Novel methods of microbiome analysis in the food industry.

The study of the food microbiome has gained considerable interest in recent years, mainly due to the wide range of applications that can be derived from the analysis of metagenomes. Among these applications, it is worth mentioning the possibility of using metagenomic analyses to determine food authenticity, to assess the microbiological safety of foods thanks to the detection and tracking of pathogens, antibiotic resistance genes and other undesirable traits, as well to identify the microorganisms responsible for food processing defects. Metataxonomics and metagenomics are currently the gold standard methodologies to explore the full potential of metagenomes in the food industry. However, there are still a number of challenges that must be solved in order to implement these methods routinely in food chain monitoring, and for the regulatory agencies to take them into account in their opinions. These challenges include the difficulties of analysing foods and food-related environments with a low microbial load, the lack of validated bioinformatics pipelines adapted to food microbiomes and the difficulty of assessing the viability of the detected microorganisms. This review summarizes the methods of microbiome analysis that have been used, so far, in foods and food-related environments, with a specific focus on those involving Next-Generation Sequencing technologies.

Sourdough starter culture microbiomes influence physical and chemical properties of wheat bread.

Sourdough fermentation is an ancient leavening method that uses wild yeasts to produce carbon dioxide, contributing to bread rise, and bacteria which produce organic acids. Sourdough starter cultures are known to be diverse in terms of the microorganisms they comprise and while specific genera and species of microorganisms have been identified from starters and associated with specific attributes, overarching relationships between sourdough starter culture microbiomes and bread quality are not well understood. The objective of this study was to characterize differences in the physical and chemical properties of breads produced with sourdough starter cultures with unique microbiomes. Sourdough starter cultures (n = 20) of known microbial populations were used to produce wheat-based dough and bread, which were analyzed for chemical and physical properties then compared to their microbial populations in order to identify relationships between microbial profiles and dough/bread qualities. All samples were also compared to bread produced only with Saccharomyces cerevisiae (baker's yeast). Significant differences among pH, titratable acidity, loaf volume, crumb firmness, crust color, free amino acids, and organic acids were observed when comparing sourdough breads to the yeast-only control (p ≤ 0.05). Furthermore, bacterial diversity of sourdough starter cultures was correlated with lactic acid and free amino acid in the dough and loaf volume and crumb firmness of baked breads. No significant correlations were found between fungal diversity and measured outcomes. These data demonstrate the importance of considering sourdough starter microbiomes as an ingredient in baked goods and they contribute to quality and safety outcomes in bread production. PRACTICAL APPLICATION: Sourdough starter cultures have diverse and dynamic populations of bacteria and yeasts, which contribute to the production of bread products. These populations can influence the physical and chemical properties of sourdough fermentation and final breads. Understanding of the relationship between sourdough starter microbiomes and bread quality parameters can lead to targeted development of sourdough bread products with specific physical and chemical properties.

Next-Generation Food Research: Use of Meta-Omic Approaches for Characterizing Microbial Communities Along the Food Chain.

Microorganisms exist along the food chain and impact the quality and safety of foods in both positive and negative ways. Identifying and understanding the behavior of these microbial communities enable the implementation of preventative or corrective measures in public health and food industry settings. Current culture-dependent microbial analyses are time-consuming and target only specific subsets of microbes. However, the greater use of culture-independent meta-omic approaches has the potential to facilitate a thorough characterization of the microbial communities along the food chain. Indeed, these methods have shown potential in contributing to outbreak investigation, ensuring food authenticity, assessing the spread ofantimicrobial resistance, tracking microbial dynamics during fermentation and processing, and uncovering the factors along the food chain that impact food quality and safety. This review examines the community-based approaches, and particularly the application of sequencing-based meta-omics strategies, for characterizing microbial communities along the food chain.

Semantics of Dairy Fermented Foods: A Microbiologist's Perspective.

Food ontologies are acquiring a central role in human nutrition, providing a standardized terminology for a proper description of intervention and observational trials. In addition to bioactive molecules, several fermented foods, particularly dairy products, provide the host with live microorganisms, thus carrying potential "genetic/functional" nutrients. To date, a proper ontology to structure and formalize the concepts used to describe fermented foods is lacking. Here we describe a semantic representation of concepts revolving around what consuming fermented foods entails, both from a technological and health point of view, focusing actions on kefir and Parmigiano Reggiano, as representatives of fresh and ripened dairy products. We included concepts related to the connection of specific microbial taxa to the dairy fermentation process, demonstrating the potential of ontologies to formalize the various gene pathways involved in raw ingredient transformation, connect them to resulting metabolites, and finally to their consequences on the fermented product, including technological, health and sensory aspects. Our work marks an improvement in the ambition of creating a harmonized semantic model for integrating different aspects of modern nutritional science. Such a model, besides formalizing a multifaceted knowledge, will be pivotal for a rich annotation of data in public repositories, as a prerequisite to generalized meta-analysis.

Harnessing lactic acid bacteria in synthetic microbial consortia.

Lactic acid bacteria (LAB) are important members in synthetic microbial consortia due to their 'generally recognized as safe' status and diverse metabolic activities. Defined communities with LAB show great potential in elucidating metabolic interactions that drive their assembly and demonstrating power to address sustainability challenges in food, environment, and health.

Microbiome Research as an Effective Driver of Success Stories in Agrifood Systems - A Selection of Case Studies.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Quorum sensing in human gut and food microbiomes: Significance and potential for therapeutic targeting.

Human gut and food microbiomes interact during digestion. The outcome of these interactions influences the taxonomical composition and functional capacity of the resident human gut microbiome, with potential consequential impacts on health and disease. Microbe-microbe interactions between the resident and introduced microbiomes, which likely influence host colonisation, are orchestrated by environmental conditions, elements of the food matrix, host-associated factors as well as social cues from other microorganisms. Quorum sensing is one example of a social cue that allows bacterial communities to regulate genetic expression based on their respective population density and has emerged as an attractive target for therapeutic intervention. By interfering with bacterial quorum sensing, for instance, enzymatic degradation of signalling molecules (quorum quenching) or the application of quorum sensing inhibitory compounds, it may be possible to modulate the microbial composition of communities of interest without incurring negative effects associated with traditional antimicrobial approaches. In this review, we summarise and critically discuss the literature relating to quorum sensing from the perspective of the interactions between the food and human gut microbiome, providing a general overview of the current understanding of the prevalence and influence of quorum sensing in this context, and assessing the potential for therapeutic targeting of quorum sensing mechanisms.

DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid.

Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

The food-gut axis: lactic acid bacteria and their link to food, the gut microbiome and human health.

Lactic acid bacteria (LAB) are present in foods, the environment and the animal gut, although fermented foods (FFs) are recognized as the primary niche of LAB activity. Several LAB strains have been studied for their health-promoting properties and are employed as probiotics. FFs are recognized for their potential beneficial effects, which we review in this article. They are also an important source of LAB, which are ingested daily upon FF consumption. In this review, we describe the diversity of LAB and their occurrence in food as well as the gut microbiome. We discuss the opportunities to study LAB diversity and functional properties by considering the availability of both genomic and metagenomic data in public repositories, as well as the different latest computational tools for data analysis. In addition, we discuss the role of LAB as potential probiotics by reporting the prevalence of key genomic features in public genomes and by surveying the outcomes of LAB use in clinical trials involving human subjects. Finally, we highlight the need for further studies aimed at improving our knowledge of the link between LAB-fermented foods and the human gut from the perspective of health promotion.

Microbiome-Guided Exploration of the Microbial Assemblage of the Exotic Beverage "Insect Tea" Native to Southwestern China.

Insect tea is a unique beverage that is native to Southwestern China and traditionally produced by local farmers in an elaborate process. It consists of insect larvae excrements that are commonly obtained from meal moths (Pyralis farinalis Linnaeus 1758) reared on a specific plant-based diet. We have reconstructed the whole production process under laboratory conditions in order to obtain microbiome-level insights into this uncommon beverage and to trace back the origin of the prevalent bacteria in the final product. The bacterial community composition was specific for each production stage, with a high proportion of Streptomycetacea, Pseudonocaridaceae, Enterococcaceae, and Enterobacteriaceae in the insect tea. A large proportion of the constituents was traced back to the producing insect (13.2%) and its excrements (43.8%), while the initial plant-based substrate for tea production was found to contribute only 0.6% of the traceable bacteria in the final product. Moreover, an enrichment of Enterobactericeae was observed during the analyzed process steps and verified with complementary analyses. The cultivation experiments indicated a high occurrence of viable bacteria in the tea at 2.7 × 105 ± 1.2 × 105 cfu g-1. The isolated bacteria included Bordetella petrii and Enterococcus spp. that were recovered from a commercial product. By implementing an integrative approach, the insect tea was shown to harbor a species-rich bacterial community that can be traced back to certain plant and insect microbiome constituents from distinct production steps. Moreover, the microbial profile of the insect tea was found to be unique for a food product so far and contained several bacterial groups that are considered from the current perspective as food contaminants or yet unreported in other beverages. Due to the high number of viable bacteria, the tea harbors a so far undescribed dynamic component that might have implications for human health.

Application of high-throughput pyrosequencing in the analysis of microbiota of food commodities procured from small and large retail outlets in a U.S. metropolitan area - A pilot study.

With the advent of high-throughput sequencing technologies, it is possible to comprehensively analyze the microbial community of foods without culturing them in the laboratory. The estimation of all microbes inhabiting a food commodity (food microbiota) therefore may shed light on the microbial quality and safety of foods. In this study, we utilized high-throughput pyrosequencing of 16S rRNA genes as well as traditional microbiological methods to evaluate the bacterial diversity and the predicted metabolic pathways associated with the bacterial communities of selected foods (romaine lettuce, cabbage, deli meat, and chicken legs, total 200 samples) procured from small and large retail outlets located in Memphis-Shelby County, Tennessee, USA. For high-throughput sequencing, microbial genomic DNA was directly extracted from the food products and subjected to genetic sequencing. Aerobic plate count of all food samples was also performed. Foods from small stores (such as corner stores) were found to contain higher bacterial counts as compared to large stores (such as supermarkets). High-throughput pyrosequencing in tandem with bioinformatics analyses revealed a comprehensive picture of the bacterial ecology of foods at different taxonomic levels. Firmicutes and Proteobacteria were the most abundant phyla across all products. At the genus level, Enterobacter and Pantoea in vegetables, and Bacillus and Aeromonas in animal products were found to be the most abundant. The bacterial predicted metabolic pathways such as inosine-5'-phosphate biosynthesis I, methylglyoxal (MG) degradation pathways, urea cycle, dTDP-l-rhamnose biosynthesis I, and mevalonate pathway I differed in foods procured from small stores as compared to large groceries or supermarkets. The results from this study revealed that the bacterial ecology (both in terms of numbers and types of bacteria) of food commodities might differ based on the vending outlet type (large vs. small) of retail stores. The overall estimation bacterial communities in foods by high-throughput sequencing method may be useful to identify potential taxa responsible for food spoilage. Moreover, the data from pyrosequencing of 16S rRNA genes can also be applied to infer major metabolic pathways in bacteria inhabiting different foods. This may reflect the role of these pathways in food-bacteria interaction and adaptation.

Regulatory Immune Mechanisms in Tolerance to Food Allergy.

Oral tolerance can develop after frequent exposure to food allergens. Upon ingestion, food is digested into small protein fragments in the gastrointestinal tract. Small food particles are later absorbed into the human body. Interestingly, some of these ingested food proteins can cause allergic immune responses, which can lead to food allergy. So far it has not been completely elucidated how these proteins become immunogenic and cause food allergies. In contrast, oral tolerance helps to prevent the pathologic reactions against different types of food antigens from animal or plant origin. Tolerance to food is mainly acquired by dendritic cells, epithelial cells in the gut, and the gut microbiome. A subset of CD103+ DCs is capable of inducing T regulatory cells (Treg cells) that express anti-inflammatory cytokines. Anergic T cells also contribute to oral tolerance, by reducing the number of effector cells. Similar to Treg cells, B regulatory cells (Breg cells) suppress effector T cells and contribute to the immune tolerance to food allergens. Furthermore, the human microbiome is an essential mediator in the induction of oral tolerance or food allergy. In this review, we outline the current understanding of regulatory immune mechanisms in oral tolerance. The biological changes reflecting early consequences of immune stimulation with food allergens should provide useful information for the development of novel therapeutic treatments.

Recent Past, Present, and Future of the Food Microbiome.

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.

A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies.

The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods.

Bacterial Communities in Boreal Forest Mushrooms Are Shaped Both by Soil Parameters and Host Identity.

Despite recent advances in understanding the microbiome of eukaryotes, little is known about microbial communities in fungi. Here we investigate the structure of bacterial communities in mushrooms, including common edible ones, with respect to biotic and abiotic factors in the boreal forest. Using a combination of culture-based and Illumina high-throughput sequencing, we characterized the bacterial communities in fruitbodies of fungi from eight genera spanning four orders of the class Agaricomycetes (Basidiomycota). Our results revealed that soil pH followed by fungal identity are the main determinants of the structure of bacterial communities in mushrooms. While almost half of fruitbody bacteria were also detected from soil, the abundance of several bacterial taxa differed considerably between the two environments. The effect of host identity was significant at the fungal genus and order level and could to some extent be ascribed to the distinct bacterial community of the chanterelle, representing Cantharellales-the earliest diverged group of mushroom-forming basidiomycetes. These data suggest that besides the substantial contribution of soil as a major taxa source of bacterial communities in mushrooms, the structure of these communities is also affected by the identity of the host. Thus, bacteria inhabiting fungal fruitbodies may be non-randomly selected from environment based on their symbiotic functions and/or habitat requirements.