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See detailShort communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments
Korsak Koulagenko, Nicolas ULg; Taminiau, Bernard ULg; Leclercq, Mathilde et al

in Journal of Dairy Science (in press)

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products ... [more ▼]

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozymaspp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. [less ▲]

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See detailSoil microbial community composition changes according to the tillage practice and plant development stage
Degrune, Florine ULg; Dufrêne, Marc ULg; Taminiau, Bernard ULg et al

Poster (2015, April)

Agricultural practices have a strong impact on soil bacterial and fungal community composition. Furthermore, microbial community composition can change with the stage of plant development. We are ... [more ▼]

Agricultural practices have a strong impact on soil bacterial and fungal community composition. Furthermore, microbial community composition can change with the stage of plant development. We are interested in exploring these effects in relation to changes induced by agriculture (conventional and reduced tillage) and plant stage (germination and flowering) in soil conditions. Here, instead of examining this impact at a high taxonomic level such as phylum and/or class, thus missing potentially relevant information from lower levels, we propose an original method: exploiting the available sequence information at the lowest taxonomic level attainable for each operational taxonomic unit. Results show that some microbial communities were impacted only by the tillage practice , while others were impacted only by the stage of plant. Changes in microbial community composition could be due to the soil conditions induced by the soil practice and the stage of plant. [less ▲]

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See detailA novel sub-phylum method discriminates better the impact of crop management on soil microbial community
Degrune, Florine ULg; Dufrêne, Marc ULg; Colinet, Gilles ULg et al

in Agronomy for Sustainable Development (2015)

Soil microorganisms such as mycorrhizae and plant-growth-promoting rhizobacteria have beneficial effects on crop productivity. Agricultural practices are known to impact soil microbial communities, but ... [more ▼]

Soil microorganisms such as mycorrhizae and plant-growth-promoting rhizobacteria have beneficial effects on crop productivity. Agricultural practices are known to impact soil microbial communities, but past studies examining this impact have focused mostly on one or two taxonomic levels, such as phylum and class, thus missing potentially relevant information from lower levels. Therefore we propose here an original, sub-phylum method for studying how agricultural practices modify microbial communities. This method involves exploiting the available sequence information at the lowest taxonomic level attainable for each operational taxonomic unit. In order to validate this novel method we assessed microbial community composition using 454 pyrosequencing of 16S and 28S rRNA genes, then we compared the results with results of a phylum-level analysis. Agricultural practices included conventional tillage, reduced tillage, residue removal and residue retention. Results show that, at the lowest taxonomic level attainable, tillage is the main factor influencing both bacterial community composition, accounting for 13% of the variation, and fungal community composition, accounting for 18% of the variation. Whereas phylum-level analysis failed to reveal any effect of soil practice on bacterial community composition, and missed the fact that different members of the same phylum responded differently to tillage practice. For instance, the fungal phylum Chytridiomycota showed no impact of soil treatment, while sub-phylum-level analysis revealed an impact of tillage practice on the Chytridiomycota sub-groups Gibberella, which includes a notorious wheat pathogen, and Trichocomaceae. This clearly demonstrates the necessity of exploiting the information obtainable at sub-phylum level when assessing the effects of agricultural practice on microbial communities. [less ▲]

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See detailDetecting microbial patterns in relation to soil agricultural practices and the plant development stage
Degrune, Florine ULg; Dufrêne, Marc ULg; Taminiau, Bernard ULg et al

Poster (2014, December 02)

Agricultural practices have a strong impact on soil bacteria and fungi. Furthermore, microbial community composition can change with the stage of plant development. We are interested in exploring these ... [more ▼]

Agricultural practices have a strong impact on soil bacteria and fungi. Furthermore, microbial community composition can change with the stage of plant development. We are interested in exploring these effects in relation to changes induced by agriculture and plant stage in soil conditions. Some bacteria are influenced only by the plant stage, which induces changes in soil humidity, pH, nitrates, and carbon. We would thus expect these bacteria to be highly sensitive to these parameters. Other bacteria are affected only by the tillage practice applied. Further study is needed to identify the soil parameters responsible for this effect. The plant stage also has a great impact on fungal community composition. [less ▲]

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See detailPredictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality
Delhalle, Laurent ULg; Ellouze, Mariem; Taminiau, Bernard ULg et al

Poster (2014, September 01)

The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods may not be relevant to understand the modifications of the microbial ecology in food products ... [more ▼]

The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods may not be relevant to understand the modifications of the microbial ecology in food products. Metagenomic analysis targeted on 16S ribosomal DNA can elucidate microbial community structures at a muche higher resolution than was previously possible. Combined with predictive microbiological models, a new approach was investigated to take into account bacterial populations dynamics in perishable foods under different environmental conditions. White pudding samples, a typical Belgian pork meat product, were packed under food wrap (atmospheric air condition). Durability studies were conducted at 4°C, 12°C and a dynamic temperature profile according to the NF V01-003 standards (4°C (1/3 of the shelf life) - 8°C (2/3 of the shelf life)) during 15 days. The effect of organic acids was also investigated using a lactic acid (1.8% w/w) treatment. At each day of the trials, classical microbiological (total flora) and 16S rDNA metagenomic analysis were carried out on all these samples. For the metagenomic analysis, a sequencing library was generated, targeting the V1-V3 region of the 16S rDNA. The two major bacterial populations were thus identified (Psychrobacter sp and Brochotrix thermosphacta) and predictive microbiology models used to assess the growth parameters. Cardinal parameters for temperature were collected on the two main bacterial species. The model was validated using the data obtained at a dynamic temperature profile. The results of the simulations for Psychrobacter sp and Brochotrix thermosphacta show a good compliance between predicted and observed data. Compared to culture based methods on selective media and previous independent culture techniques, metagenomic analysis combined with predictive microbiology gives more valuable information, and could be considered as a technological breakthrough to control the quality or for accurately determining shelf life. [less ▲]

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See detailModelling the development of potential spoilage and biopreservative microorganisms on precooked pasta in different conditions of temperature based on classical microbiology and 16S rDNA metagenomic
Gand, Mathieu ULg; Cargnel, Mickaël ULg; Kergourlay, Gilles ULg et al

Poster (2014, September)

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food ... [more ▼]

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food products, it is not always the case with the consumers. Indeed, a few persons reach the right temperature level required for a safe storage of foodstuffs in their refrigerator. Besides, the food can sometimes spend a few hours in ambient temperature between the buying in the supermarket and the storage in cold temperature. In this study, we propose to model the growth of microorganisms on precooked pasta, stored in different conditions of temperature that reflect the situations described above (constant 4°C, constant 8°C, constant 12°C, 1/3 4°C – 2/3 8°C, 1/3 4°C – breach during 4h at 20°C – 2/3 8°C and 1/3 8°C – breach during 4h at 20°C – 2/3 8°C. The product was surface inoculated with potential spoilage and biopreservative strains (Lactococcus piscium, Leuconostoc mesenteroides and Leuconostoc citreum). Analyses by classical microbiology and V1-V3 16S rDNA metagenomics were done each day until the out of date of the food matrix. The transition from 4 to 8°C and the breach at 20°C during 4h have clearly boosted the growth of the microorganisms. The metagenomic analysis was a powerful tool to follow separately each population in each condition of storage. The results of this communication show the importance of keeping the foodstuffs in 4°C or lower in the refrigerator with the goal to avoid the spoilage or the development of pathogens and the potential of metagenomics for selection of biopreservative strains. [less ▲]

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See detailModelling the development of potential spoilage and biopreservative microorganisms on white pudding in different conditions of temperature based on classical microbiology and 16S rDNA metagenomic
Gand, Mathieu ULg; Cauchie, Emilie ULg; Kergourlay, Gilles ULg et al

Poster (2014, September)

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food ... [more ▼]

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food products, it is not always the case with the consumers. Indeed, a few persons reach the right temperature level required for a safe storage of foodstuffs in their refrigerator. Besides, the food can sometimes spend a few hours in ambient temperature between the buying in the supermarket and the storage in cold temperature. In this study, we propose to model the growth of microorganisms on white pudding, stored in different conditions of temperature that reflect the situations described above (constant 4°C, constant 8°C, constant 12°C, 1/3 4°C – 2/3 8°C, 1/3 4°C – breach during 4h at 20°C – 2/3 4°C and 1/3 4°C – breach during 4h at 20°C – 2/3 8°C. The product was surface inoculated with potential spoilage and biopreservative strains (Raoultella terrigena, Serratia quinivorans, Carnobacterium maltaromaticum, Lactobacillus oligofermentans, Lactobacillus nenjiangensis, Lactobacillus fuchuensis, Leuconostoc mesenteroides, lactococcus lactis and Lactobacillus graminis). Analyses by classical microbiology and V1-V3 16S rDNA metagenomics were done each day until the out of date of the food matrix. The transition from 4 to 8°C and the breach at 20°C during 4h have clearly boosted the growth of the microorganisms. The metagenomic analysis was a powerful tool to follow separately each population in each condition of storage. The results of this communication show the importance of keeping the foodstuffs in 4°C or lower in the refrigerator with the goal to avoid the spoilage or the development of pathogens and the potential of metagenomics for selection of biopreservative strains. [less ▲]

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See detailHigh throughput sequencing analysis reveals genetic variability and selection pressure in different murine norovirus genomic regions during in vitro replication
Mauroy, Axel ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2014, July)

Murine norovirus (MuNoV), a single stranded positive sense RNA virus belonging to the Caliciviridae family, is considered as a representative model for human norovirus infections, one of the most ... [more ▼]

Murine norovirus (MuNoV), a single stranded positive sense RNA virus belonging to the Caliciviridae family, is considered as a representative model for human norovirus infections, one of the most important etiological cause of both epidemic and sporadic gastroenteritis cases worldwide. Four open reading frames are described into its genome: ORF1 codes the non-structural (NS) proteins, including the viral RNA dependent RNA polymerase (RdRp); ORF2 codes the single capsid protein (VP1), wherein two domains are present: a relatively conserved domain (“shell”) and a more variable domain (“protruding”); ORF3 codes a minor structural protein; and ORF4, currently only found in viruses genetically related to MuNoV codes a virulence factor. In this study, we demonstrated by high throughput sequencing that, during serial passages of MuNoV in cell culture, the substitution rates, estimated by Bayesian inferences, did not significantly differ across the five targeted genomic regions except one. These rates were similar in four genomic regions encompassing partial non-structural 1-2 protein (NS1-2)-, NS5-, NS6-, NS7 (RdRp)- and VP1-coding sequences (coding the conserved part of the protein also including the ORF4 region). In the partial minor structural protein-coding region, this substitution rate was however estimated to be at least one log higher when expressed as substitution/site/day. The precise localisation of the detected nucleotide point mutations (substitution, deletion and insertion) were reported as well as the quantitative increase or decrease of the sequences harbouring them along ten cell culture passages. The non-silent amino acid mutations were also depicted in 3D models for four out of the five studied regions. These results have important implications for different norovirus research fields, especially in terms of diagnosis, classification methodology and genetic evolution. [less ▲]

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See detailStudy of the microbial flora of steak tartare by metagenomic approach
Korsak Koulagenko, Nicolas ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2014, May 06)

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See detailL'analyse par séquençage à haut débit révèle la variabilité génétique et la pression de sélection dans différentes régions génomiques du norovirus murin durant sa réplication in vitro
Mauroy, Axel ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Conference (2014, March)

Le norovirus murin (MuNoV), un virus à ARN de polarité positive appartenant à la famille des Caliciviridae, est considéré comme un modèle adéquat pour les infections humaines à norovirus, une des causes ... [more ▼]

Le norovirus murin (MuNoV), un virus à ARN de polarité positive appartenant à la famille des Caliciviridae, est considéré comme un modèle adéquat pour les infections humaines à norovirus, une des causes étiologiques les plus importantes dans les cas de gastroentérite épidémique ou sporadique dans le monde entier. Quatre cadres de lecture ouverts (ORF) sont décrits au sein de son génome : l’ORF1 code les protéine non structurales (NS), dont l’ARN polymérase ARN dépendante virale (RdRp) ; l’ORF2 code l’unique protéine de capside (VP1), dans laquelle sont décrites deux régions : une relativement conservée (domaine « shell ») et une autre beaucoup plus variable (domaine « protruding ») ; l’ORF3 code une protéine structurale mineure ; et l’ORF4, actuellement uniquement décrit chez les virus génétiquement apparentés au MuNoV, code un facteur de virulence. Dans cette étude, nous démontrons par séquençage à haut débit que, durant des passages successifs du MuNoV en culture cellulaire, les taux de substitution, estimés par inférences Bayésiennes, n’ont pas significativement différé au travers des cinq régions génomiques ciblées à l’exception d’une région bien précise. Ces taux étaient similaires pour quatre régions englobant des séquences partielles codant les protéines non structurales NS1-2, NS5, NS6 et NS7 (RdRp) et VP1 dans sa région conservée (incluant également l’ORF4). Dans la région codant partiellement la protéine structurale mineure, ce taux de substitution, exprimé en substitution/site/jour, a été cependant estimé être plus élevée d’au moins une unité logarithmique. La localisation précise des mutations ponctuelles détectées (substitution, délétion et insertion) est rapportée ainsi que l’augmentation ou la diminution quantitative du nombre des séquences qui les présentaient au cours de dix passages successifs en culture cellulaire. Les localisations des mutations non silencieuses ont aussi été représentées dans une modélisation tridimensionnelle de quatre des cinq régions étudiées. Ces résultats ont d’importantes implications pour différents champs de recherche sur les norovirus, spécialement en termes de diagnostic, de méthodologie de classification et d’évolution génétique. [less ▲]

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See detailPsychrotrophic lactic acid bacteria associated with production batch recalls and sporadic cases of early spoilage in Belgium between 2010 and 2014
Pothakos, Vasileios; Taminiau, Bernard ULg; Huys, Geert et al

in International Journal of Food Microbiology (2014), 191

Between 2010 and 2014 several spoilage cases in Belgium occurring in retail foodstuffs prior to the end of shelf-life have been reported to our laboratory. Overall, seven cases involved strictly ... [more ▼]

Between 2010 and 2014 several spoilage cases in Belgium occurring in retail foodstuffs prior to the end of shelf-life have been reported to our laboratory. Overall, seven cases involved strictly psychrotrophic lactic acid bacteria (LAB) contamination in packaged and chilled-stored food products. The products derived either fromrecalls of entire production batches or as specimens of sporadic spoilage manifestations. Some of these samples were returned to the manufacturing companies by consumers who observed the alterations after purchasing the products. The products covered a wide range of foodstuffs (i.e. meat, dairy, vegetable, egg products and composite food) and denoted different spoilage defects. However, the microbiota determined by means of 16S rRNA gene high throughput sequencing analysis underpin few LAB genera (i.e. Leuconostoc, Lactobacillus, Weissella and Lactococcus), which are frequently encountered nowadays as specific spoilage organisms (SSO) albeit overlooked by mesophilic enumeration methods due to their strictly psychrotrophic character. The present study confirms the spreading of psychrotrophic LAB in Belgian food processing environments leading to unexpected spoilage, corroborating their spoilage dynamics and prevalence in all kinds of packaged and refrigerated foodstuffs in Northern Europe. [less ▲]

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See detailMicrobiota characterization of a protected designation of origin Belgian cheese: Herve cheese, using metagenomic analysis.
Delcenserie, Véronique ULg; Taminiau, Bernard ULg; Delhalle, Laurent ULg et al

in Journal of Dairy Science (2014), 97

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or ... [more ▼]

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclettetype cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese. [less ▲]

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See detailImpact of agricultural practices on soil microbial communities in Belgium
Degrune, Florine ULg; Taminiau, Bernard ULg; Dufrêne, Marc ULg et al

Poster (2013, December 11)

The use of fertilizers in agricultural soils is becoming a real environmental issue (an obvious example is eutrophication caused by leaching of phosphorus and nitrates). Much research has focused on ... [more ▼]

The use of fertilizers in agricultural soils is becoming a real environmental issue (an obvious example is eutrophication caused by leaching of phosphorus and nitrates). Much research has focused on finding ways to reduce the use of chemicals, and investigating microbial life may lead to solutions. We know that bacteria and fungi are deeply involved in nutrient cycles. Recently the emergence of massive parallel sequencing has enabled us to realize that microbial diversity is huger than we expected. With such a tool it should be possible to study how soil management practices affect the microbial diversity of agricultural soils. A few such studies have been conducted, most of them focusing on bacteria. For Belgium in particular, there is a lack of data on this topic. Here the aim was to see how residue management and tillage practices affect communities of both bacteria and fungi in Belgian agricultural soils. For this we used 454 pyrosequencing of 16S bacterial and 28S fungal rRNA genes. Soil samples came from an experiment in which faba beans were grown with four soil management practices (tillage and no tillage, with and without crop residues), each repeated four times in a Latin square. Several chemical and physical characteristics were measured on each sample. The results show that fungi and bacteria are both impacted by Tillage practices. The main soil drivers are Magnesium and Phosphorus for Fungi communities, and Phosphorus and Potassium for bacteria communities. Finally, the fungi variance observed between plots is explained at 38% by Tillage, Magnesium and phosphorus. And the bacteria variance is explained at 28% by Tillage, Phosphorus and Potassium. [less ▲]

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See detailEvaluation of the bacterial diversity and its evolution during storage of fresh beef from British and Belgian origins under different atmosphere and temperature conditions
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Conference (2013, December 10)

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature, by using a metagenomic ... [more ▼]

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature, by using a metagenomic approach. Metagenomic analyzes revealed that the origin, atmosphere and temperature conditions influenced the selection of the predominant flora. Vacuum-packaged British samples presented higher concentrations of Lactobacillus algidus at the begging of the experiment than Belgian samples. Furthermore, the development of Lactobacillus algidus was favored in British and Belgian samples preserved under vacuum at −1 °C, while a predominance of Lactococcus piscium was observed for samples stored at +4 °C. These microorganisms have already been isolated from beef, but taking into account that the knowledge about these two species is currently limited, it is still not possible to state if the conservability of the tested samples was influenced by the presence of these bacteria. Moreover, storage under modified atmosphere favored the development of Leuconostoc gasicomitatum in both British and Belgian samples. This specie is often associated with spoilage of cold-stored modified-atmosphere-packaged (MAP) nutrient-rich foods. This result can partially explain the short shelf-life of the samples once they are stored under this condition. Metagenomics showed to be a useful tool to study the microbial population of a complex matrix since some of the identified genera could not have grown or have grown slowly in culture media commonly used. In addition, it helped to clarify the evolution of the bacterial ecosystem associated to meat during its storage. [less ▲]

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See detailBacterial diversity and its evolution during storage of fresh beef from different origins under different atmosphere and temperature conditions
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Poster (2013, October 11)

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature. Two batches of three vacuum ... [more ▼]

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature. Two batches of three vacuum packed striploins from United Kingdom and Belgium were obtained from a food wholesaler located in the Walloon Region. Fifteen days after slaughter, the striploins were sliced and individually kept under vacuum for 30 days: i) at −1 °C; ii) at +4 °C and iii) at −1 °C for 15 days and then at +4 °C for 15 days. The bacterial diversity was evaluated by metagenomic approach 15, 30 and 45 days after slaughter. Furthermore, each 15 days part of the vacuum packed striploin slices were repacked under modified atmosphere (70 % O2/30 % CO2), stored at +4 °C for 2 days and at +8 °C for 5 days, and then analyzed. Metagenomic analysis revealed a selection of the initial flora depending on atmosphere and temperature conditions. The development of Lactobacillus algidus was favored in samples preserved under vacuum at −1 °C, while a predominance of Lactococcus piscium was observed for samples stored at +4 °C. Moreover, storage under modified atmosphere favored the development of Leuconostoc gasicomitatum. These microorganisms have already been isolated from beef, but no study has evaluated their role in food conservation. The next step of this study will be to isolate and characterize strains of Lactobacillus algidus from meat and to assess their bioprotective potential. [less ▲]

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See detailPredictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Ellouze, Mariem et al

in Ellouze, Mariem; Tenenhaus-Aziza, Fanny (Eds.) Proceedings of thge 8th International Conference on predictive microbiology in foods (ICPMF8) (2013, September 18)

OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These ... [more ▼]

OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These techniques are simple to implement but may not be relevant to understand the modifications of the microbial ecology which occur in the food product in response to different changes in the environmental conditions. Metagenomic analysis targeted on 16S ribosomal DNA can bring about a solution to this new need and elucidate microbial community structures, including the identification and quantification of culturable and non-culturable organisms, at a much higher resolution than was previously possible with culture-based methods to provide a picture of the microbial community. Combined with predictive microbiological models, a new approach was investigated to take into account the dynamics of the evolutions of the microbial community in food products. This work describes the application of a metagenomic analysis and predictive microbiology in order to study bacterial populations dynamics in perishable foods under different environmental conditions. METHODS White pudding samples, a typical Belgian pork meat product, were packed under food wrap (atmospheric air condition). Durability studies were conducted at 4°C, 12°C and a dynamic temperature profile according to the NF V01-003 standards (4°C (1/3 of the shelf life) - 8°C (2/3 of the shelf life)) during 15 days. The effect of organic acids was also investigated using a lactic acid / diacetic acid mix (1.8% w/w) treatment. At each day of the trials, classical microbiological (total flora, lactic acid bacteria) and 16S rDNA metagenomic analysis were carried out on all these samples. For the metagenomic analysis, a sequencing library was generated, targeting the V1-V3 region of the 16S rDNA. Libraries were sequenced on a GS junior sequencer using Titanium technology. The Bio- informatic pipeline using Mothur, Blast and Stamp was used to assign a taxonomical identity to the sequences and to obtain the bacterial population proportions of the samples (Schloss, Westcott et al. 2009). The major bacterial populations were thus identified and predictive microbiology models (Baranyi and Roberts 1994; Augustin, Zuliani et al. 2005) were used to assess the growth parameters. The model was validated using the data obtained at a dynamic temperature profile. RESULTS The metagenomic analysis of the samples shows that the bacterial populations from the day 0 sample to the post-shelf life sample have important modifications. Brochothrix and Psychrobacter were identified as the dominant flora. As expected, the storage temperature had a strong impact on the  bacterial evolutions. Moreover, the use of lactic acid/diacetic acid reveals the sensitivity of the different populations to the treatment. For the storage at 4°C, the initial dominance of Pseudomonas and Shewanella is slightly reduced during storage until shelf life, after which it drops to be replaced by Brochothrix and Psychrobacter. The addition of the preservation treatment has a statistical negative impact on the Psychrobacter and Acinetobacter populations. During the ageing assay (2 days at 4°C followed by 10 days at 8°C), the analysis underlines the influence of the temperature change on the onset of the Brochothrix and Psychrobacter dominance compared to the entire 4°C storage. Again, the preservation treatment delays this onset. Finally, at an abusive 12°C temperature, samples are quickly dominated by the Psychrobacter/Brochothrix pair after 2 days of storage. In this case, the lactic acid mix does not appear to be of any effective use. Adjustment of primary model was made on the major bacterial populations and simulation was made based on estimated growth rate. The simulations of the three major populations seem to be sufficient for this food product to predict 80 -90 % of the bacterial population at the end of the shelf life in function of the environmental conditions. CONCLUSIONS AND IMPACT OF THE STUDY Compared to culture based methods on selective media and previous independent culture techniques, metagenomic analysis combined with predictive microbiology gives more valuable information, and its use could be considered as a technique for quality control or for accurately determining shelf life. REFERENCES Augustin, J. C., V. Zuliani, et al. (2005). "Growth rate and growth probability of Listeria monocytogenes in dairy, meat and seafood products in suboptimal conditions." J Appl Microbiol 99(5): 1019-­‐1042. Baranyi, J. and T. A. Roberts (1994). "A dynamic approach to predicting bacterial growth in food." International Journal of Food Microbiology 23(3-­‐4): 277-­‐294. Schloss, P. D., S. L. Westcott, et al. (2009). "Introducing mothur: open-­‐source, platform-­‐independent, community-­‐supported software for describing and comparing microbial communities." Appl. Environ. Microbiol. 75(23): 7537-­‐7541. [less ▲]

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See detailMetagenomic analysis targeted on the 16S ribosomal DNA to study the quality of meat : a example with raw minced beef meat
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2013, July 01)

Introduction: Steak tartare is a popular meat dish in Belgium and some other European countries. This meat preparations due to their raw nature, is highly sensitive to bacterial spoilage. A better ... [more ▼]

Introduction: Steak tartare is a popular meat dish in Belgium and some other European countries. This meat preparations due to their raw nature, is highly sensitive to bacterial spoilage. A better understanding of the bacterial content of this product will thus be insightful to control the risk of spoilage. Metagenomics targeted on the 16S ribosomal DNA has appeared as a powerful tool to study bacterial composition of food samples. The aim of this study is to identify the bacterial population sof steak tartare from different origin along their shelf life. Material and methods: A total of 59 samples were analysed from seven butcheries, six restaurants, six sandwich bars, 8 supermarkets without intern butcheries and 8 supermarkets with intern butcheries. Samples where directly analysed the day of receipt (day 0) and at the end their shelf life after storage at 4°C (day 2), except for six restaurants and sandwich bars who were analysed only at day 0. Classical microbiological analyses were performed in order to determine psychotrophic aerobic colony counts using modified ISO 4833 method. Metagenomic analysis targeting the 16S rDNA was performed using the Roche GS junior. Raw sequences were treated by bioinformatics in order to obtain identification and proportion of bacteria in food sample. Results: Remarkable differences appear between the origins of steaks tartare. The bacterial concentration is between 3 and 7 log CFU/g depending of the origin and the day of analysis. The samples from the butcheries are mainly composed of Lactobacillus populations and to a lesser extend of environmental contaminants like Xanthomonas campestris. On the opposite, the samples from some of the restaurants are contaminated with an estimated level of 6 to 7 log CFU/g of Brochotrix thersmosphacta, Leuconostocaceae like Leuconostoc carnosum or an uncultured Weissella sp., or, with a lesser extend, with some contaminants like Pseudomonas sp. or Psychrobacter sp. These last samples were characterized with some spoilage characteristics (slime, off odor) that can thus be put in relation with the identified bacterial populations. The samples from sandwich bars were characterized by a lower level of bacterial population (3-4 log CFU/g), but with a greater diversity in the microflora along with a higher number of environmental contaminants that are not usually found in meat products. The products at the end of the shelf life have a higher bacterial concentration but with a lower diversity with spoiled bacteria as Brochotrix thermosphacta. Significance: Compared to culture based methods on selective media and previous independent culture techniques, metagenomic analysis combined with the enumeration of psychrotrophic flora gives more valuable information, and its use should be considered as a technique for quality control or for accurately determining the shelf life and the quality of the meat. [less ▲]

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See detailLa diversité bactérienne et son évolution pendant la conservation de viandes bovines fraîches de différentes origines conditionnées sous vide
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Poster (2012, December)

Le but de cette étude à été d'évaluer la diversité bactérienne et son évolution pendant la conservation de viandes bovine fraîches sous vide, en fonction de leur origine et du respect ou non d’une ... [more ▼]

Le but de cette étude à été d'évaluer la diversité bactérienne et son évolution pendant la conservation de viandes bovine fraîches sous vide, en fonction de leur origine et du respect ou non d’une température proche du point de congélation. Les dénombrements réalisés ont mis en évidence que les viandes d’origines britannique et belge testées présentent un écosystème microbien différent. Les analyses par approche métagénomique permettront d’éclaircir ces différences, surtout en ce qui concerne la présence de bactéries pouvant jouer un rôle "bioprotecteur" permettant d’améliorer la conservabilité des viandes. [less ▲]

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See detailValidation de la qualité de la viande hachée de porc par une approche métagénomique ciblée
Taminiau, Bernard ULg; Nezer, Carine; Adolphe, Ysabelle ULg et al

Conference (2012, November 13)

Food products represent great biotopes for bacteria. The optimization of foodstuffs conservation pass by a better understanding of those biotopes and their spoilage. The current techniques of new ... [more ▼]

Food products represent great biotopes for bacteria. The optimization of foodstuffs conservation pass by a better understanding of those biotopes and their spoilage. The current techniques of new generation sequencing give a new dimension to the microbial ecology, through the metagenomic analysis of individuals' large number, within a mixed microbial population. Our aim is to demonstrate that this methodology can be successfully applied to the validation of the quality of foodstuffs during storage. This study was carried out on pork minced meat with shelf-life tests in various conditions of preservation (temperature and packaging). The analysis was performed in parallel with standardized microbiological methods and with massive sequencing of two hypervariables regions of the rDNA 16S. The results show an excellent correlation between the two approaches and underline the tremendous utility of metagenomic analysis for in-depth characterization of the potential altering bacteria in fresh meat. [less ▲]

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See detailViandes bovines à longue durée de conservation conditionnées sous vide : isolement et caractérisation de souches de Carnobacterium
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Baptista Rodrigues, Ana Lucia ULg et al

Poster (2012, November 13)

The lactic acid bacteria Carnobacterium divergens and Carnobacterium maltaromaticum are often associated to meat and meat products and may be used as a protective culture, improving the microbial ... [more ▼]

The lactic acid bacteria Carnobacterium divergens and Carnobacterium maltaromaticum are often associated to meat and meat products and may be used as a protective culture, improving the microbial stability and the safety of these products. In this context, the aim of this study was to isolate and characterize Carnobacterium from long shelf-life vacuum-packed beef. LAB counts after culture at +22°C remained below 2.0 log UFC/cm², even at the end of shelf life. On the other hand, the ecosystem evaluation performed by metagenomics revealed the predominance of Carnobacterium and Lactobacillus on the samples. After spreading of a peptone water suspension obtained from the samples on PCA, pure isolates were collected and identified by API 50 CHL galleries. Seventy-eight % of isolates were C. maltaromaticum, 3 % C. divergens and 19 % could not be identified. The next step of this work will consist in performing a genotypic and functional characterization of these Carnobacterium isolates. [less ▲]

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