The development of a multi-omics approach has provided a new approach to the investigation of microbial communities allowing an integration of data, which can be used to better understand the ...behaviour of and interactions between community members. Metagenomics, metatranscriptomics, metaproteomics and metabolomics have the potential of producing a large amount of data in a very short time, however an important challenge is how to exploit and interpret these data to assist risk managers in food safety and quality decisions. This can be achieved by integrating multi-omics data in microbiological risk assessment.
In this paper we identify limitations and challenges of the multi-omics approach, underlining promising potentials, but also identifying gaps, which should be addressed for its full exploitation. A view on how this new way of investigation will impact the traditional microbiology schemes in the food industry is also presented.
•Omics represent valuable tools to produce behavioural and interactions data in microbial communities.•NGS produces data about ecology (metataxonomics), gene presence (metagenomics) and its transcription (metatranscriptomics).•Metagenomic libraries can allow the reconstruction of pathogens genome then used for Genome Scale Metabolic Models (GSMM).•Integrating omics data into MRA will have an impact in food safety, but also to better control spoilage processes.
Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind ...the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in 27 fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three interleukins (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.
Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target ...nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America.
The information collected using DGGE has made it possible to confirm the existing knowledge on food fermentation and spoilage. However, in some cases, new evidence that helps scientists to fully comprehend a specific microbial ecosystem has emerged.
In this review, the roadmap of culture independent methods in food microbiology will be summarized, focusing on the DGGE technique. Examples of how this approach is useful to obtain a better understanding of microbial diversity are reported for several kinds of fermented food, such as fermented sausage, cheese and wine.
The future of culture independent methods in food microbiology, with the increasing availability of next generation sequencing techniques, is also discussed.
•Culture-independent methods are able to better profile complex microbial ecosystems than traditional microbiological methods•The last 20 years have seen an extensive use of DGGE in food microbiology•New insights have been obtained by applying DGGE in food fermentations and food spoilage processes•New generation sequencing will allow to go further in the understanding of microbial ecology in food microbiology
To date, only a few studies have investigated the complex microbiota of table olives in order to identify new probiotic microorganisms, even though this food matrix has been shown to be a suitable ...source of beneficial lactic acid bacteria (LAB). Two hundred and thirty eight LAB, belonging to Lactobacillus plantarum, Lactobacillus pentosus and Leuconostoc mesenteroides species, and isolated from Nocellara Etnea table olives, have been screened in this survey through an in vitro approach. A simulation of transit tolerance in the upper human gastrointestinal tract, together with autoaggregation and hydrophobicity, have been decisive in reducing the number of LAB to 17 promising probiotics. None of the selected strains showed intrinsic resistances towards a broad spectrum of antibiotics and were therefore accurately characterized on an undifferentiated and 3D functional model of the human intestinal tract made up of H4-1 epithelial cells. As far as the potential colonization of the intestinal tract is concerned, a high adhesion ratio was observed for Lb. plantarum O2T60C (over 9%) when tested in the 3D functional model, which closely mimics real intestinal conditions. The stimulation properties towards the epithelial barrier integrity and the in vitro inhibition of L. monocytogenes adhesion and invasion have also been assessed. Lb. plantarum S1T10A and S11T3E enhanced trans-epithelial electrical resistance (TEER) and therefore the integrity of the polarized epithelium in the 3D model. Moreover, S11T3E showed the ability to inhibit L. monocytogenes invasion in the undifferentiated epithelial model. The reduction in L. monocytogenes infection, together with the potential enhancement of barrier integrity and an adhesion ratio that was above the average in the 3D functional model (6.9%) would seem to suggest the Lb. plantarum S11T3E strain as the most interesting candidate for possible in vivo animal and human trials.
The field of microbiome research has evolved rapidly over the past few decades and has become a topic of great scientific and public interest. As a result of this rapid growth in interest covering ...different fields, we are lacking a clear commonly agreed definition of the term "microbiome." Moreover, a consensus on best practices in microbiome research is missing. Recently, a panel of international experts discussed the current gaps in the frame of the European-funded MicrobiomeSupport project. The meeting brought together about 40 leaders from diverse microbiome areas, while more than a hundred experts from all over the world took part in an online survey accompanying the workshop. This article excerpts the outcomes of the workshop and the corresponding online survey embedded in a short historical introduction and future outlook. We propose a definition of microbiome based on the compact, clear, and comprehensive description of the term provided by Whipps et al. in 1988, amended with a set of novel recommendations considering the latest technological developments and research findings. We clearly separate the terms microbiome and microbiota and provide a comprehensive discussion considering the composition of microbiota, the heterogeneity and dynamics of microbiomes in time and space, the stability and resilience of microbial networks, the definition of core microbiomes, and functionally relevant keystone species as well as co-evolutionary principles of microbe-host and inter-species interactions within the microbiome. These broad definitions together with the suggested unifying concepts will help to improve standardization of microbiome studies in the future, and could be the starting point for an integrated assessment of data resulting in a more rapid transfer of knowledge from basic science into practice. Furthermore, microbiome standards are important for solving new challenges associated with anthropogenic-driven changes in the field of planetary health, for which the understanding of microbiomes might play a key role. Video Abstract.
Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamics ...were determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolates were identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45°C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeasts were screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarum and C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.
•Box and heap cocoa bean fermentations were followed in West Africa.•Saccharomyces cerevisiae, Lactobacillus fermentum and Acetobacter pasteurianus were the main species isolated.•Molecular characterization allowed to identify fermentation-specific biotypes of the technological microorganisms.•Through the use of technological tests it was possible to select potential strains as starters for cocoa bean fermentation.
Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of
and
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).
,
,
,
, and
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 (
< 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (
< 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 (
< 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.
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.
The ecology of fermented sausages is complex and includes different species and strains of bacteria, yeasts and molds. The developments in the field of molecular biology, allowed for new methods to ...become available, which could be applied to better understand dynamics and diversity of the microorganisms involved in the production of sausages. Methods, such as denaturing gradient gel electrophoresis (DGGE), employed as a culture-independent approach, allow to define the microbial dynamics during the fermentation and ripening. Such approach has highlighted that two main species of lactic acid bacteria, namely
Lactobacillus sakei and
Lb. curvatus, are involved in the transformation process and that they are accompanied by
Staphylococcus xylosus, as representative of the coagulase-negative cocci. These findings were repeatedly confirmed in different regions of the world, mainly in the Mediterranean countries where dry fermented sausages have a long tradition and history. The application of molecular methods for the identification and characterization of isolated strains from fermentations highlighted a high degree of diversity within the species mentioned above, underlining the need to better follow strain dynamics during the transformation process. While there is an important number of papers dealing with bacterial ecology by using molecular methods, studies on mycobiota of fermented sausages are just a few. This review reports on how the application of molecular methods made possible a better comprehension of the sausage fermentations, opening up new fields of research that in the near future will allow to unravel the connection between sensory properties and co-presence of multiple strains of the same species.
► Molecular methods allow for a better comprehension of sausage fermentation. ► Culture-independent methods are able to avoid biases related to cultivation. ► DGGE is a method often used to study the ecology of fermented sausages. ►
Lb. sakei and
Lb. curvatus dominate in several types of Mediterranean sausages. ► Molecular characterization indicates if inoculated strains take over the fermentation. ► RAPD and Rep-PCR underlined high biodiversity in naturally fermented sausages.
Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape ...juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two
strains displaying metabolic properties, compared to the reference yeast
The analysis of growth and fermentation parameters provided a comprehensive picture of
preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed in
regarding rates, final population and CO
production. In particular, Lys and His supported substantial
growth and fermentation contrary to
while only 3 nitrogen sources, Arg, NH4
and Ser, promoted
growth more efficiently than that of
strains. Furthermore
strains displayed a higher fermentative activity than
during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of
regarding nitrogen sources preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species.
Mixed fermentations combining non-
and
strains are increasingly implemented in the wine sector as they offer promising opportunities to diversify the flavour profile of end-products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of
, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation, through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources, and consequently potential issues during fermentation. The data of this study highlight the importance of an appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-
yeasts.
The aims of this study were to compare the effects of sealing forage corn with a new oxygen barrier film with those obtained by using a conventional polyethylene film. This comparison was made during ...both ensilage and subsequent exposure of silage to air and included chemical, microbiological, and molecular (DNA and RNA) assessments. The forage was inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium and ensiled in polyethylene (PE) and oxygen barrier (OB) plastic bags. The oxygen permeability of the PE and OB films was 1,480 and 70 cm3 m–2 per 24 h at 23°C, respectively. The silages were sampled after 110 days of ensilage and after 2, 5, 7, 9, and 14 days of air exposure and analyzed for fermentation characteristics, conventional microbial enumeration, and bacterial and fungal community fingerprinting via PCR-denaturing gradient gel electrophoresis (DGGE) and reverse transcription (RT)-PCR-DGGE. The yeast counts in the PE and OB silages were 3.12 and 1.17 log10 CFU g–1, respectively, with corresponding aerobic stabilities of 65 and 152 h. Acetobacter pasteurianus was present at both the DNA and RNA levels in the PE silage samples after 2 days of air exposure, whereas it was found only after 7 days in the OB silages. RT-PCR-DGGE revealed the activity of Aspergillus fumigatus in the PE samples from the day 7 of air exposure, whereas it appeared only after 14 days in the OB silages. It has been shown that the use of an oxygen barrier film can ensure a longer shelf life of silage after aerobic exposure.
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