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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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See detailL’analyse métagénomique ciblée au service de la microbiologie des aliments : applications concrètes
Taminiau, Bernard ULg; Nezer, Carine; Delhalle, Laurent ULg et al

Conference (2012, September 21)

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See detailMetagenomic analysis as a tool to better characterize the bacterial content of food and food preparations.
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Conference (2012, September 04)

Metagenomic analysis is a new culture-independent approach for assigning a taxonomic, genic or functional identity to bacterial DNA fragments of unknown origin. Its power and utility is increasingly ... [more ▼]

Metagenomic analysis is a new culture-independent approach for assigning a taxonomic, genic or functional identity to bacterial DNA fragments of unknown origin. Its power and utility is increasingly rising thanks to the next generation sequencing techniques. It is now mature and cheap enough to be transposed to more applied fields like the food microbiology. We demonstrated in several studies the extraordinary potential of the targeted metagenomic analysis to different problematics related to food products. First, this approach is highly useful for the validation of the shelf life of food products. We analyzed standardized pork minced meat and meat product samples packaged either under modified atmosphere (MAP - 30% CO2, 70% O2) or under permeable atmosphere packaging, stored at different temperature (4°C, 4-8°C and 12°C) until the end of shelf-life. The metagenomic analysis allowed to identify species of all the sub-dominant bacterial populations. This approach showed why MAP can improve meat quality by favoring certain species rather than others. As a second example, we sought to identify the potential spoiling bacteria in several food products like raw fish, rind cheese or vacuum packed beef meats in order to illustrate the usefulness of metagenomics for the quality control of food preparations. Samples from various food matrices were screened to identify the bacterial contaminants. We combined the bioinformatics analysis with a classical approach to generate effective quantitative data for the various bacterial populations detected. This analysis characterizes the samples both on the identity of the potential spoiling bacteria present and on the quantification level of the contaminants. Finally, the metagenomic analysis reveals the presence of numerous uncultured and uncharacterized bacteria. The use of a carefully designed analysis pipeline has been used to ensure to label the bacterial population with a precise taxonomic identity and to determine whether the targeted population corresponds to a known species or not. This way, even if the nearest known homologous sequence is an environmental sample, its relatedness to known species can be deduced. This represents a new tool to trace yet uncharacterized food spoiling bacteria. [less ▲]

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See detailMolecular identification of the bacterial populations of steak tartare, a raw consumed meat preparation: a practical use of targeted metagenomic analysis
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2012, September 04)

Steak tartare is a popular meat dish in Belgium and other european countries. It is often consumed with french fries or as sandwich spread. This product, due to its raw nature, is highly sensitive to ... [more ▼]

Steak tartare is a popular meat dish in Belgium and other european countries. It is often consumed with french fries or as sandwich spread. This product, due to its raw nature, is highly sensitive to bacterial alteration. A better understanding of the bacterial content of this meat product will thus be insightful to master the alteration hazards. Throughout a targeted metagenomic analysis we characterized the bacterial populations of several steak tartare samples. These samples were bought and analyzed during the same day, from three different commercial sources: butchery, sandwich vendor and restaurant. A classic microbiological analysis was performed in parallel. The metagenomic analysis was targeted on two different hypervariable regions of the bacterial 16S rDNA, in order to compare the bacterial identification efficiency. A total of 60,500 sequences for 12 samples were submitted to a metagenomic analysis. The best hypervariable region enabled us to identify 356 different bacterial species. Lactobacillus algidus is the leading bacterial species, representing 52% of the total analyzed sequences, followed by an uncultured Pseudomonas sp. (8.43%) and Photobacterium phosphoreum (7.92%). The analysis of the results shows that remarkable differences appear between the three sources of steak tartare. First, the samples from the butchery are mainly composed of Lactobacillus populations and to a lesser extend of environmental contaminants like Xanthomonas campestris. On the opposite, the samples from the restaurant are contaminated with higher level of Leuconostocaceae like Leuconostoc carnosum or an uncultured Weissella sp., or with gamma-proteobacteria like Pseudomonas sp. or Psychrobacter sp. These last samples were characterized with some alteration (slime, off odor) that can thus be put in relation with the bacterial populations identified. Combining a broad-range sequencing effort to a rigorous computer analysis gives a powerful tool for the microbiology of food products. Its application can be virtually extended to every food product be readily transposed to the food industry. [less ▲]

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See detailThe extraordinary potential of metagenomic tools for food microbiology: an example with bacterial microbiota of raw and pasteurized milk cheeses
Delhalle, Laurent ULg; Nezer, Carine; Taminiau, Bernard ULg et al

Poster (2012, September 03)

Among the culture-independent techniques, ultra-sequencing has contributed to place metagenomic analysis as the best alternative to study complex microbiota. During the last three years, metagenomic ... [more ▼]

Among the culture-independent techniques, ultra-sequencing has contributed to place metagenomic analysis as the best alternative to study complex microbiota. During the last three years, metagenomic studies were used essentially for environmental samples but it could be used also to analyse bacterial populations of food samples. This work describes the application of this technique to study the bacterial population of different types of soft cheeses. Among these, three of them are a typical Belgian soft cheese with washed rind (two with raw milk and the third with pasteurized milk). The fourth is a French creamy soft cheese made with raw milk. Classical microbiological and 16S rDNA metagenomic analysis were carried out in the core and on the rind of the four cheeses, giving a total of 8 samples. In total, 48 genus and 163 species were identified for all samples. As expected Lactoccocus lactis and/or cremoris are the most representative species in the core of the four cheeses. On the rind of cheeses, the predominant bacterial species are Psychrobacter glacinola, Staphylococcus equorum, Corynebacterium casei and Marinilactibacillus psychrotolerans. Brevibacterium spp and Psychroflexus spp are important for the rind of washed rind cheeses. All these species are present in different proportions following the origin and the cheese making process and they are well known for their organoleptic properties on the rind of cheese. The two Belgian soft cheese made with raw milk are composed of many more different bacterial species. While the cheese made from pasteurized milk contains less species mainly composed by Lactococcus lactis (97,6%) in the core. An unexpected result is the low diversity of the a French creamy soft cheese made with raw milk with only two predominent species : Lactoccocus cremoris and Leuconostoc citreum are present in the core (94,9% and 4,9% , respectively) and on the rind (93,8% and 5% , respectively). Compared with the other cheeses made with raw milk, this result is surprising. The bacterial cheese microbiota plays a central role in cheese-making. The subtleties of cheese character, as well as their shelf-life, are largely determined by the evolution of their microbiota. [less ▲]

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See detailA new approach of food microbiology with the metagenomic tools: an application on fish
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2012, September 03)

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. Its interest has started to appear in food microbiology but only on the study of very ... [more ▼]

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. Its interest has started to appear in food microbiology but only on the study of very particular bacterial populations of fermented food. 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). Regardless the packaging and the fish origin, significant variations of the initial flora were noted. The important growth of some Gram negative populations could indicate a risk of spoilage. Thus, the metagenomic approach could be used to adequately determine the duration of shelf-life. 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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See detailLes modèles de microbiologie prévisionellepour la maitrise de la sécurité des aliments (synthèse bibliographique)
Delhalle, Laurent ULg; Daube, Georges ULg; Adolphe, Ysabelle ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2012), 16(3), 369-381

Predictive microbiology aims to predict the evolution of microorganisms in foods with mathematical models. Several models have been published and the complexity of some of them makes their use difficult ... [more ▼]

Predictive microbiology aims to predict the evolution of microorganisms in foods with mathematical models. Several models have been published and the complexity of some of them makes their use difficult for the uninitiated. However, the use of this discipline will become widespread in coming years. These models provide, for example, additional tools to ensure the microbiological safety of food, to establish the contamination flow in a food chain, to develop and to assist the quality assurance systems. The development of new computer software and database will enable stakeholders in the food chain to have a better control of microbiological hazards. The aim of this summary is to give an overview of existing models of predictive microbiology and their applications. A first approach of the primary, secondary and tertiary models is given. The modelling of latency, integrated models and growth tests are also discussed. [less ▲]

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See detailEtude de la flore bactérienne de steacks tartares prélevés en boucherie, sandwicherie et restaurant par analyse métagénomique ciblée sur l’ADN ribosomial 16S.
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2012, May 24)

Etude de la flore bactérienne de steacks tartares prélevés en boucheries, sandwicheries et restaurants par analyse métagénomique ciblée sur l’ADN ribosomial 16S. Taminiau B.1, Delhalle L 2, Nezer C.2 ... [more ▼]

Etude de la flore bactérienne de steacks tartares prélevés en boucheries, sandwicheries et restaurants par analyse métagénomique ciblée sur l’ADN ribosomial 16S. Taminiau B.1, Delhalle L 2, Nezer C.2, Daube G 1 1 Université de Liège, Faculté de Médecine Vétérinaire, Département des Sciences des Denrées Alimentaires ; Sart Tilman B43 Bis, 4000 Liège, Belgique ; T+32 (0)4 366 40 29 / F+32 (0)4 366 40 44 2 Quality Partner sa, 62 Rue Hayeneux, 4040 Herstal, Belgique ; T+32 (0)4 240 75 00 / F+32 (0)4 240 75 10 Bernard.taminiau@ulg.ac.be Le steak tartare est une préparation à base de viande hachée de boeuf crue assaisonnée et souvent additionnée de condiments. Cette préparation est souvent accompagnée de frites ou étalée dans un sandwich. L’utilisation de viande crue et la composition de cet aliment rend cette préparation très sensible à l’altération d’origine bactérienne. Une meilleure compréhension de la flore bactérienne composant ce produit est indispensable pour contrôler et comprendre les phénomènes d'altération. L'analyse métagénomique ciblée a permis de caractériser les populations bactériennes de plusieurs échantillons de steak tartare achetés dans deux boucheries, deux sandwicheries et deux restaurants et analysés le jour-même. Une analyse classique microbiologique a été réalisée en parallèle. L'analyse métagénomique a été ciblée sur deux régions différentes de l'ADNr 16S bactérien (V&-V3 et V5-V6), afin de comparer l'efficacité d'identification des bactéries présentes. L’analyse métagénomique a permis de collecter un total de 60.500 séquences pour les 6 échantillons analysés par les deux approches et 356 espèces bactériennes différentes ont pu être identifiées via la région V1-V3. Lactobacillus algidus est la principale espèce présente (52% des séquences totales analysées), suivie par Pseudomonas sp. (8,43%) et Photobacterium phosphoreum (7,92%). L'analyse des résultats montre des différences remarquables entre les trois sources commerciales de steak tartare. Les échantillons provenant des deux boucheries et d’un restaurant étaient principalement composés de populations de Lactobacillus et, dans une moindre mesure, de contaminants environnementaux, comme Xanthomonas campestris. Les échantillons provenant de l'un des deux restaurants étaient fortement contaminés par des Leuconostocaceae comme Leuconostoc carnosum ou Weissella sp., ou avec des gamma-protéobactéries comme Pseudomonas sp. ou Psychrobacter sp. Ces derniers échantillons présentaient aussi des signes d’altérations (mauvaise odeur, aspect) qui peuvent ainsi être mis en relation avec la nature et le niveau des populations bactériennes identifiées. Les échantillons provenant des sandwicheries et probablement issues de filières de production industrielles étaient faiblement contaminés mais avec une grande variabilité dans les flores identifiées. Beaucoup d’entre elles ne sont pas classiquement décrites dans les viandes crues et doivent provenir des autres ingrédients. La combinaison du séquençage haut débit couplé à la bioinformatique est désormais un outil puissant pour l’analyse microbiologique des denrées alimentaires. Son application peut être étendue à pratiquement tous les aliments. Enfin, cette technologie ouvre de nouvelles perspectives aux industries agro alimentaires pour améliorer leurs procédés de fabrication, leurs recettes et leurs méthodes de conservation. [less ▲]

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See detailL’analyse métagénomique ciblée au service de la microbiologie des aliments : applications concrètes
Taminiau, Bernard ULg; Nezer, Carine; Delhalle, Laurent ULg et al

Conference (2012, May 23)

La métagénomique est une discipline récente qui s’attache à attribuer une identité, taxonomique, génique ou fonctionnelle, à des fragments d’ADN d’origine inconnue. Son développement et son utilité ... [more ▼]

La métagénomique est une discipline récente qui s’attache à attribuer une identité, taxonomique, génique ou fonctionnelle, à des fragments d’ADN d’origine inconnue. Son développement et son utilité bénéficient grandement de l’essor des techniques de séquençage de seconde génération permettant d’obtenir d’un échantillon de grandes quantités de séquences d’ADN. L’écologie microbienne et la santé intestinale sont les thématiques ayant le plus approfondi la métagénomique et les possibilités qu’elle offre, mais d’autres domaines peuvent grandement bénéficier de cette nouvelle façon d’investiguer les flores microbiennes. Cette étude démontre, à travers plusieurs exemples concrets, que cette méthodologie peut rapidement être adaptée aux exigences spécifiques de la microbiologie des aliments. En effet, les denrées alimentaires périssables, comme la viande et les préparations à base de viande, représentent des biotopes de choix pour les bactéries. L’optimisation de la conservation de ces denrées, importante tant du point de vue économique que de la santé publique, passe par une meilleure connaissance de l’évolution de ces flores et des processus de détérioration des qualités du produit. Les microbiologistes ont depuis longtemps abordé ce problème en utilisant différentes approches dépendantes ou non de la culture microbienne, mais à petite échelle. L’analyse métagénomique est donc une rupture technologique qui allie analyse à grande échelle et large spectre. Un premier exemple a permis de démontrer la capacité de l’analyse métagénomique à identifier les populations bactériennes sous-dominantes dans des produits de viande (viande hachée de porc et boudin blanc). La sensibilité de l’analyse effectuée permet d’identifier les populations microbiennes dès la sortie de production et de comprendre leurs évolutions en fonction de différentes conditions de conservation lors de tests de vieillissement. De plus, la possibilité d’utiliser des échantillons standardisés a servi à comparer plusieurs zones hypervariables de l’ARNr 16S ainsi que d’étudier la reproductibilité de l’analyse. Ensuite, l’impact de la conservation sous-vide de longue durée de la viande bovine a été investigué pour des échantillons d’origines diverses. L’analyse a révélé des différences très marquées malgré un conditionnement apparemment identique. Enfin, nous avons exploré la capacité de cette méthodologie à identifier les bactéries potentiellement responsables d’altérations dans trois matrices alimentaires : le steak tartare, le poisson cru et le fromage. Si l’identification des germes à large spectre est actuellement efficace et reproductible comme nous l’avons démontré ; plusieurs axes de recherche restent à approfondir pour améliorer son utilité dans l’industrie agro-alimentaire. Parmi ceux-ci, il y a la capacité à transposer cette méthodologie pour d’autres micro-organismes comme les moisissures et les champignons, la détermination du degré de profondeur d’analyse utile en fonction du but recherché, et enfin, l’organisation de la manne d’informations qui sont générées par la métagénomique. [less ▲]

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See detailL’extraordinaire potentiel de l’approche métagénomique en microbiologie des aliments: analyse du microbiote de fromages au lait cru et pasteurisé
Delhalle, Laurent ULg; Nezer, Carine; Darcis, Amélie et al

Poster (2012, May 22)

Parmi les techniques culture indépendante, le développement de l’ultra-séquençage a permis de positionner l’analyse métagénomique comme étant la meilleure alternative pour l’étude de microbiotes complexes ... [more ▼]

Parmi les techniques culture indépendante, le développement de l’ultra-séquençage a permis de positionner l’analyse métagénomique comme étant la meilleure alternative pour l’étude de microbiotes complexes. Durant ces trois dernières années, les études métagénomiques ont été consacrées essentiellement à l’analyse d’échantillons environnementaux. Ce travail décrit l’application de cette technique à l’étude des populations bactériennes de quatre types de fromages à pâte molle. Parmi ceux-ci, trois d’entre eux sont des fromages belges typiques à croute lavée (deux sont fabriqués avec du lait cru et le troisième avec du lait pasteurisé). Le quatrième est un fromage français crémeux à base de lait cru. Des analyses microbiologiques classiques et métagénomiques ciblant l’ADNr 16S ont été réalisées dans le cœur et sur la croute des quatre fromages, donnant un total de 8 échantillons. Au total, 48 genres et 163 espèces bactériennes ont été identifiées pour tous les échantillons. Comme attendu, Lactoccocus lactis subsp lactis et/ou cremoris sont les espèces les plus représentées dans le cœur des quatre fromages. Concernant la croute des fromages, les espèces bactériennes les plus abondantes sont Psychrobacter glacinola, Staphylococcus equorum, Corynebacterium casei et Marinilactibacillus psychrotolerans. A noter la présence de Brevibacterium spp et Psychroflexus spp qui permettent d’obtenir la couleur orangée de la croute. Toutes ces espèces sont présentes en différentes proportions suivant l’origine et le processus de fabrication et sont connues pour leurs propriétés technologiques et/ou organoleptiques. Les deux fromages belges au lait cru sont composés de beaucoup d’espèces bactériennes différentes. Tandis que le fromage au lait pasteurisé contient moins d’espèces, principalement Lactococcus lactis (97,6%) dans le cœur. Un résultat inattendu concerne la faible diversité bactérienne du fromage français crémeux à base de lait cru. Seulement deux espèces étaient majoritairement présentes : Lactoccocus lactis subsp cremoris et Leuconostoc citreum avec respectivement 94,9% et 4,9% dans le cœur et 93,8% et 5% dans la croute). Comparé avec les résultats des autres fromages à base de lait cru, ce résultat est particulièrement surprenant. Le microbiote des fromages joue un rôle central sur le processus de fabrication, les qualités organoleptiques et la durée de vie commerciale des produits. L’analyse métagénomique est devenu un outil puissant, rapide et abordable pour identifier, comprendre et maitriser les flores impliquées [less ▲]

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See detailUne nouvelle approche de la microbiologie alimentaire avec l’analyse métagénomique : un exemple avec une analyse de filets de poisson
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2012, May 22)

L’analyse métagénomique est un outil puissant pour étudier la flore bactérienne d’échantillons issus de divers environnements. Cette technologie commence à être appliquée à des aliments mais seulement sur ... [more ▼]

L’analyse métagénomique est un outil puissant pour étudier la flore bactérienne d’échantillons issus de divers environnements. Cette technologie commence à être appliquée à des aliments mais seulement sur l’étude de produits asiatiques fermentés. Ce travail décrit l’application de cette technologie pour étudier la population bactérienne de deux types de filets de poissons : un issu d’un poisson d’eau douce chaude (pangasius) et un de poisson d’eau de mer (églefin). Les échantillons ont été directement analysés le jour de la réception. D’autres échantillons ont été analysés à la fin de leur durée de vie après stockage à 4°C (1/3 de la durée de vie) et 8°C (2/3 de la durée de vie). Ces échantillons ont été conditionnés en barquette de polystyrène et film étirable sous air atmosphérique et en barquette et film PP-EVOH sous atmosphère modifiée (50% N2 / 50% CO2). Des analyses microbiologiques classiques et métagénomiques ciblant l’ADNr 16S ont été réalisés sur tous les échantillons. L’évolution des populations microbiennes des filets de poissons conservés sous différents types de conditionnement et de température a été étudiée. Quarante espèces bactériennes différentes ont été identifiées pour les deux types de poissons. Les bactéries Gram négatives sont majoritaires que ce soit au début ou à la fin de la durée de vie des produits et quel que soit le type de conditionnement. Au début de la conservation, les bactéries Gram négatives présentes sont essentiellement des Moraxellaceae (Acinetobacter spp, Psychrobacter sp.), Pseudomonadaceae (Pseudomonas spp), et Shewanella spp et les bactéries Gram positives sont des Lactobacillaceae (Carnobacterium spp), Brochothrix thermosphacta et Planococcus donghaensis (seulement pour le pangasius). Suivant le type de conditionnement et le type de poisson, des variations importantes de la flore initiale ont été observées. La croissance de certaines espèces Gram négatives pourrait être un indicateur d’altération. Par conséquent, l’analyse métagénomique pourrait être un outil supplémentaire pour fixer adéquatement la date limite de consommation. Pour le pangasius, Planococcus donghaensis est présent uniquement avant le conditionnement et sa présence pourrait être un indicateur de la fraîcheur du poisson. Cette étude a permis d’appliquer l’analyse métagénomique pour identifier et mesurer les proportions relatives des espèces bactériennes dans les filets de poissons au cours du temps. [less ▲]

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See detailDevelopment of a quantitative risk assessment for cheese made from raw goat milk contaminated by Listeria monocytogenes
Delhalle, Laurent ULg; Ellouze, Mariem; Clinquart, Antoine ULg et al

Poster (2011, September)

A retrospective study was performed to assess the potential risk of human listeriosis following a contamination by L. monocytogenes of cheeses made from goat raw milk reported by the Belgian Federal ... [more ▼]

A retrospective study was performed to assess the potential risk of human listeriosis following a contamination by L. monocytogenes of cheeses made from goat raw milk reported by the Belgian Federal Agency for the Safety of the Food Chain in 2005. The source of the contamination was related to a shedder goat, excreting 2.6 log cfu (colonies forming units) L. monocytogenes / ml without any clinical symptom. On the basis of the collected data, a quantitative microbial risk assessment model was developed covering the production chain from the milking of goats until the consumed products. Predictive microbiology models were used to simulate the growth of L. monocytogenes during the process of cheeses made from goat raw milk. The modular exposure assessment model showed a significant growth of L. monocytogenes during chilling and storage of the milk collected the day before the cheese production (increase of 1.7 log cfu/ml for the median) and during the step of starter and rennet adjunction to milk (increase of 0.8 log cfu/ml for the median). The median estimated final result (in the fresh cheese) was equal to 3.5 log cfu/g. The model estimates (expressed as median final result issued from the exposure assessment) were realistic compared to the number of L. monocytogenes measured in the fresh cheese (3.6 log cfu/g) reported during the cheese contamination period. The average number of expected cases of human listeriosis was between 0 and 1 for a high-risk sub-population and 0 for a low-risk healthy sub-population. Scenario analysis was finally performed to identify the most significant factors and aid in developing priorities for risk mitigation. Thus, by using quantitative risk assessment and predictive microbiology models, this study provided valuable information to identify and to control critical steps in a local production chain of goat cheese made from raw milk. [less ▲]

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See detailCase study of contamination by Listeria monocytogenes in raw goat milk cheese: development of a quantitative risk assessment model of the production chain
Korsak Koulagenko, Nicolas ULg; Delhalle, Laurent ULg; Daube, Georges ULg

Conference (2011, August 03)

Introduction: Quantitative Risk assessment could be applied in industries as a tool to control and manage the safety of food products. Purpose: A contamination by Listeria monocytogenes of cheeses made ... [more ▼]

Introduction: Quantitative Risk assessment could be applied in industries as a tool to control and manage the safety of food products. Purpose: A contamination by Listeria monocytogenes of cheeses made from raw milk was reported by the Belgian food agency. This contamination was caused by the presence of an asymptomatic “shedder” goat in the herd. With field and laboratory collected data, a quantitative risk assessment model of the production chain was developed. Methods: A modular risk model was built to simulate the food production pathway covering the milking of goats until the final product for the customers. A dynamic square root model was used to predict the growth rate in relation with the temperature, the pH and the water activity along the production chain with predictive microbiology modules. Results: The shedder goat was identified from the herd and milk samples were taken from the two different parts of the mammary gland with 2.6 log cfu (colonies forming units) Listeria monocytogenes/ml for the right part and absence in 25 ml for the left part of the mammary gland. Numbering of Listeria monocytogenes was carried out on the final products with 3.6 log cfu/g in the fresh not ripened cheeses. The modular risk assessment shows a significant growth of Listeria monocytogenes during chilling and storage of the milk collected the day before the cheese production (increase of 0.6 log cfu/ml) and during adjunction of ferment and rennet to milk (increase of 0.8 log cfu/ml). The model confirms the results obtained in the final products. Significance: The modular risk model gives valuable informations to identify and to control critical steps in the food production chain of goat cheese made from raw milk. Only one shedder goat can cause a high risk to become ill for the consumers of contaminated products. [less ▲]

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