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Caseinophosphopeptides (CPPs) are bioactive peptides originating from proteolysis of caseins, the main proteins of milk. In this study, the generation of CCPs during Beaufort cheese making and by in vitro simulated gastro-intestinal hydrolysis using pepsin and pancreatin were assessed using selective precipitation and liquid chromatography–electrospray ionisation tandem mass spectrometry. Seventy-two water-soluble CPPs, mainly originating from β-casein, were identified in Beaufort cheese, while 79 CPPs, mainly generated from α s1 -caseins, were obtained from enzymatic hydrolysates. Most of the peptides generated by the action of digestive enzymes were monophosphorylated; however, 17 out of the 23 polyphosphorylated CPPs identified from digested Beaufort cheese still contained the characteristic cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu, providing calcium-binding properties to CPPs. The content of CPPs in Beaufort cheese before and after the action of digestive enzymes was estimated to be 230 and 275 mg per 100 g of fresh cheese, respectively. The action of proteolytic digestive enzymes on CPPs is discussed.

We analyzed 178 Streptococcus thermophilus strains isolated from diverse products, from around the world, over a 60-year period with a new multilocus sequence typing (MIST) scheme. This collection included isolates from two traditional cheese-making sites with different starter-use practices, in sampling campaigns carried out over a three years period. The nucleotide diversity of the S. thermophilus population was limited, but 116 sequence types (ST) were identified. Phylogenetic analysis of the concatenated sequences of the six housekeeping genes revealed the existence of groups confirmed by eBURST analysis. Deeper analyses performed on 25 strains by CRISPR and whole-genome analysis showed that phylogenies obtained by MLST and whole-genome analysis were in agreement but differed from that inferred by CRISPR analysis. Strains isolated from traditional products could cluster in specific groups indicating their origin, but also be mixed in groups containing industrial starter strains. In the traditional cheese-making sites, we found that S. thermophilus persisted on dairy equipment, but that occasionally added starter strains may become dominant. It underlined the impact of starter use that may reshape S. thermophilus populations including in traditional products. This new MLST scheme thus provides a framework for analyses of S. thermophilus populations and the management of its biodiversity.

Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α s1 -casein. The aim of this work was to investigate the proteolytic activity of 2 L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α s1 - and α s2 -caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis.

Understanding the origin of the overconsumption of too high levels of sucrose, sodium chloride and lipids in foods raises the question of the influence of the hedonics for these sensations. To better understand this relationship, a sensory tool that enables measurement of liking towards sweet, salty or fatty sensations is required. This liking towards a sensation has to be understood as an overall attractiveness of the sensation. Instruments already existing were unsatisfactory as including a limited number of foods not representative of the overall sensation. A set of hedonic tests, named PrefSens, was developed to measure an overall liking for fatty, salty or sweet sensations. Each test consisted in the rating on a 9-point hedonic scale of a food product at 5 different levels of lipids, sodium chloride or sucrose. To build the PrefSens test, a total of 144 food ranges were tested during the development step ( n = 341 subjects) including various food categories, matrices and serving temperatures. Then, based on the technical feasibility and ability to discriminate, 32 food ranges were selected (10 for fatty, 10 for salty and 12 for sweet). The perceived intensity of fatty, salty or sweet “sensations” of these selected foods was evaluated by a trained panel ( n = 12) using Spectrum® scales. During the application study, the hedonic evaluation of the selected foods was assessed in 6 tasting sessions by 567 subjects over 8 laboratories spread out in France. For each subject and each product, hedonic ratings ( y ) were fitted versus the level of fatty, sweet or salty ( x ) in a quadratic regression, from which the predicted optimal level (Lpref) was derived based on the x -coordinate of the parabola’s maximum weighted by the correlation coefficient between observed and predicted data. The overall liking score for fatty, salty or sweet sensations was the average Lpref over the compounding food ranges. The sensory profile showed that within each sensation the mean perceived intensity significantly increased in a linear manner with the nutrient level. An exception was for the two lowest levels of lipids which were not significantly perceived differently. Concomitantly, the overall liking scores followed an inverted U-shape centered on the middle level of lipids, sodium chloride or sucrose. The distribution was close to normality. Internal validity and consistency of the items compounding the sensations of fatty, salty or sweet were demonstrated. PrefSens is an internally valid and original tool that can be applied to a population sample in order to better understand the determinants of dietary behaviors.

Oiling-off is essential for the culinary functional properties of hard- and semi-hard cheeses. However, the parameters implied in the mechanisms leading to oiling-off are not well known. In Emmental cheese, a marked change in the supramolecular organization of fat occurs during the pressing of the warm curd and could be related to the oiling-off of cheese. To check this hypothesis and to determine the relative effects of the temperature and pressing strength, twin-cheeses were submitted to various cooling rates after the curd have been dipped from the vat. These cheeses were either pressed or non-pressed. Using this strategy, we showed that a higher heat load yields to larger inclusions of fat in the cheese matrix, which has been characterised using confocal laser scanning microscopy, and to higher oiling-off. The heat load applied to the curd (integral of the temperature vs. time curve) and oiling-off of cheeses aged of 5 days were linearly related with r2 = 0.89. In contrast, the pressing strength did not influence oiling-off. As a conclusion, the heat load applied during pressing and acidification could be a tool to modulate the oiling-off of Swiss cheese.

The aim of this study was to investigate the mechanism of the observed inhibition of Listeria monocytogenes by the natural biofilm microflora (BM) on wooden shelves used in the ripening of a soft and smear cheese. For this, BM was harvested and we conducted a series of experiments in which two strains of L. monocytogenes were co-cultured with BM on glass fiber filters deposited on model cheeses. Compared to monoculture, L. monocytogenes growth rate in co-culture was not reduced but the growth of the pathogen stopped as soon as BM entered the stationary phase. This reduction in maximum population density can be explained by nutrient consumption and exhaustion by BM as no production of inhibitors by BM has been detected. This mechanism of pathogen inhibition has been previously described as the "Jameson effect".

This review is respectfully dedicated to the memory of Michel Ollivon, Research Director at CNRS (Châtenay-Malabry, France), outstanding physico-chemist specialist of lipid organization, recipient of the Hilditch Memorial Lecture award, who was the initiator of the network RMT LISTRAL. We are also sadly paying tribute to Jean-Luc Vendeuvre, Food Engineer at the French Pork and Pig Institute (IFIP, Maisons-Alfort, France), outstanding expert in meat products who participated actively in RMT LISTRAL and provided unpublished data for figures in the present review, who passed away during review submission. RMT LISTRAL: Mixed Technological Network combining academic and industrial partners, devoted to the enhancement and divulgation of knowledge regarding structured dietary lipids.; International audience; On a nutritional standpoint, lipids are now being studied beyond their energy content and fatty acid (FA) profiles. Dietary FA are building blocks of a huge diversity of more complex molecules such as triacylglycerols (TAG) and phospholipids (PL), themselves organised in supramolecular structures presenting different thermal behaviours. They are generally embedded in complex food matrixes. Recent reports have revealed that molecular and supramolecular structures of lipids and their liquid or solid state at the body temperature influence both the digestibility and metabolism of dietary FA. The aim of the present review is to highlight recent knowledge on the impact on FA digestion, absorption and metabolism of: (i) the intramolecular structure of TAG; (ii) the nature of the lipid molecules carrying FA; (iii) the supramolecular organization and physical state of lipids in native and formulated food products and (iv) the food matrix. Further work should be accomplished now to obtain a more reliable body of evidence and integrate these data in future dietary recommendations. Additionally, innovative lipid formulations in which the health beneficial effects of either native or recomposed structures of lipids will be taken into account can be foreseen.

Dossier technique ; Enils = Ecoles Nationales de l'Industrie Laitière; National audience

Dossier Technique n° 26 d' ACTILAIT; Lactobacillus helveticus possède un équipement protéolytique complexe incluant non seulement une multitude de peptidases intracellulaires bien connues, mais aussi une ou plusieurs protéases de paroi plus méconnues. Quel est l’impact de ces enzymes et de leur biodiversité sur la protéolyse et les propriétés fonctionnelles des fromages à pâte pressée ? C’est l’une des thématiques majeures de l’Unité Mixte de Technologie Caséolis de Rennes. Voici quelques résultats qui illustrent comment les protéases de paroi des lactobacilles peuvent modifier les propriétés filantes des fromages.

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log 10 CFU · g −1 ) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

Chapitre 1.; National audience

Proteolytic milk-clotting enzymes are extracted from various sources (animals, plants, fungi) and processed according to various methods that are specific to each manufacturer or cheese-maker. Chemical composition and polypeptide patterns of 24 milk-clotting preparations from animal and fungal sources: 10 commercial rennets, 9 artisanal calf rennets, 2 recombinant chymosin preparations and 3 microbial preparations, were compared in order to identify differences according to both their manufacturing process and their source. The preparation from Cryphonectria parasitica had the highest ammonia and small peptide content. Commercial rennets and preparations from Rhizomucor miehei had the highest NaCl and pH values while artisanal rennets had the lowest and recombinant chymosins were intermediate. In comparison with the other commercial preparations, commercial rennets had the highest variability in chemical composition and their polypeptide profiles showed numerous protein bands ranging from 15 kDa to 197 kDa, like artisanal rennets. Protein composition of commercial rennets revealed the presence of bovine serum albumin, either native or degraded, and degraded chymosin. The results indicated that the source of coagulating enzymes and the conditions applied for enzyme extraction led to specific chemical compositions, polypeptide patterns and protein composition which are described in this article. (C) 2012 Elsevier Ltd. All rights reserved.

Au cours des procédés de transformation des produits laitiers, l'hydrolyse des protéines est un des phénomènes biochimiques essentiels pour obtenir les caractéristiques finales tant sensorielles, nutritionnelles que santé, via les peptides produits. Ces peptides sont produits sous l'action de multiples enzymes, d'origine variée, présentes dans le lait (plasmine), ou ajoutées comme auxiliaire technologique (chymosine) ou issues des flores bactériennes. Si l'action des protéases telles la plasmine et la chymosine ont d'ores et déjà été bien étudiées, des études sont en cours pour mieux connaitre les protéases bactériennes localisées en surface et les peptidases intracellulaires qui sont relarguées lors de la mort des bactéries. Nous proposons au cours de cette conférence de montrer en quoi ces peptides interviennent de manière cruciale dans les propriétés sensorielle (texture et flaveur), et sur la santé humaine au travers des peptides bioactifs, et quelles sont les limites pour contrôler la production de ces peptides in situ. Ces peptides étant de mieux en mieux caractérisés, nous présenterons les dernières avancées en termes d'identification et de quantification des peptides dans des produits laitiers au moyen de techniques émergentes de type "omic" qui permettront à terme de comprendre l'élaboration et l'évolution de la qualité des produits laitiers.

Over decades, the peptide fractions produced during cheese ripening have been more and more deeply described(HPLC, tandem mass spectrometry) even if their precise assessment remains challenging. As reviewed here, thisfraction is absolutely crucial in many cheese properties like flavor and/or texture, ecosystem regulation, and humanhealth via bioactive peptides. Actually, besides their well-known role in increasing bacterial growth rate, peptides areimplied in cell-cell communications between bacteria, in a positive (synergy) or negative (antimicrobial peptides) way.They also participate in building the casein network in the dairy matrices and are involved in their structural changesduring ripening, conferring various final textural properties like stretching. Finally, they are the most important sourcesof bioactive potentialities.However, the control of both the in situ release of peptides from caseins by proteinases, and their further hydrolysisby peptidases, is far from being achieved. Many proteinases, either from milk and coagulant agent or from bacterialorigins, are required to produce the first peptides in cheese. Plasmin and chymosin activities are well-studied, howevermany questions remain on the action of the bacterial proteinases. Moreover, the evolution of the peptide profile afterbacterial consumption during growth is only partially understood, in balance with the activity of intracellular peptidasesreleased through lysis.Actually, many bottlenecks exist. It is impossible to have a complete view of the peptides present in situ at each ripeningtime and to correlate this fraction with specific properties. It would require first the quantification of each peptideand second to take into account the interactions between them and the casein network. Lastly, very few are knownalso regarding their diffusion in dairy matrices. Peptidomics, metabolomics and new quantitative proteomic methods(iTRAQ) could be a future opportunity to bridge the gap between identification, quantification of peptides and certaincheese functionalities.

La conception hygiénique au service de la sécurité sanitaire de l'hygiène des matériaux

Textes issus des travaux du programme Casdar "Innovation et Partenariat" de 2007 et présentés lors d'un colloque le 4 décembre 2012, sous l'égide du GIS Relance Agronomique; National audience; The practice of using native ferments, in particular the use of whey in lactic technology (transplanting), is a guarantee of the specificity of the products of the farm and is therefore considered by transformers producers as essential to be maintained. But, multifactorial management relying upon empirical practices, is difficult, and sometimes encourages producers to give up this practice. The references acquired in this project resulted in an approach of diagnosis and advice to perpetuate the use of whey. Investigations and experiments of this program showed the essential role of acidification to these problems of transplanting. They also revealed the crucial importance of milk microbial diversity and of microbial balance management. A USB key contains all documents from this program in three operational parts for technicians: providing a technical support, training producers and some "to go further" which include literature syntheses but above all the syntheses of the results obtained in the different actions of the project.; La pratique consistant à utiliser des ferments indigènes, en particulier l’utilisation de lactosérum en technologie lactique (repiquage), est garante de la spécificité des produits de la ferme et est donc considérée par les éleveurs transformateurs comme essentielle à maintenir. Or, sa maîtrise, de nature multifactorielle et reposant sur des pratiques empiriques, est difficile, incitant parfois les producteurs à abandonner cette pratique. Les références acquises dans ce projet ont permis d’aboutir à une démarche de diagnostic et de conseil visant à pérenniser l’utilisation du lactosérum. Les enquêtes et expérimentations de ce programme ont montré le rôle essentiel de la conduite de l’acidification face à ces problèmes de repiquage. Elles ont aussi révélé l’importance déterminante qu’ont la diversité microbienne des laits et la gestion des équilibres microbiens. Une clé USB regroupe les documents issus de ce programme en 3 parties opérationnelles pour les techniciens : réaliser un appui technique, former les producteurs, et une partie « pour aller plus loin » qui regroupe des synthèses bibliographiques mais surtout les synthèses des résultats obtenus dans les différentes actions du projet.

La surconsommation d'aliments gras, salés ou sucrés est nuisible à la santé, et, même si elles sont entendues du public, les recommandations nutritionnelles visant à éviter ces comportements à risque restent trop partiellement suivies. On peut alors s’interroger sur les déterminants de ces comportements alimentaires et plus spécifiquement sur le plaisir apporté par la consommation des aliments considérés comme gras, salés ou sucrés. En effet, les matières grasses, le sucre et le sel induisent les propriétés sensorielles de l’aliment (goût, texture, palatabilité) et contribuent au plaisir lors de la consommation, parfois en contradiction avec les préoccupations nutritionnelles. Entre le plaisir et le respect des règles de nutrition, et sous l’influence des pouvoirs publics, les industries agro-alimentaires s’engagent dans des chartes nutritionnelles pour réduire la teneur en gras, sel, sucre, mais restent très prudentes. L’enjeu est de parvenir à réduire ces niveaux, lorsque la technologie le permet, sans détériorer les propriétés organoleptiques des produits, et en restant en accord avec la dimension plaisir de la consommation. La connaissance des préférences des consommateurs en matière de gras, salé et sucré est indispensable dans une telle démarche. Mais les outils épidémiologiques habituels, en se basant sur les niveaux de consommation, offrent plus un reflet et de l’offre du marché que des préférences. Ils proposent des méthodes fondées sur des questionnaires, afin de mettre en évidence les surconsommations de gras, sel, ou sucre, mais aucunes permettant la mesure quantitative du plaisir lié à la consommation de ces produits...

For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log(10) loss of culturability whereas qPCR on pelleted cells revealed only one log(10) of decrease for both of these species. 10(9) ST and 10(8) LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10(6) copies of mRNAs per 10(9) cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

Textes issus des travaux du programme Casdar "Innovation et Partenariat" de 2007 et présentés lors d'un colloque le 4 décembre 2012, sous l'égide du GIS Relance Agronomique; National audience; The practice of using native ferments, in particular the use of whey in lactic technology (transplanting), is a guarantee of the specificity of the products of the farm and is therefore considered by transformers producers as essential to be maintained. But, multifactorial management relying upon empirical practices, is difficult, and sometimes encourages producers to give up this practice. The references acquired in this project resulted in an approach of diagnosis and advice to perpetuate the use of whey. Investigations and experiments of this program showed the essential role of acidification to these problems of transplanting. They also revealed the crucial importance of milk microbial diversity and of microbial balance management. A USB key contains all documents from this program in three operational parts for technicians: providing a technical support, training producers and some "to go further" which include literature syntheses but above all the syntheses of the results obtained in the different actions of the project.; La pratique consistant à utiliser des ferments indigènes, en particulier l’utilisation de lactosérum en technologie lactique (repiquage), est garante de la spécificité des produits de la ferme et est donc considérée par les éleveurs transformateurs comme essentielle à maintenir. Or, sa maîtrise, de nature multifactorielle et reposant sur des pratiques empiriques, est difficile, incitant parfois les producteurs à abandonner cette pratique. Les références acquises dans ce projet ont permis d’aboutir à une démarche de diagnostic et de conseil visant à pérenniser l’utilisation du lactosérum. Les enquêtes et expérimentations de ce programme ont montré le rôle essentiel de la conduite de l’acidification face à ces problèmes de repiquage. Elles ont aussi révélé l’importance déterminante qu’ont la diversité microbienne des laits et la gestion des équilibres microbiens. Une clé USB regroupe les documents issus de ce programme en 3 parties opérationnelles pour les techniciens : réaliser un appui technique, former les producteurs, et une partie « pour aller plus loin » qui regroupe des synthèses bibliographiques mais surtout les synthèses des résultats obtenus dans les différentes actions du projet.

International audience

absent

National audience

Nutritional recommendations concern the reduction of the consumption of certain saturated fatty acids (SFA), which are associated with the risk of cardio-vascular diseases, in favour of increased amounts of unsaturated fatty acids (UFA) in the human diet. In this context, adapting the FA profile of high-fat content dairy products such as cheeses is of considerable interest for industrials and consumers. The objectives of this study were i) to produce an experimental UFA-enriched milk by adapting the cows diet (corn silage + grass silage + rapeseed) and to collect control milk in a local dairy plant during winter season (corn silage based diet), ii) to evaluate the possibility of manufacturing mould-ripened soft cheeses, iii) to characterize the physico-chemical and sensory properties of the UFA-enriched and control cheeses. Camembert-type cheeses have been chosen since they are highly appreciated by French consumers and about 108.000 tons are produced each year (CNIEL 2009).These experiments have been performed in the project “AGILAIT” (ANR 06-PNRA-012).

Proteolytic milk-clotting enzymes are extracted from various sources (animals, plants, fungi) and processed according to various methods that are specific to each manufacturer or cheese-maker. Chemical composition and polypeptide patterns of 24 milk-clotting preparations from animal and fungal sources: 10 commercial rennets, 9 artisanal calf rennets, 2 recombinant chymosin preparations and 3 microbial preparations, were compared in order to identify differences according to both their manufacturing process and their source. The preparation from Cryphonectria parasitica had the highest ammonia and small peptide content. Commercial rennets and preparations from Rhizomucor miehei had the highest NaCl and pH values while artisanal rennets had the lowest and recombinant chymosins were intermediate. In comparison with the other commercial preparations, commercial rennets had the highest variability in chemical composition and their polypeptide profiles showed numerous protein bands ranging from 15 kDa to 197 kDa, like artisanal rennets. Protein composition of commercial rennets revealed the presence of bovine serum albumin, either native or degraded, and degraded chymosin. The results indicated that the source of coagulating enzymes and the conditions applied for enzyme extraction led to specific chemical compositions, polypeptide patterns and protein composition which are described in this article. (C) 2012 Elsevier Ltd. All rights reserved.

Cette publication est également parue dans la revue "Journal of Animal Science", Vol.89, E-Suppl.1.; In contrast to other lactic acid bacteria, Lactobacillus helveticus can possess two cell-envelope proteinases (CEPs) called PrtH2 and PrtH (Genay et al 2009, Appl. Env. Microbiol.. 75 (10), 3238-3249; Sadat et al 2011a, in press Int. J. Food Microbiol.). These CEPs exhibit different cleavage specificity on pure s1 -casein (Sadat-Mekmene et al., 2011b, Appl. Env. Microbiol. 77(1) 179-186). The aim of this work was to investigate the proteolytic activity of L. helveticus strains in cheese matrix according to the number of their CEPs and the cheese stretchability. Two strains were selected, ITGLH77 and ITGLH1 which possess one CEP, PrtH2, and two CEPs, PrtH and PrtH2, respectively. The proteolysis was monitored during ripening:i) casein hydrolysis by urea-PAGE; ii) peptide pattern of the cheese aqueous extracts first by SDS-PAGE and second by RP-HPLC. Three chromatographic fractions were collected and peptides identified by RP-HPLC coupled on-line with tandem mass spectrometry. In parallel, the dynamic of stretchability of Emmental cheese was measured in order to determine the contribution of CEPs in its generation, since L. helveticus is known to enhance stretching properties in contrast to other lactobacilli, such as L. delbrueckii subsp. lactis . The microstructure of the strands of Emmental cheeses was observed by confocal laser scanning microscopy. The stretchability of Emmental was significantly higher in cheese manufactured with strain ITGLH77. By using Principal Component Analyses, the stretching properties were found to be correlated with the presence of peculiar peptides derived from primary proteolysis, hydrophobic peptides, containing more than 20 amino acid residues and derived from s1 -casein, which were accumulated in cheese manufactured using strain ITGLH77. The presence of these peptides suggest their less hydrolysis by this strain, in agreement with results observed in vitro (Sadat-Mekmene et al., 2011b), namely different cleavage specificity of CEPs PrtH et PrtH2 on pure s1 -casein.

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus , isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α s1 - and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of α s1 -casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α s1 -casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

International audience; To know and to control the fine milk composition is an important concern in the dairy industry. The mid-infrared (MIR) spectrometry method appears to be a good, fast and cheap method for assessing milk fatty acid profile with accuracy. Although partial least squares (PLS) regression is a very useful and powerful method to determine fine milk composition from spectra, the estimations are often less accurate on new samples coming from different spectrometers. Therefore a genetic algorithm (GA) combined with a PLS was used to produce models with a reduced number of wavelengths and a better accuracy. Number of wavelengths to consider is reduced substantially by 5 or 10 according the number of steps in the genetic algorithm. The accuracy is increased on average by 9% for fatty acids of interest.

International audience; The new challenges of the dairy industry require an accurate estimation of fine milk composition. The mid-infrared (MIR) spectrometry method appears to be a good, fast and cheap method for assessing milk fatty acid profile. Although partial least squares (PLS) regression is a very useful and powerful method to determine fine milk composition from the spectra, the estimations are not always very accurate and stable over time. Therefore a genetic algorithm (GA) combined with a PLS regression was used to produce models with a reduced number of wavelengths and a better accuracy. The results are a little sensitive to the choice of parameters in the algorithm. The number of wavelengths to consider is reduced substantially by 4 and accuracy is increased on average by 15%.

International audience; Aims: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non-E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H-, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H-, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19 center dot 75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.

Revue réalisée en hommage à Michel Ollivon (Directeur de Recherche au CNRS, UMR8612, Châtenay-Malabry), décédé en juin 2007, qui a été à l’origine de la création du RMT Listral. Ce Réseau Mixte Technologique avec pour thématique les Lipides Structurés Alimentaires est coordonné par l’ACTIA et animé par Actilait. Les autres partenaires du réseau sont : F Sobolewski & JP Bodin (Enilia-Ensmic), K Duhem & JP Guyonnet (Cniel), JP Bergé & JP Gouygou (Ifremer Nantes), G Nassy (Ifip), Lebois & D Majou (Actia), P Gaborit & R Richoux (Actilait).; National audience; From the nutrition point of view, lipids are primarily considered as energy suppliers, their dietary intake having to be limited. In fact, lipids are sources of various fatty acids, and dietary recommendations include the required daily intakes in different fatty acids (fatty acid profile). Beyond these quantitative aspects, fatty acids are part of larger molecules, mainly triacylglycerols (TAG), that are organized in supramolecular structures as for example fat crystals and lipid droplets. Recent advances in nutrition research have demonstrated that these structures, and lipid organization in food matrices, influence digestibility and metabolism of fatty acids. Therefore, the members of the technological network Listral propose a synthesis of the knowledge about the influence of molecular and supramolecular structures of lipids on digestion and metabolic fate of dietary fatty acids originating from for the main food industry sectors. Fatty acids are mainly provided in the diet in the form of TAG, or phospholipids (PL) where they are esterified in external (sn-1, sn-3) or internal (sn-2) position of the glycerol backbone. They can also be found as ethyl esters (EE) in specific formulations. These molecular characteristics of the lipid molecules affect their hydrolysis and their absorption rate but also their metabolism in indifferent organs, as assessed in studies often using inter esterified fat sources. The results depend on the types of fatty acids (saturated or polyunsaturated fatty acids) but also on the model used for the studies: in vitro or in vivo studies ; animal model, human newborn or adult, either healthy or exhibiting some diseases. Among others, it was found that the crystal form and fat melting point as affected by the fatty acid profiles and the lipid molecular structures directly impact fatty acid absorption kinetics through modifications of lipolytic enzyme activities. However, in these studies, the relative effect of the thermal properties of lipids and the proportion of saturated fatty acids chains in sn-2 position were hardly distinguished. Supramolecular structures, namely type and size of the fat droplets and their interfacial composition, of lipids, either in their native forms or obtained after food processes, also affect the digestibility and absorption of lipids. This aspect is reviewed with specific focus on dairy, marine, meat, oil plant and egg products. The impact of other nutritional components present in these food matrices on the absorption of lipids is also discussed.

Enterohemorrhagic Escherichia coli (EHEC) strains are a subset of Shiga toxin (Stx)-producing E. coli (STEC) strains that are isolated from human patients and are responsible for severe clinical symptoms, such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS) (28, 29). Although most outbreaks of HC and HUS have been attributed to serotype O157:H7/H−, an increasing number of human infections are caused by other serotypes, such as O26:H11/H−, O103:H2, O111:H8/H−, and O145:H28/H− (13, 20, 28). O157:H7/H− and these four serotypes were classified into seropathotypes A and B, respectively, which occur most frequently in human diseases and outbreaks and in patients with severe symptoms (30). Shiga toxins, the main virulence factors contributing to pathogenicity, consist of two major types, Stx1 and Stx2, each including several variants (47). In addition to Stx, typical EHEC strains carry on their chromosomes the locus for enterocyte effacement (LEE), a large pathogenicity island that is shared with enteropathogenic E. coli (EPEC). The LEE is responsible for attaching and effacing (A/E) lesions on enterocytes (34); it encodes several virulence factors, including the outer membrane adhesin intimin and its translocated receptor Tir, as well as components of a type III secretion machinery and its effector proteins. Intimin is involved in the tight attachment of bacteria to the enterocytes. It is encoded by the eae gene, 18 types and 9 subtypes of which have been described on the basis of its variable 3′ region (25). The EHEC serotypes O157:H7 and O145:H28 are known to be associated with the eae-γ1 subtype, whereas EHEC O26:H11, O103:H2, and O111:H8 harbor the eae-β1, eae-ɛ, and eae-γ2/θ subtypes, respectively (7, 11, 37, 51). Additional candidate pathogenicity islands, or “O islands” (OI), that may contribute to virulence have been identified. They include OI-71 and OI-122, which contain a number of type III non-LEE-encoded effector (nle) genes whose presence correlates with outbreak and HUS potential (17). Finally, EHEC O157:H7 contains a large plasmid with putative virulence genes encoding the EHEC-hemolysin (EHEC-hlyA), a serine protease (espP), and a catalase peroxidase (katP). However, strains lacking one or more of these genes have been involved in HUS, and high variability in the gene composition of this large plasmid has also been reported in various STEC serotypes (14). Domestic ruminants have long been identified as a major reservoir for STEC (29). Although STEC transmission to humans is frequently associated with consumption of raw or undercooked meat, raw milk and dairy products have also been implicated in human disease (4). Detection and isolation of STEC in foodstuffs by traditional culture methods is rather laborious and time-consuming and is complicated by the lack of common biochemical characteristics distinguishing most STEC from other E. coli strains. Development of rapid methods for the detection of the most pathogenic STEC strains is essential to ensure the safety of food products. A sequential approach based on real-time PCR assays specific for stx, wzxO26, wzxO103, wbdIO111, ihp1O145, and rfbEO157 genetic markers has been described for the detection of STEC O26, O103, O111, O145, and O157 in foods (3, 40). A similar strategy that includes an additional eae amplification step has been proposed by working group 6 of the European Committee for Standardization (CEN) TC275 for a technical specification (currently under International Organization for Standardization [ISO] evaluation) regarding the detection of these five STEC serogroups in foods (21). Recently, we developed real-time PCR assays for detection of the eae variants and fliC alleles associated with the five most common EHEC serotypes (32). The aim of the present study was to investigate whether these newly developed assays could further refine the diagnostic result obtained from PCR analysis of foods. To this end, the prevalence of stx, O group genetic markers, eae variants, and fliC alleles typical of EHEC O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 was investigated in 400 raw-milk cheeses. STEC and E. coli strains belonging to the five targeted serotypes were then isolated for confirmation of the PCR results, and their virulence traits were characterized. As the term EHEC includes a clinical connotation (28, 29), the stx-positive E. coli isolates recovered from food are designated STEC (not EHEC) here, irrespective of the serotype.

Communication Orale"Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access "; absent

Controlling lipolysis in cheese is necessary to ensure the formation of desirable flavor. To get a better understanding of the mechanism of lipolysis in Swiss cheese, cheeses were manufactured with and without (control) the addition of Propionibacterium freudenreichii. Products of lipolysis were quantified throughout ripening. Half of the free fatty acids (FFA) released in milk (3.66 mg/g fat), in particular the short-chain FFA, were lost in the whey during curd drainage, whereas diglycerides and monoglycerides were retained within the curd. P. freudenreichii was responsible for the release of most FFA during ripening (10.84 and 0.39 mg/g fat in propionibacteria-containing and control cheeses, respectively). Indices of lipolysis displayed low specificity. All types of FFA were released, but butyric and palmitic acids more significantly, which could be due to a low sn-1,3 regioselectivity. All glycerides were hydrolyzed in the following order: monoglycerides > diglycerides > triglycerides. The results of this study show the quantitative and qualitative contributions of the different lipolytic agents to Swiss cheese lipolysis.

1 page Diffusion du document : Abstracts"Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access "; absent

Book Abstracts N°16; absent

International audience; Aims: To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. Methods and Results: Primers and probes specific to fliC(H2), fliC(H7), fliC(H8), fliC(H11), fliC(H28), eae-beta 1, eae-gamma 1, eae-epsilon and eae-theta were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of < 5 CFU per 25 g after overnight enrichment using the PCR assays. Conclusions: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. Significance and Impact of the Study: Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.

International audience

International audience

National audience; Specific cases of goat and sheep milks Small ruminants show specificities in terms of production of milk, protein and fat contents, and lipolysis levels, which are inherent in breeding systems, the number and size of animals, their genetic heritage, feeding and seasonality. Specific responses to feeding have been noticed especially on the profiles of fatty acids of nutritional interest (CLA, omega 3...). Nevertheless, whatever the answers, milk fat from small ruminants is characterized by significant levels of short-and medium-chain saturated fatty acids, with present nutritional and health benefits, and also different sizes of fat globules. The high polymorphism of the caprine s1 casein, which directly affects the protein content, also impacts the fat content, the size of fat globules and the lipolysis level. With regard to the use of milk from small ruminants, 98 % of the volume is turned into cheeses, most often without standardization of the fat. The fatty acid profiles from milk and cheese are thus similar implying that any improvement on the quality of the milk fat is directly transfered to the cheese. However, the specific technology, including storage, related to the seasonality can contribute to alter the nutritional and sensorial quality of fat (e.g. oxidation). The relationship between the structure of milk fat and its nutritional qualities is complex and constitute one of the most important current research thematic, especially when one takes into account the matrix effects on the bioavailability of the fat.; Les petits ruminants présentent des particularités en terme de production de lait, de taux protéiques et butyreux et de niveaux de lipolyse, qui sont inhérentes aux modes de conduite d'élevage, à leur potentiel génétique, à l'alimentation et à la saisonnalité. Des réponses particulières à l'alimentation ont été enregistrées notamment sur les profils d'acides gras d'intérêt nutritionnel (CLA, omega 3...). Néanmoins, quelles que soient ces réponses, la matière grasse des laits de petits ruminants se caractérise par des teneurs importantes en acides gras saturés à courte et moyenne chaîne, présentant des bénéfices nutritionnels et santé et des tailles de globules gras très variables. Le fort polymorphisme de la caséine s1 caprine, qui influe directement sur les taux protéiques, a également un impact sur les taux de matière grasse, la taille des globules gras et les niveaux de lipolyse. Pour ce qui est de l'utilisation du lait de petits ruminants, 98 % de la production est transformé en fromage, la plupart du temps sans standardisation de la matière grasse. Les profils d'acides gras entre lait et fromage sont donc conservés et toute modification qualitative de la matière grasse du lait est directement répercutée sur les fromages. Néanmoins, des spécificités technologiques, de report notamment, liées à la saisonnalité peuvent contribuer à modifier la qualité de la matière grasse (l'oxydation notamment), nutritionnelle et sensorielle. La relation entre la structuration de la matière grasse laitière et ses qualités nutritionnelles est complexe et constitue une des pistes de recherche, en particulier avec la prise en compte des effets matrice sur la biodisponibilité de la matière grasse.

2nd Edition; International audience

National audience

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National audience

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 103 copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese.

National audience

International audience

Format du poster : 21 X29,7; Microbial risk assessment in coagulase negative staphylococci isolated from fermented foodstuffs using a dedicated diagnostic microarray. 21. International ICFMH " Evolving microbial Food quality and safety", 1st-4th september 2008, Aberdeen

International audience; Increasing the knowledge on dietary fat composition, mainly the minor components, will improve the nutritional value of foods and their labeling. In this study, we examined the trans-octadecenoic acid (C18:1) composition of Emmental cheeses enriched in unsaturated fatty acids (FA) and manufactured with milks produced by cows selected to produce small and large fat globules. The FA composition of the milks was not significantly (P > 0.05) different from the FA composition of the corresponding Emmental cheeses. Increasing the unsaturated FA content of the cheeses using dietary manipulations lead to an increase in the trans-C18:1 and changed their isomeric profiles. In milk fat produced with the linseed-enriched diet, the trans-10 C18:1 concentration was greater than trans-11 C18:1 (vaccenic acid), which is classically the major trans-C18:1 in milk fat. The content in trans-C18:1 and more particularly in trans-10 C18:1 was negatively correlated with the size of fat globules (r2 ) 0. 82 and 0.87, respectively) and related to milk fat depression. The trans-C18:1 content was negatively correlated with the saturated FA (slope ) -0.35; r2 ) 0.81) and positively correlated with the unsaturated (slope ) 0.29; r2 ) 0.85) and monounsaturated (slope ) 0.32; r2 ) 0.81) FA. Focusing on the health-related considerations of fat in food products, further nutritional studies are needed to elucidate the role of trans-C18:1 isomers.

National audience

National audience