Microbial contamination pathways in a poultry abattoir provided clues on the distribution and persistence of Arcobacter spp.

The consumption of contaminated poultry meat is a significant threat for public health, as it implicates in foodborne pathogen infections, such as those caused by Arcobacter. The mitigation of clinical cases requires the understanding of contamination pathways in each food process and the characterization of resident microbiota in the productive environments, so that targeted sanitizing procedures can be effectively implemented. Nowadays these investigations can benefit from the complementary and thoughtful use of culture- and omics-based analyses, although their application in situ is still limited. Therefore, the 16S-rRNA gene-based sequencing of total DNA and the targeted isolation of Arcobacter spp. through enrichment were performed to reconstruct the environmental contamination pathways within a poultry abattoir, as well as the dynamics and distribution of this emerging pathogen. To that scope, broiler's neck skin and caeca have been sampled during processing, while environmental swabs were collected from surfaces after cleaning and sanitizing. Metataxonomic survey highlighted a negligible impact of fecal contamination and a major role of broiler's skin in determining the composition of the resident abattoir microbiota. The introduction of Arcobacter spp. in the environment was mainly conveyed by this source rather than the intestinal content. Arcobacter butzleri represented one of the most abundant species and was extensively detected in the abattoir by both metataxonomic and enrichment methods, showing higher prevalence than other more thermophilic Campylobacterota. In particular, Arcobacter spp. was recovered viable in the plucking sector with high frequency, despite the adequacy of the sanitizing procedure.IMPORTANCEOur findings have emphasized the persistence of Arcobacter spp. in a modern poultry abattoir and its establishment as part of the resident microbiota in specific environmental niches. Although the responses provided here are not conclusive for the identification of the primary source of contamination, this biogeographic assessment underscores the importance of monitoring Arcobacter spp. from the early stages of the production chain with the integrative support of metataxonomic analysis. Through such combined detection approaches, the presence of this pathogen could be soon regarded as hallmark indicator of food safety and quality in poultry slaughtering.

Effects of a prothioconazole- and tebuconazole-based fungicide on the yield, silage characteristics, and fungal mycobiota of corn harvested and conserved as whole-crop and high-moisture ear silages.

AIMS: To analyze the effect of a prothioconazole- and tebuconazole-based fungicide on the yield and silage characteristics of whole-crop corn (WCC) and high-moisture ear corn (HMC) silages and on the fungal community dynamics from the harvest to aerobic exposure. METHODS AND RESULTS: Corn were untreated (NT) or treated (T) with a prothioconazole- and tebuconazole-based fungicide and harvested as WCC and HMC. Silages were conserved for 60 and 160 d and subjected to an aerobic stability test. The fungicide increased the yield per hectare however, it did not affect the main nutritional characteristics of WCC or HMC. The main chemical, fermentative and microbial characteristics, dry matter (DM) losses and aerobic stability were mainly affected by the conservation time, regardless of the treatment. Fusarium, Alternaria, Aspergillus, and Penicillium genera were identified as dominant before ensiling, but Aspergillus and Penicillium became dominant after silo opening and aerobic exposure. Yeast population during ensiling and aerobic deterioration resulted in a simplification, with Pichia and Kazachstania genera being dominant. CONCLUSIONS: The application of fungicide improved the DM, starch, and net energy for lactation (NEL) yield per hectare but had no consistent effect on the microbial and fermentative silage quality and aerobic stability.

Selection of food cultures with protective properties for cooked ham.

Sliced cooked ham stored in modified atmosphere packaging (MAP) can be spoiled by lactic acid bacteria (LAB) which are dominating under psychrotrophic conditions. Depending on the strains, the colonization can result in a premature spoilage characterized by off-flavors, gas and slime production, discoloration, and acidification. The purpose of this study was the isolation, identification and characterization of potential food culture with protective properties, able to prevent or delay spoilage in cooked-ham. The first step was to identify by means of microbiological analysis, the microbial consortia both in unspoiled and in spoiled lots of sliced cooked ham by the use of media for the detection lactic acid bacteria and total viable count. Counts ranged from values lower than 1 Log CFU/g to 9 Log CFU/g in spoiled and unflawed samples. The interaction between consortia was then studied in order to screen for strains able to inhibit spoilage consortia. Strains showing antimicrobial activity were identified and characterized by molecular methods and tested for their physiological features. Among a total of 140 strains isolated, nine were selected for their ability to inhibit a large number of spoilage consortia, to grow and ferment at 4 °C and to produce bacteriocins. The effectiveness of the fermentation made by food culture was evaluated, through challenge tests in situ, analysing the microbial profiles of artificially inoculated cooked-ham slices during storage by high throughput 16 S rRNA gene sequencing. The native population in situ resulted competitive against the inoculated strains and only one strain was able to significantly reduce the native populations reaching about 46.7% of the relative abundance. The results obtained in this study provide information about the selection of autochthonous LAB on the base of their action against spoilage consortia, in order to select protective potential cultures able to improve the microbial quality of sliced cooked ham.

Impact of Different Drying Methods on the Microbiota, Volatilome, Color, and Sensory Traits of Sea Fennel (Crithmum maritimum L.) Leaves.

Sea fennel (Crithmum maritimum L.) is a strongly aromatic herb of the Apiaceae family, whose full exploitation by the modern food industry is of growing interest. This study aimed at investigating the microbiological quality, volatile profile, and sensory traits of sea fennel spices produced using room-temperature drying, oven drying, microwave drying, and freeze drying. All the assayed methods were able to remove moisture up until water activity values below 0.6 were reached; however, except for microwave drying, none of the assayed methods were effective in reducing the loads of contaminating microorganisms. The metataxonomic analysis highlighted the presence of phytopathogens and even human pathogens, including members of the genera Bacillus, Pseudomonas, Alternaria, and Cryptococcus. When compared to fresh leaves, dried leaves showed increased L* (lightness) and c* (chroma, saturation) values and reduced hue angle. Dried leaves were also characterized by decreased levels of terpene hydrocarbons and increased levels of aldehydes, alcohols, and esters. For the sensory test, the microwave-dried samples obtained the highest appreciation by the trained panel. Overall, the collected data indicated microwave drying as the best option for producing sea fennel spices with low microbial loads, brilliant green color, and high-quality sensory traits.

The need for an integrated multi-OMICs approach in microbiome science in the food system.

Microbiome science as an interdisciplinary research field has evolved rapidly over the past two decades, becoming a popular topic not only in the scientific community and among the general public, but also in the food industry due to the growing demand for microbiome-based technologies that provide added-value solutions. Microbiome research has expanded in the context of food systems, strongly driven by methodological advances in different -omics fields that leverage our understanding of microbial diversity and function. However, managing and integrating different complex -omics layers are still challenging. Within the Coordinated Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), a project supported by the European Commission, the workshop "Metagenomics, Metaproteomics and Metabolomics: the need for data integration in microbiome research" gathered 70 participants from different microbiome research fields relevant to food systems, to discuss challenges in microbiome research and to promote a switch from microbiome-based descriptive studies to functional studies, elucidating the biology and interactive roles of microbiomes in food systems. A combination of technologies is proposed. This will reduce the biases resulting from each individual technology and result in a more comprehensive view of the biological system as a whole. Although combinations of different datasets are still rare, advanced bioinformatics tools and artificial intelligence approaches can contribute to understanding, prediction, and management of the microbiome, thereby providing the basis for the improvement of food quality and safety.

Campylobacter spp. prevalence and mitigation strategies in the broiler production chain.

This study aims to discuss the microbial ecology of the broiler gut environment, Campylobacter prevalence across the broiler production chain with a follow-up focus on a possible mitigation strategy, based on the use of bacteriophages. Scientific literature published from the last two decades was reviewed and data were collected to establish the ranges of Campylobacter loads from different samples. Results showed that the pathogen load in the sample is likely to increase from the different stages of the production chain. Contamination of water and feed represents the most notable source of contamination during the primary production, while cross-contamination of broiler carcasses, skin, and meat occurs during the slaughter, dressing, and processing via machinery, work surfaces, water, and air partially due to the leaking of contaminated feces from visceral rupture. Knowledge gaps were identified and included: a lack of studies detecting Campylobacter in broilers in most of the European countries over the last decade and a low number of studies determining the bacterial load in crates used to transport broilers to the slaughterhouse. Determining the prevalence of Campylobacter in the broiler industry will enable us to set critical control points to produce broiler flocks and meat products with a low risk of Campylobacter contamination.

Functional pangenome analysis reveals high virulence plasticity of Aliarcobacter butzleri and affinity to human mucus.

Aliarcobacter butzleri is an emerging pathogen that may cause enteritis in humans, however, the incidence of disease caused by this member of the Campylobacteriaceae family is still underestimated. Furthermore, little is known about the precise virulence mechanism and behavior during infection. Therefore, in the present study, through complementary use of comparative genomics and physiological tests on human gut models, we sought to elucidate the genetic background of a set of 32 A. butzleri strains of diverse origin and to explore the correlation with the ability to colonize and invade human intestinal cells in vitro. The simulated infection of human intestinal models showed a higher colonization rate in presence of mucus-producing cells. For some strains, human mucus significantly improved the resistance to physical removal from the in vitro mucosa, while short time-frame growth was even observed. Pangenome analysis highlighted a hypervariable accessory genome, not strictly correlated to the isolation source. Likewise, the strain phylogeny was unrelated to their shared origin, despite a certain degree of segregation was observed among strains isolated from different segments of the intestinal tract of pigs. The putative virulence genes detected in all strains were mostly encompassed in the accessory fraction of the pangenome. The LPS biosynthesis and in particular the chain glycosylation of the O-antigen is harbored in a region of high plasticity of the pangenome, which would indicate frequent horizontal gene transfer phenomena, as well as the involvement of this hypervariable structure in the adaptive behavior and sympatric evolution of A. butzleri. Results of the present study deepen the current knowledge on A. butzleri pangenome by extending the pool of genes regarded as virulence markers and provide bases to develop new diagnostic approaches for the detection of those strains with a higher virulence potential.

Influence of Taxonomic and Functional Content of Microbial Communities on the Quality of Fermented Cocoa Pulp-Bean Mass.

Microbial metabolism drives changes in the physicochemical properties and, consequently, the sensory characteristics of fermented cocoa beans. In this context, information regarding the structure, function, and metabolic potential of microbial communities' present during cocoa pulp-bean mass fermentation is limited, especially concerning the formation of aromatic compounds. To bridge the gap, the metagenome of fermented cocoa pulp-bean mass (Criollo and Forastero) has been investigated using shotgun metagenomics coupled with physicochemical, microbiological, quality, and sensory analyses to explore the impact of microbial communities on the quality of fermented cocoa pulp-bean mass on one farm in one season and in one region under the same environmental conditions. Our findings showed that the metagenomic diversity in cocoa, the fermentation length, and the diversity and function of metagenome-assembled genomes (MAGs) greatly influence the resulting distinctive flavors. From the metabolic perspective, multiple indicators suggest that the heterolactic metabolism was more dominant in Criollo fermentations. KEGG genes were linked with the biosynthesis of acetic acid, ethanol, lactic acid, acetoin, and phenylacetaldehyde during Criollo and Forastero fermentations. MAGs belonging to Lactiplantibacillus plantarum, Limosilactobacillus reuteri, and Acetobacter pasteurianus were the most prevalent. Fermentation time and roasting are the most important determinants of cocoa quality, while the difference between the two varieties are relatively minor. The assessment of microbiological and chemical analysis is urgently needed for developing fermentation protocols according to regions, countries, and cocoa varieties to guarantee safety and desirable flavor development. IMPORTANCE Monitoring the composition, structure, functionalities, and metabolic potential encoded at the level of DNA of fermented cocoa pulp-bean mass metagenome is of great importance for food safety and quality implications.

Spatiotemporal Distribution of the Environmental Microbiota in Food Processing Plants as Impacted by Cleaning and Sanitizing Procedures: the Case of Slaughterhouses and Gaseous Ozone.

Microbial complexity and contamination levels in food processing plants heavily impact the final product fate and are mainly controlled by proper environmental cleaning and sanitizing. Among the emerging disinfection technologies, ozonation is considered an effective strategy to improve the ordinary cleaning and sanitizing of slaughterhouses. However, its effects on contamination levels and environmental microbiota still need to be understood. For this purpose, we monitored the changes in microbiota composition in different slaughterhouse environments during the phases of cleaning/sanitizing and ozonation at 40, 20, or 4 ppm. Overall, the meat processing plant microbiota differed significantly between secondary processing rooms and deboning rooms, with a greater presence of psychrotrophic taxa in secondary processing rooms because of their lower temperatures. Cleaning/sanitizing procedures significantly reduced the contamination levels and in parallel increased the number of detectable operational taxonomic units (OTUs), by removing the masking effect of the most abundant human/animal-derived OTUs, which belonged to the phylum Firmicutes Subsequently, ozonation at 40 or 20 ppm effectively decreased the remaining viable bacterial populations. However, we could observe selective ozone-mediated inactivation of psychrotrophic bacteria only in the secondary processing rooms. There, the Brochothrix and Pseudomonas abundances and their viable counts were significantly affected by 40 or 20 ppm of ozone, while more ubiquitous genera like Staphylococcus showed a remarkable resistance to the same treatments. This study showed the effectiveness of highly concentrated gaseous ozone as an adjunct sanitizing method that can minimize cross-contamination and so extend the meat shelf life.IMPORTANCE Our in situ survey demonstrates that RNA-based sequencing of 16S rRNA amplicons is a reliable approach to qualitatively probe, at high taxonomic resolution, the changes triggered by new and existing cleaning/sanitizing strategies in the environmental microbiota in human-built environments. This approach could soon represent a fast tool to clearly define which routine sanitizing interventions are more suitable for a specific food processing environment, thus limiting the costs of special cleaning interventions and potential product loss.

Discovering microbiota and volatile compounds of surströmming, the traditional Swedish sour herring.

In this study, the microbiota of ready-to-eat surströmming from three Swedish producers were studied using a combined approach. The pH values of the samples ranged between 6.67 ± 0.01 and 6.98 ± 0.01, whereas their aw values were between 0.911 ± 0.001 and 0.940 ± 0.001. The acetic acid concentration was between 0.289 ± 0.009 g/100 g and 0.556 ± 0.036 g/100 g. Very low concentrations of lactic acid were measured. Viable counting revealed the presence of mesophilic aerobes, mesophilic lactobacilli and lactococci as well as halophilic lactobacilli and lactococci, coagulase-negative staphylococci, halophilic aerobes and anaerobes. Negligible counts for Enterobacteriaceae, Pseudomonadaceae and total eumycetes were observed, whereas no sulfite-reducing anaerobes were detected. Listeria monocytogenes and Salmonella spp. were absent in all samples. Multiplex real-time PCR revealed the absence of the bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP) genes, which encode botulinic toxins, in all the samples analyzed. Metagenomic sequencing revealed the presence of a core microbiota dominated by Halanaerobium praevalens, Alkalibacterium gilvum, Carnobacterium spp., Tetragenococcus halophilus, Clostridiisalibacter spp. and Porphyromonadaceae. Psychrobacter celer, Ruminococcaceae, Marinilactibacillus psychrotolerans, Streptococcus infantis and Salinivibrio costicola were detected as minor OTUs. GC-MS analysis of volatile components revealed the massive presence of trimethylamine and sulphur compounds. Moreover, 1,2,4-trithiolane, phenols, ketones, aldehydes, alcohols, esters and long chain aliphatic hydrocarbons were also detected. The data obtained allowed pro-technological bacteria, which are well-adapted to saline environments, to be discovered for the first time. Further analyses are needed to better clarify the extent of the contribution of either the microbiota or autolytic enzymes of the fish flesh in the aroma definition.

Short communication: High-throughput sequencing approach to investigate Italian artisanal cheese production.

In this study, high-throughput sequencing (HTS) was used to investigate the microbiota of Robiola di Roccaverano production, an artisanal Protected Designation of Origin soft cheese made with raw goat milk by addition of a natural milk starter (NMS), from the Piedmont region of Italy. Different steps of production of Robiola di Roccaverano cheese at one artisanal dairy were monitored. Matched samples of milk, NMS, curd, and 5-d and 15-d matured cheeses were collected at different periods of the year. The DNA sequences obtained by HTS belonged to 5 phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Tenericutes. In milk, Proteobacteria and Firmicutes were mainly found, and several operational taxonomic units (OTU) belonging to contaminant bacteria such as Pseudomonas, Serratia, and Staphylococcus were observed. However, in NMS, curd, and 5- and 15-d cheeses, Firmicutes were principally observed where OTU of Lactococcus lactis were predominant, followed by Leuconostoc mesenteroides OTU. The results of the analysis showed high bacterial diversity in milk samples compared with NMS, curd, and 5- and 15-d cheeses, suggesting strong action of NMS in driving the characteristics of the final products.

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR < 0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR < 0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community. HIGHLIGHTS Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds. Differences were found between infected and healthy animals in gut microbiota. Candidatus Arthromitus was closely associated with infected birds. M. ornithogaster can affect intestinal microbiota composition of canaries.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

Shotgun Metagenomics and Volatilome Profile of the Microbiota of Fermented Sausages.

Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of Lactobacillus sakei and Staphylococcus xylosus), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of Enterobacteriaceae and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 µg/kg), ethyl acetate (251.58 µg/kg), and acetoin (1,100.19 µg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid.IMPORTANCE We present an advance in the understanding of meat fermentation by coupling DNA sequencing metagenomics and metabolomics approaches to describe the microbial function during this process. Very few studies using this global approach have been dedicated to food, and none have examined sausage fermentation, underlying the originality of the study. The starter culture drastically affected the organoleptic properties of the products. This finding underlines the importance of starter culture selection that takes into consideration the functional characteristics of the microorganism to optimize production efficiency and product quality.

Dynamics and Biodiversity of Bacterial and Yeast Communities during Fermentation of Cocoa Beans.

Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.

Modulation of intestinal microbiota, morphology and mucin composition by dietary insect meal inclusion in free-range chickens.

BACKGROUND: Gut health in poultry depends on the balance between the host, intestinal microbiota, intestinal microscopic features and diet. The effects of insect meal (a promising alternative protein source for poultry feed) on chicken gut morphology have recently been reported, but no data about intestinal microbiota and mucin composition modulation are available. The present study evaluated the effects of dietary Tenebrio molitor (TM) meal inclusion on gut health of free-range chickens by intestinal microbiota, morphology and mucin composition characterization. RESULTS: One hundred forty female medium-growing hybrids were divided into 2 dietary treatments (control feed [C] and 7.5% TM inclusion, with 5 replicate pens/treatment and 14 birds/pen) and slaughtered at 97 days of age (2 birds/pen for a total of 10 chickens/diet). The gut microbiota assessment on cecal content samples by 16S rRNA amplicon based sequencing showed higher alpha (Shannon, P < 0.05) and beta (Adonis and ANOSIM, P < 0.001) diversity in birds fed TM diet than C. In comparison with C group, TM birds displayed significant increase and decrease, respectively, of the relative abundances of Firmicutes and Bacteroidetes phyla, with higher Firmicutes:Bacteroidetes ratios (False Discovery Rate [FDR] < 0.05). The relative abundance of Clostridium, Oscillospira, Ruminococcus, Coprococcus and Sutterella genera was higher in TM chickens than C (FDR < 0.05). On the contrary, TM birds displayed significant decrease of the relative abundance of Bacteroides genus compared to the C group (FDR < 0.05). Gut morphology evaluation by morphometric analysis on small intestine revealed similar villus height, crypt depth and villus height to crypt depth ratio between C and TM birds. Characterization of gut mucin composition by periodic-acid Schiff, Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine showed unaffected mucin staining intensity in TM chickens when compared to C group. CONCLUSIONS: Dietary TM meal inclusion may positively modulate the gut microbiota of the free-range chickens without influencing the intestinal morphology and mucin composition. Since the rapid growth of chickens directly depends on morphological and functional integrity of the digestive tract, the gut health assessment by a post mortem multidisciplinary approach appears to be fundamental.

A bioinformatics pipeline integrating predictive metagenomics profiling for the analysis of 16S rDNA/rRNA sequencing data originated from foods.

The recent advances in molecular biology, such as the advent of next-generation sequencing (NGS) platforms, have paved the way to new exciting tools which rapidly transform food microbiology. Nowadays, NGS methods such as 16S rDNA/rRNA metagenomics or amplicon sequencing are used for the taxonomic profiling of the food microbial communities. Although 16S rDNA/rRNA NGS-based microbial data are not suited for the investigation of the functional potential of the identified operational taxonomic units as compared to shotgun metagenomics, advances in the bioinformatics discipline allow now the performance of such studies. In this paper, a bioinformatics workflow is described integrating predictive metagenomics profiling with specific application to food microbiology data. Bioinformatics tools pertinent to each sub-module of the pipeline are suggested as well. The published 16S rDNA/rRNA amplicon data originated from an Italian Grana-type cheese, using an NGS platform, was employed to demonstrate the predictive metagenomics profiling approach. The pipeline identified the microbial community and the changes that occurred in the microbial profile during manufacture of the food product studied (taxonomic profiling). The workflow also indicated significant changes in the functional profiling of the community. The tool may help to investigate the functional potential, alterations, and interactions of a microbial community. The proposed workflow may also find an application in the investigation of the ecology of foodborne pathogens encountered in various food products.

Microbiota dynamics and volatilome profile during stink bean fermentation (Sataw-Dong) with Lactobacillus plantarum KJ03 as a starter culture.

Sataw-Dong is a fermented stink bean in brine, made with Parkia speciosa subjected to spontaneous fermentation. This study aimed to investigate the impact of Lactobacillus plantarum KJ03 as a starter culture during Sataw-Dong fermentation and to determine its effect on the volatilome profile. Two fermentations were performed: spontaneous and inoculated with starter. The surface of the beans and the brines were separately analyzed throughout fermentation period for 15 days. Inoculated samples clearly showed a significantly higher acidification of the brine, reaching a pH of 3.98 within 5 days of fermentation. The microbiota was investigated through 16S amplicon based sequencing and revealed the dominance of Lactobacillus plantarum and Lactobacillus sanfranciscensis throughout the fermentation in both brine and bean samples. The starter used clearly influenced volatile organic compounds (VOCs) profiles. Inoculated samples showed the lowest abundance of Brevundimonas, Corynebacterium, Enterobacteriaceae, Methylobacterium and Klebsiella, compared to the spontaneous fermentation. Correlation between OTUs and VOCs revealed that acids, aldehydes, and alcohols exhibited a positive correlation with L. plantarum and L. sanfranciscencis. Overall aldehydes were mostly produced at the beginning, while acids, alcohols, and ketones at the middle until the end of the fermentation. Sataw-Dong produced with the starter significantly perceived a positive response in the overall acceptance. As shown through microbiological changes, acidification, VOCs and sensory analysis, the successful and accelerated Sataw-Dong fermentation was achieved when using a functional starter strain.

Study of Lactococcus lactis during advanced ripening stages of model cheeses characterized by GC-MS.

Lactococcus lactis, is extensively used as starter culture in dairy products. Nevertheless, it has recently been detected in cheese, as metabolically active cells, in advanced ripening stages. In this study, we assessed the viability of L. lactis subsp. lactis in model cheeses up to 180 days of ripening by both culture-dependent and -independent methods. In addition, we studied the expression of metC and als genes involved in the production of aroma compounds detected by Gas Chromatography-Mass Spectrometry (GC-MS). Three L. lactis subsp. lactis commercial starters were inoculated in pasteurized milk and model cheeses were manufactured and ripened for six months. Samples were analysed at manufacturing and ripening steps, in terms of viability of L. lactis by both traditional plating and direct analysis of RNA by reverse transcription quantitative PCR (RT-qPCR), and in terms of aroma profile by GC-MS. Relatively to RT-qPCR analysis, L. lactis was found viable throughout the whole process of cheesemaking and aging, with final average loads of 3-4 Log CFU/g at 180 days. On the contrary, the microorganism was not detected, in ripened samples, by traditional plating on M17 medium, suggesting its entering in a viable but not cultivable (VBNC) state. The aroma profiles of the cheeses highlighted the presence of volatile compounds related to cheese flavor as acetoin, diacetyl, 2,3-butanediol and dimethyl disulfide, whose presence was partially correlated to metC and als genes expression. These results add new insights on the capability of L. lactis to persist during late cheese ripening and suggest a potential contribution of the microorganism to cheese flavor formation.

Staphylococcus aureus undergoes major transcriptional reorganization during growth with Enterococcus faecalis in milk.

Previous studies have demonstrated the antagonistic potential of lactic acid bacteria (LAB) present in raw milk microbiota over Staphylococcus aureus, albeit the molecular mechanisms underlying this inhibitory effect are not fully understood. In this study, we compared the behavior of S. aureus ATCC 29213 alone and in the presence of a cheese-isolated LAB strain, Enterococcus faecalis 41FL1 in skimmed milk at 30 °C for 24 h using phenotypical and molecular approaches. Phenotypic analysis showed the absence of classical staphylococcal enterotoxins in co-culture with a 1.2-log decrease in S. aureus final population compared to single culture. Transcriptional activity of several exotoxins and global regulators, including agr, was negatively impacted in co-culture, contrasting with the accumulation of transcripts coding for surface proteins. After 24 h, the number of transcripts coding for several metabolite responsive elements, as well as enzymes involved in glycolysis and acetoin metabolism was increased in co-culture. The present study discusses the complexity of the transcriptomic mechanisms possibly leading to S. aureus attenuated virulence in the presence of E. faecalis and provides insights into this interspecies interaction in a simulated food context.