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See detailExploring the bacterial diversity of Belgian steak tartare using metagenetics and qPCR analysis
Delhalle, Laurent ULg; Korsak Koulagenko, Nicolas ULg; Taminiau, Bernard ULg et al

in Journal of Food Protection (2016), 79(2), 200-229

Steak tartare is a popular meat dish in Belgium. It is prepared with raw ground minced beef and eaten with sauce, vegetables, and spiced. Since it contains raw meat, steak tartare is highly prone to ... [more ▼]

Steak tartare is a popular meat dish in Belgium. It is prepared with raw ground minced beef and eaten with sauce, vegetables, and spiced. Since it contains raw meat, steak tartare is highly prone to bacterial spoilage. The objective of this study was to explore the bacterial flora diversity in steak tartare in Belgium according to the source and to determine which bacteria are able to grow during the shelf life. A total of 58 samples from butchers’ shops, restaurants, sandwich shops and supermarkets were collected. These samples were analyzed using 16S rDNA metagenetics, a classical microbiological technique, and quantitative real-time PCR (qPCR) targeting the Lactobacillus genus. Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops analyzed only at the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. But only seven bacterial species were predominant in the samples, depending on the source: Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp. and Xanthomonas oryzae. With this work, an alternative method is proposed to evaluate the total flora in food samples based on the number of reads from metagenetic analysis and the results of qPCR. The degree of underestimation of aerobic plate counts (APCs) at 30°C estimated with the classical microbiology method was demonstrated in comparison with the proposed culture independent method. Compared to culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information for characterizing the bacterial flora of raw meat. [less ▲]

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See detailMetagenomic analysis of samples
Daube, Georges ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Patent (2015)

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See detailMethod of testing for animal-derived ingredients in foods
Daube, Georges ULg; Burteau, Sophie; Nezer, Carine et al

Patent (2015)

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See detailEvaluation of the microbiota of foods with metagenetics
Delhalle, Laurent ULg

Conference (2015, March 12)

An overivew of the -omics technologies applied for the agri food sector

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See detailEvualation of the microbiota in foods with metagenetics
Delhalle, Laurent ULg

Conference (2015)

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See detailCecal drop reflects the chickens' cecal microbiome, fecal drop does not.
Pauwels, J.; Taminiau, Bernard ULg; Janssens, Guy ULg et al

in Journal of microbiological methods (2015)

Microbiota in the gastro-intestinal tract are closely related to both the intestinal and overall health of the host. Experimental chickens have always been euthanized in order to identify and quantify the ... [more ▼]

Microbiota in the gastro-intestinal tract are closely related to both the intestinal and overall health of the host. Experimental chickens have always been euthanized in order to identify and quantify the bacteria in cecal content. In this study, quantification and identification of the microbial populations in cecal drop, cecal content and fecal drop samples from chickens showed that cecal drop contains a bacterial community that is very similar (concerning bacterial diversity, richness and species composition) to cecal content, as opposed to the bacterial community found in fecal drop. Cecal drop analysis thus allows for longitudinal experiments on chickens' cecal bacteria. The varying results in the analysis of fecal samples questions the method's reliability in reflecting the true cecal microbiota in chickens. [less ▲]

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See detailThermophilic and cellulolytic consortium isolated from composting plants improves anaerobic digestion of cellulosic biomass: toward a microbial resource management approach
Kinet, Romain ULg; Destain, Jacqueline ULg; Hiligsmann, Serge ULg et al

in Bioresource Technology (2015)

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas ... [more ▼]

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas production, was observed for the cultures inoculated with the exogenous consortium. Metagenomics analyses pointed out a weak richness (related to the number of OTUs) of the exogenous consortium induced by the selective pressure (cellulose as sole carbon source) met during the initial isolation steps. Main microbial strains determined were strictly anaerobic and belong to the Clostridia class. During cellulose anaerobic degradation, pH drop induced a strong modification of the microbial population. Despite the fact that richness and evenness were very weak, the exogenous consortium was able to adapt and to maintain the cellulolytic degradation potential. This important result point out the fact that simplified microbial communities could be used in order to increase the robustness of mixed cultures involved in environmental biotechnology. [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 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 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 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 detailRetrospective Analysis of a Listeria monocytogenes Contamination Episode in Raw Milk Goat Cheese Using Quantitative Microbial Risk Assessment tools
Delhalle, Laurent ULg; Ellouze, Mariem; Yde, Marc et al

in Journal of Food Protection (2012), 75(12), 2122-2135

In 2005, the Belgian authorities reported a Listeria monocytogenes contamination episode in cheese made from raw goat's milk. The presence of an asymptomatic shedder goat in the herd caused this ... [more ▼]

In 2005, the Belgian authorities reported a Listeria monocytogenes contamination episode in cheese made from raw goat's milk. The presence of an asymptomatic shedder goat in the herd caused this contamination. On the basis of data collected at the time of the episode, a retrospective study was performed using an exposure assessment model covering the production chain from the milking of goats up to delivery of cheese to the market. Predictive microbiology models were used to simulate the growth of L. monocytogenes during the cheese process in relation with temperature, pH, and water activity. The model showed significant growth of L. monocytogenes during chilling and storage of the milk collected the day before the cheese production (median increase of 2.2 log CFU/ml) and during the addition of starter and rennet to milk (median increase of 1.2 log CFU/ml). The L. <br /><br />monocytogenes concentration in the fresh unripened cheese was estimated to be 3.8 log CFU/g (median). This result is consistent with the number of L. monocytogenes in the fresh cheese (3.6 log CFU/g) reported during the cheese contamination episode. A variance-based method sensitivity analysis identified the most important factors impacting the cheese contamination, <br /><br />and a scenario analysis then evaluated several options for risk mitigation. Thus, by using quantitative microbial risk assessment tools, this study provides reliable information to identify and control critical steps in a local production chain of cheese made from raw goat's milk. [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 detailStudy of the microbial flora of freshwater and seawater fish filets in different packaging conditions by metagenomic analysis targeted on the 16S ribosomal DNA
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Conference (2012, October 19)

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. This work describes the application of this technique to study the bacterial population of two ... [more ▼]

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. This work describes the application of this technique to study the bacterial population of two fresh fish filets. The two fish species are from freshwater (pangasius) and seawater (haddock), respectively. Samples where directly analyzed the day of receipt. Others samples were analyzed at the end their shelf life after storage at 4°C (1/3 of their shelf life) and 8°C (2/3 of their shelf life). For these samples, packagings were made in plastic wrap for atmospheric air condition and in trays under modified atmosphere. Classical microbiological and 16S rDNA metagenomic analysis were carried out on all these samples. The composition and evolution of microbial populations of fish filet stored under different packaging conditions and temperatures of storage were investigated with identification of bacteria species. A total of 40 different species were identified for both fish types. Gram-negative bacteria are always predominated among the initial flora and at the end of the shelf life in all the trials. At the beginning of storage, the predominant Gram-negative microflora consisted of Moraxellaceae (Acinetobacter spp, Psychrobacter spp.), Pseudomonadaceae (Pseudomonas spp), and Shewanella spp and the Gram-positive flora was identified as Lactobacillaceae (Carnobacterium spp), Brochothrix thermosphacta and Planococcus donghaensis (only for pangasius). For the pangasius, Planococcus donghaensis is only present before the fish is packed and its dominant presence could provide an indication of the freshness of the fish. The metagenomic analysis is a useful tool to identify and to measure the relative proportions of bacterial species in fish filet samples. [less ▲]

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