted in the most divergent results in terms of both alpha and beta diversity, compared to open reference methods. Nevertheless, when OTUs were collapsed at the genus level, a high correlation was obtained among the estimated abundances of taxa for most studies. Aggregation of samples by their nature and occurrence of food spoilage or fermentation resulted in a very similar classification using two beta diversity analysis methods. We conclude that comparisons of data obtained from different studies are feasible at the genus level, when the same OTU picking strategy is used. Finally, we provide a new version of FoodMicrobionet (Parente et al., 2016), including data from 26 recent studies on food bacterial communities, together with R scripts allowing both the extraction of data in formats which can be used in several analysis tools (including the R package phyloseq and the Cytoscape app CoNet) and the statistical and graphical analysis using common alpha- and beta-diversity analysis methods.

This article addresses general concepts surrounding the use of starter cultures in cheesemaking. Starter cultures are essential to the manufacture of many cheese types. Whether prepared from the natural flora of milk, propagated as cultured whey, or selected from a preserved collection of defined single strains or undefined mixtures, starter cultures must be matched to the cheese type being made. Cheese starter cultures are predominantly composed of lactic acid bacteria, although other bacteria and yeasts may also be involved. In cheese manufacture, the primary role of starter cultures is the production of lactic acid from lactose at a predictable and controlled rate. Controlling acid production during cheesemaking is key to achieving control over curd pH, moisture, and lactose level. These factors in turn have a major influence on the microbial, chemical, and biochemical environment of the maturing cheese. Metabolism by starter cultures and the enzymes they produce contribute to the development of cheese flavor. The preparation and use of bulk starter cultures and culture concentrates are discussed.

The antimicrobial activity of hydro-alcoholic extract of Myrtus communis L. was investigate in vitro and in situ against different meat spoilage biotypes of Pseudomonas fragi and Brochothrix thermosphacta. Water-ethanol myrtle extract (WEME) was prepared from myrtle leaves and used to study the antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Naturally contaminated ground beef was used to evaluate in situ antibacterial activity of freeze-dried myrtle extract at different concentrations. In vitro antimicrobial activity of WEME was significantly more effective against B. thermosphacta than P. fragi strains. MIC and MLC values were in the range of 12.5–50 and 25–100 mg DM/ml, respectively, for B. thermosphacta and P. fragi strains. In situ results showed that the microbial population, except for Pseudomonas spp., decreased significantly in ground meat with added 5 % of freeze-dried myrtle extract. In conclusion, the findings demonstrate the effectiveness of myrtle extract to control or prevent the proliferation of meat spoilage bacteria.

The polymorphism of 5 stress response genes (hrcA, ctsR, clpP, ftsH, dnaK) in 32 Lactobacillus plantarum strains was evaluated by multilocus restriction typing (MLRT) and by sequence analysis of ctsR, hrcA and clpP genes. Both these approaches allowed the discrimination of the subspecies L. plantarum ssp. plantarum and L. plantarum ssp. argentoratensis. HrcA sequence analysis also allowed discrimination at the species and subspecies level of several species of lactic acid bacteria, thus confirming that it can be used as a valuable taxonomic marker. No significant relationship was found between stress response gene polymorphism and resistance to heat treatments. The effect of temperature on growth kinetics and the protein expression were investigated for selected strains carrying different mutations in hrcA. L. plantarum ssp. argentoratensis NCIMB12120 and L. plantarum ssp. plantarum DPC2159, both of which had mutations in domains of HrcA which are important for the repressor functionality, had a reduced growth rate at all temperatures tested (25, 30, 37, 40, and 42 °C) compared to L. plantarum WCFS1. In L. plantarum DPC2159, protein expression upon temperature shifts from 25 to 40 °C or growth at 40 °C was altered compared to L. plantarum WCFS1, but further study is needed to unequivocally confirm the relationship with mutations in hrcA.

Aims: The aim of the study is to gain further insight on the respiratory behaviour of Lactobacillus plantarum and its consequences on stress tolerance. Methods and Results: We investigated the effect of temperature and respiration on the growth and stress (heat, oxidative, freezing, freeze-drying) response of Lact. plantarum C17 during batch cultivations. Temperature as well as respiration clearly affected the physiological state of cells, and generally, cultures grown under respiratory conditions exhibited improved tolerance of some stresses (heat, oxidative, freezing) compared to those obtained in anaerobiosis. Our results revealed that the activities in cell-free extracts of the main enzymes related to aerobic metabolism, POX (pyruvate oxidase) and NPR (NADH peroxidase), were significantly affected by temperature. POX was completely inhibited at 37°C, while the activity of NPR slightly increased at 25°C, indicating that in Lact. plantarum, the temperature of growth may be involved in the activation and modulation of aerobic/respiratory metabolism. Conclusions: We confirmed that respiration confers robustness to Lact. plantarum cells, allowing a greater stress tolerance and advantages in the production of starter and probiotic cultures. Significance and Impact of the Study: This is the first study on respiratory metabolism on a strain other than the model strains WCFS1; novel information on the role of temperature in the modulation of aerobic/respiratory metabolism in Lact. plantarum is presented. © 2013 The Society for Applied Microbiology.

Summary Partial least square regression analysis was used to study the correlation between X variables (semolina quality, hydration level and mixing time) and Y variables, which were, in a first model, dough consistency during mixing, and, in a second model, dough properties after mixing (strength, elasticity, density) and leavening (maximum volume). The first model showed a predictive residual sum of squares (PRESS) of 2.98 and a predictive R2 (Q2) of 0.92, and highlighted the key role of hydration and mixing time on dough consistency. The second model had the best PRESS (8.25) and Q2 (0.94) values for dough volume and indicated that the volume increased with increasing mixing time until the dough consistency decreased of 20–30%. Dough volume was primarily affected by hydration. The model indicated that maximum volume after leavening, corresponding to optimum mixing time, was obtained with a soft and elastic dough, with a low-density value.

Lactobacilli have been used as adjunct cultures in the manufacture of different cheeses with the objective of accelerating ripening and/or improving cheese quality, but no studies have been conducted with strains from non-dairy origins. A miniature cheddar-type cheese model was used to screen ten dairy and non-dairy Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus strains for their performances as adjuncts in cheese manufacture. All strains were able to grow and survive in the cheese environment and produced only minor, although statistically significant, changes in gross cheese composition. Adjuncts affected secondary proteolysis causing differences in the levels of free amino groups, total free amino acids and reversed-phase HPLC (RP-HPLC) profiles of pH 4.6-soluble extract. Three strains were selected on the basis of differences in proteolysis pattern and used in a pilot-plant production of cheddar cheese, which was ripened for 180 days. The results confirmed that use of L. plantarum adjuncts significantly affected secondary proteolysis as measured by free amino acid production with minor impact on gross composition and primary starter performance, but the impact on RP-HPLC profiles of pH 4.6-soluble extracts was not statistically significant. The use of a strain originally isolated from olive brine fermentation, L. plantarum P1.5, resulted in significantly improved preference scores over the control.

This review outlines the recent advances in the knowledge on aerobic and respiratory growth of lactic acid bacteria, focusing on the features of respiration-competent lactobacilli. The species of the genus Lactobacillus have been traditionally classified as oxygen-tolerant anaerobes, but it has been demonstrated that several strains are able to use oxygen as a substrate in reactions mediated by flavin oxidases and, in some cases, to synthesize a minimal respiratory chain. The occurrence of genes related to aerobic and respiratory metabolism and to oxidative stress response apparently correlates with the taxonomic position of lactobacilli. Members of the ecologically versatile Lactobacillus casei, L. plantarum and L. sakei groups are apparently best equipped to deal with aerobic/respiratory growth. The shift from anaerobic growth to aerobic (oxygen) and/or respiratory promoting (oxygen, exogenous haem and menaquinone) conditions offers physiological advantages and affects the pattern of metabolite production in several species. Even if this does not result in dramatic increases in biomass production and growth rate, cells grown in these conditions have improved tolerance to heat and oxidative stresses. An overview of benefits and of the potential applications of Lactobacillus cultures grown under aerobic or respiratory conditions is also discussed.

Summary This review describes the recent advances in the study of food microbial ecology, with a focus on food fermentations. High-throughput sequencing (HTS) technologies have been widely applied to the study of food microbial consortia and the different applications of HTS technologies were exploited in order to monitor microbial dynamics in food fermentative processes. Phylobiomics was the most explored application in the past decade. Metagenomics and metatranscriptomics, although still underexploited, promise to uncover the functionality of complex microbial consortia. The new knowledge acquired will help to understand how to make a profitable use of microbial genetic resources and modulate key activities of beneficial microbes in order to ensure process efficiency, product quality and safety.

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. © 2016 Elsevier B.V.

Aims To evaluate the effect of cultivation (anaerobiosis vs respiration) and Tween 80 supplementation on the production of metabolites and on the composition of membrane fatty acids (FAs) in Lactobacillus casei N87. Methods and Results Anaerobic and respiratory growth, with or without Tween 80 supplementation, was carried out in a chemically defined medium. Production of biomass, organic acids, volatile organic compounds (VOCs), consumption of amino acids and changes in membrane FAs were investigated. Respiration altered the central metabolism rerouting pyruvate away from lactate accumulation, while Tween 80 had a minor effect on metabolic pathways. VOCs were mainly affected by growth conditions and significant amounts of diacetyl were produced by respiratory cultures. Respiration increased desaturation of membrane lipids and Tween 80 improved the production of essential polyunsaturated FAs. Palmitic acid decreased in Tween-supplemented aerated cultures. Conclusions Combination of Tween 80 and respiratory growth promoted production of biomass and aroma compounds and affected the composition of membrane FAs in Lact. casei N87. Significance and Impact of the Study Respiration might be exploited in Lact. casei as a natural strategy for the enhanced production of aroma compounds.

Model undefined thermophilic starter cultures were produced from raw milk of nine pasta-filata cheesemaking plants using a selective procedure based on pasteurization and incubation at high temperature with the objective of studying the microbial community dynamics and the variability in performances under repeated (7-13) reproduction cycles with backslopping. The traditional culture-dependent approach, based on random isolation and molecular characterization of isolates was coupled to the determination of pH and the evaluation of the ability to produce acid and fermentation metabolites. Moreover, a culture-independent approach based on amplicon-targeted next-generation sequencing was employed. The microbial diversity was evaluated by 16S rRNA gene sequencing (V1-V3 regions), while the microdiversity of Streptococcus thermophilus populations was explored by using novel approach based on sequencing of partial amplicons of the phosphoserine phosphatase gene (serB). In addition, the occurrence of bacteriophages was evaluated by qPCR and by multiplex PCR. Although it was relatively easy to select for a community dominated by thermophilic lactic acid bacteria (LAB) within a single reproduction cycle, final pH, LAB populations and acid production activity fluctuated over reproduction cycles. Both culture-dependent and -independent methods showed that the cultures were dominated by either S. thermophilus or Lactobacillus delbrueckii subsp. lactis or by both species. Nevertheless, subdominant mesophilic species, including lactococci and spoilage organisms, persisted at low levels. A limited number of serB sequence types (ST) were present in S. thermophilus populations. L. delbrueckii and Lactococcus lactis bacteriophages were below the detection limit of the method used and high titres of cos type S. thermophilus bacteriophages were detected in only two cases. In one case a high titre of bacteriophages was concurrent with a S. thermophilus biotype shift in the culture. This study largely confirms previous data on the composition of undefined thermophilic starters used for the production of traditional cheeses in Italy but it is the first one to systematically address the dynamics of the cultures under a repeated reproduction regime with backslopping.