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Anne Thierry - One of the best experts on this subject based on the ideXlab platform.
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Identification of a secreted lipolytic esterase in Propionibacterium freudenreichii
2010Co-Authors: Julien Dherbecourt, Julien Jardin, Frédérique Barloy-hubler, Hélène Falentin, Anne ThierryAbstract:Free fatty acids are important flavour compounds in Cheese, where they bring “pungent”, “rancid”, “Cheese”, and “fruity” notes. They mainly result from the lipolytic activity of Cheese micro-organisms. In Emmental Cheese, the main agent of lipolysis is Propionibacterium freudenreichii, a species used as a ripening culture. By combining in silico prediction of subcellular localisation, zymography, and identification by mass spectrometry, we recently demonstrated that P. freudenreichii CIRM1T possess one secreted lipolytic esterase, PF279. PF279 possesses an atypical motif TXSXG instead of the classical GXSXG of esterases, and shares only weak homology with the proteins of NCBI nr database. It is constitutively expressed during Pf growth and is active on milk fat. Transformed P. freudenreichii CIRM1T clones over-expressing PF279 showed ~5 times more lipolytic activity on milk fat than the wild strain. PF279 is likely a key component in Emmental Cheese lipolysis.
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Identification of a secreted lipolytic esterase in Propionibacterium freudenreichii, a ripening process bacterium involved in Emmental Cheese lipolysis.
Applied and Environmental Microbiology, 2010Co-Authors: Julien Dherbecourt, Julien Jardin, Frédérique Barloy-hubler, M.-b. Maillard, F. Baglinière, Anne ThierryAbstract:Lipolysis plays an important role in the formation of Cheese flavor. In Emmental Cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii, a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in Cheese lipolysis by P. freudenreichii. Since Cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico. The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in Cheese lipolysis by P. freudenreichii.
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Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout Emmental Cheese manufacture
International Journal of Food Microbiology, 2010Co-Authors: Hélène Falentin, Romain Richoux, Anne Thierry, Florence Postollec, Sandrine Parayre-breton, Nadine Hénaff, Pierre Le Bivic, Danièle SohierAbstract: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.
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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental Cheese
Microbial Cell Factories, 2008Co-Authors: Julien Dherbecourt, Hélène Falentin, Stéphane Canaan, Anne ThierryAbstract:Background Lipolysis is an important process of Cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental Cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027^T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental Cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. Results The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027^T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. Conclusion We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental Cheese.
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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental Cheese
Microbial cell factories, 2008Co-Authors: Julien Dherbecourt, Hélène Falentin, Stéphane Canaan, Anne ThierryAbstract:Lipolysis is an important process of Cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental Cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental Cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental Cheese.
Christelle Lopez - One of the best experts on this subject based on the ideXlab platform.
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Combined temperature–time parameters during the pressing of curd as a tool to modulate the oiling-off of Swiss Cheese
Food Research International, 2008Co-Authors: Romain Richoux, Grégory Roset, Valérie Briard-bion, Lydie Aubert, René Kerjean, Christelle LopezAbstract: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.
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Multiscale characterization of the organization of triglycerides and phospholipids in Emmental Cheese: From the microscopic to the molecular level
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Christelle Lopez, Valérie Briard-bion, Eric Beaucher, Michel OllivonAbstract:Multiscale characterization of the organization of triglycerides and phospholipids in Emmental Cheese: From the microscopic to the molecular level
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development of the milk fat microstructure during the manufacture and ripening of Emmental Cheese observed by confocal laser scanning microscopy
International Dairy Journal, 2007Co-Authors: Christelle Lopez, Benedicte Camier, Jean-yves GassiAbstract:Abstract Changes in the physico-chemical properties and microstructure of milk fat globules were investigated during the manufacture and ripening of Emmental Cheese. The measurement of fat globule size and apparent zeta-potential showed that they were slightly affected during Cheese milk preparation, i.e. storage of Cheese milk overnight at 4 °C and pasteurisation. After rennet-induced coagulation and heating of curd grains, coalescence caused the formation of large fat globules (i.e.>10 μm). The structure of fat in Emmental Cheese was characterised in situ using confocal laser scanning microscopy (CLSM). The rennet-induced coagulation lead to the formation of a continuous network of casein strands in which fat globules of various sizes were entrapped. Heating of curd grains induced the formation of fat globule aggregates. Pressing of the curd grains resulted in the greatest disruption of milk fat globules, their coalescence, the formation of non-globular fat (free fat) and the release of the milk fat globule membrane (MFGM) material. This study showed that milk fat exists in three main forms in ripened Emmental Cheese: (i) small fat globules enveloped by the MFGM; (ii) aggregates of partially disrupted fat globules and (iii) free fat, resulting from the disruption of the MFGM and allowing free triacylglycerols to fill voids in the protein matrix. The curd grain junctions formed in Emmental Cheese were also characterised using CLSM: they are compact structures, rich in protein and devoid of fat globules.
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Functionality of smaller vs control native milk fat globules in Emmental Cheeses manufactured with adapted technologies
Food Research International, 2007Co-Authors: Marie-caroline Michalski, Marie-hélène Famelart, Valérie Briard-bion, Benedicte Camier, Jean-yves Gassi, Nadine Leconte, Christelle LopezAbstract:Abstract Emmental Cheeses were produced with control native milk fat globules (CFG) or smaller ones (SFG) selected from the same milk by microfiltration. Either the same regular technology was used for both Cheeses (so-called SFGreg and CFGreg), or two different adapted technologies to obtain Cheeses with the same moisture (so-called SFGadapt and CFGadapt). SFGreg Cheeses were more humid and presented lower firmness and longness than CFGreg Cheeses. Yellow index was greater for SFGadapt than CFGadapt Cheeses while melting coefficient and extrusion force were similar. SFGadapt Cheeses exhibited improved sensory characteristics. Stretching and elasticity increase were always greater for SFG than for the corresponding CFG Cheeses; eyes were always smaller in SFG Cheeses. Confocal micrographs revealed larger inclusions of non-globular fat in CFG Cheeses; there were more fat globules and aggregates in SFG Cheeses. The ultrastructure of milk fat played a role in the functional and sensory properties of Emmental Cheese and the use of smaller native globules may be advantageous.
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Lipolysis during ripening of Emmental Cheese considering organization of fat and preferential localization of bacteria.
Journal of agricultural and food chemistry, 2006Co-Authors: Christelle Lopez, Valérie Briard-bion, Benedicte Camier, Marie-bernadette Maillard, John A. HannonAbstract:This study followed the progression of lipolysis in Emmental Cheese by quantifying the concentrations of individual free fatty acids (FFA) released during ripening in each of the different rooms: 12 days at 12 degrees C, 28 days at 21 degrees C, and 8 days at 4 degrees C. Lipolysis, which corresponded to 1.56% of fat, mainly occurred in the 21 and 4 degrees C rooms, with 68 and 16.5% of total FFA, respectively. The nonselectivity of lipolytic enzymes was evidenced: all fatty acids were released with level of > or =1%. Differential scanning calorimetry experiments showed that the thermal properties of Cheese were affected by (i) lipolysis of fat, that is, the monoacylglycerols, diacylglycerols, and FFA that may be localized at the fat/whey interface, and/or by (ii) hydrolysis of high-melting-point triacylglycerols constituted mainly by long-chain saturated fatty acids (e.g., palmitic acid). Analysis of the Cheese microstructure was performed using confocal laser scanning microscopy. Fat globules were mainly disrupted after pressing of curd grains, leading to the release of the milk fat globule membrane (MFGM); fat inclusions were surrounded by pockets of whey, delimited by casein strands. Moreover, colonies of bacteria were preferentially localized in situ at the fat/protein interface. This study showed that both the localization of bacteria and the supramolecular organization of fat which was not protected by the MFGM can help the accessibility of milk fat to lipolytic enzymes and then contribute to the quality of Cheese.
Valérie Briard-bion - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance
2016Co-Authors: Valérie Gagnaire, Julien Jardin, Houem Rabah, Valérie Briard-bionAbstract:Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii, is generally recognized as safe and used both as probiotic and as Cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle. Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress toler-ance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental Cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermaniiwas observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as over-expression of various early stress adaptation proteins in Emmental juice, compared to chem-ically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis of valine, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard Cheeses as delivery vehicle for probiotics with enhanced efficacy
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Initial composition in free amino acids of the EJ and the CdM.
2015Co-Authors: Valérie Gagnaire, Valérie Briard-bion, Julien Jardin, Houem Rabah, Gwénaël JanAbstract:The concentrations in free amino acids were measured in triplicate.CdM: Chemically defined medium;EJ: Emmental Cheese aqueous phaseInitial composition in free amino acids of the EJ and the CdM.
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Emmental Cheese Environment Enhances [i]Propionibacterium freudenreichii[/i] Stress Tolerance
PLoS ONE, 2015Co-Authors: Valerie Gagnaire Soumet, Valérie Briard-bion, Julien Jardin, Houem Rabah, Gwénaël Jan¨Abstract:Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii , is generally recognized as safe and used both as probiotic and as Cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle.Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress toler- ance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental Cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermanii was observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as over- expression of various early stress adaptation proteins in Emmental juice, compared to chem-ically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis ofvaline, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard Cheeses as delivery vehicle for probiotics with enhanced efficacy
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Multiscale characterization of the organization of triglycerides and phospholipids in Emmental Cheese: From the microscopic to the molecular level
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Christelle Lopez, Valérie Briard-bion, Eric Beaucher, Michel OllivonAbstract:Multiscale characterization of the organization of triglycerides and phospholipids in Emmental Cheese: From the microscopic to the molecular level
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Combined temperature–time parameters during the pressing of curd as a tool to modulate the oiling-off of Swiss Cheese
Food Research International, 2008Co-Authors: Romain Richoux, Grégory Roset, Valérie Briard-bion, Lydie Aubert, René Kerjean, Christelle LopezAbstract: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.
Jean-yves Gassi - One of the best experts on this subject based on the ideXlab platform.
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development of the milk fat microstructure during the manufacture and ripening of Emmental Cheese observed by confocal laser scanning microscopy
International Dairy Journal, 2007Co-Authors: Christelle Lopez, Benedicte Camier, Jean-yves GassiAbstract:Abstract Changes in the physico-chemical properties and microstructure of milk fat globules were investigated during the manufacture and ripening of Emmental Cheese. The measurement of fat globule size and apparent zeta-potential showed that they were slightly affected during Cheese milk preparation, i.e. storage of Cheese milk overnight at 4 °C and pasteurisation. After rennet-induced coagulation and heating of curd grains, coalescence caused the formation of large fat globules (i.e.>10 μm). The structure of fat in Emmental Cheese was characterised in situ using confocal laser scanning microscopy (CLSM). The rennet-induced coagulation lead to the formation of a continuous network of casein strands in which fat globules of various sizes were entrapped. Heating of curd grains induced the formation of fat globule aggregates. Pressing of the curd grains resulted in the greatest disruption of milk fat globules, their coalescence, the formation of non-globular fat (free fat) and the release of the milk fat globule membrane (MFGM) material. This study showed that milk fat exists in three main forms in ripened Emmental Cheese: (i) small fat globules enveloped by the MFGM; (ii) aggregates of partially disrupted fat globules and (iii) free fat, resulting from the disruption of the MFGM and allowing free triacylglycerols to fill voids in the protein matrix. The curd grain junctions formed in Emmental Cheese were also characterised using CLSM: they are compact structures, rich in protein and devoid of fat globules.
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Functionality of smaller vs control native milk fat globules in Emmental Cheeses manufactured with adapted technologies
Food Research International, 2007Co-Authors: Marie-caroline Michalski, Marie-hélène Famelart, Valérie Briard-bion, Benedicte Camier, Jean-yves Gassi, Nadine Leconte, Christelle LopezAbstract:Abstract Emmental Cheeses were produced with control native milk fat globules (CFG) or smaller ones (SFG) selected from the same milk by microfiltration. Either the same regular technology was used for both Cheeses (so-called SFGreg and CFGreg), or two different adapted technologies to obtain Cheeses with the same moisture (so-called SFGadapt and CFGadapt). SFGreg Cheeses were more humid and presented lower firmness and longness than CFGreg Cheeses. Yellow index was greater for SFGadapt than CFGadapt Cheeses while melting coefficient and extrusion force were similar. SFGadapt Cheeses exhibited improved sensory characteristics. Stretching and elasticity increase were always greater for SFG than for the corresponding CFG Cheeses; eyes were always smaller in SFG Cheeses. Confocal micrographs revealed larger inclusions of non-globular fat in CFG Cheeses; there were more fat globules and aggregates in SFG Cheeses. The ultrastructure of milk fat played a role in the functional and sensory properties of Emmental Cheese and the use of smaller native globules may be advantageous.
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Milk fat thermal properties and solid fat content in Emmental Cheese : a differential scanning calorimetry study
Journal of Dairy Science, 2006Co-Authors: Christelle Lopez, Valérie Briard-bion, Benedicte Camier, Jean-yves GassiAbstract:The experiments reported in this study give deeper insight into the crystallization of milk fat in Emmental Cheese, which is the most widely consumed hard Cheese in France. Differential scanning calorimetry (DSC) was used to monitor the thermal properties of milk fat after the main stages involved during manufacture of Emmental Cheese. By heating the samples to 60°C to eliminate their thermal history and cooling them at 2°C/min, the liquid → solid phase transition of fat was investigated. Confocal laser scanning microscopy was used to characterize in situ the supramolecular organization of milk fat dispersed in the casein matrix. The destabilization of fat globules by aggregation or coalescence and the formation of free fat during the manufacture altered the thermal properties of milk fat by increasing the initial temperature of crystallization and by the formation of 2 overlapping exotherms. The melting properties of the crystalline structures formed by fat at the temperatures used for ripening (12, 21, and 4°C) were examined. Differential scanning calorimetry was used to determine the ratio of solid to liquid fat; that is, the amount of fat that is crystallized, by dividing the partial enthalpy of melting of the fat for ripening temperature by the total enthalpy of melting of the same fat extracted from Cheese
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Milk fat thermal properties and solid fat content in Emmental Cheese : a differential scanning calorimetry study
Journal of Dairy Science, 2006Co-Authors: Christelle Lopez, Valérie Briard-bion, Benedicte Camier, Jean-yves GassiAbstract:The experiments reported in this study give deeper insight into the crystallization of milk fat in Emmental Cheese, which is the most widely consumed hard Cheese in France. Differential scanning calorimetry (DSC) was used to monitor the thermal properties of milk fat after the main stages involved during manufacture of Emmental Cheese. By heating the samples to 60°C to eliminate their thermal history and cooling them at 2°C/min, the liquid → solid phase transition of fat was investigated. Confocal laser scanning microscopy was used to characterize in situ the supramolecular organization of milk fat dispersed in the casein matrix. The destabilization of fat globules by aggregation or coalescence and the formation of free fat during the manufacture altered the thermal properties of milk fat by increasing the initial temperature of crystallization and by the formation of 2 overlapping exotherms. The melting properties of the crystalline structures formed by fat at the temperatures used for ripening (12, 21, and 4°C) were examined. Differential scanning calorimetry was used to determine the ratio of solid to liquid fat; that is, the amount of fat that is crystallized, by dividing the partial enthalpy of melting of the fat for ripening temperature by the total enthalpy of melting of the same fat extracted from Cheese. This study shows, for the first time, that milk fat is partially crystallized in Emmental Cheese: about 55.7 ± 3.5% of fat is solid at 4°C at the end of ripening. Polymorphic phase transitions of milk fat are also suggested during ripening of Emmental Cheese. Key words: triacylglycerol, polymorphism, fat crys
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Milk Fat Thermal Properties and Solid Fat Content in Emmental Cheese: A Differential Scanning Calorimetry Study
Journal of dairy science, 2006Co-Authors: Christelle Lopez, Valérie Briard-bion, Benedicte Camier, Jean-yves GassiAbstract:Abstract The experiments reported in this study give deeper insight into the crystallization of milk fat in Emmental Cheese, which is the most widely consumed hard Cheese in France. Differential scanning calorimetry (DSC) was used to monitor the thermal properties of milk fat after the main stages involved during manufacture of Emmental Cheese. By heating the samples to 60°C to eliminate their thermal history and cooling them at 2°C/min, the liquid → solid phase transition of fat was investigated. Confocal laser scanning microscopy was used to characterize in situ the supramolecular organization of milk fat dispersed in the casein matrix. The destabilization of fat globules by aggregation or coalescence and the formation of free fat during the manufacture altered the thermal properties of milk fat by increasing the initial temperature of crystallization and by the formation of 2 overlapping exotherms. The melting properties of the crystalline structures formed by fat at the temperatures used for ripening (12, 21, and 4°C) were examined. Differential scanning calorimetry was used to determine the ratio of solid to liquid fat; that is, the amount of fat that is crystallized, by dividing the partial enthalpy of melting of the fat for ripening temperature by the total enthalpy of melting of the same fat extracted from Cheese. This study shows, for the first time, that milk fat is partially crystallized in Emmental Cheese: about 55.7±3.5% of fat is solid at 4°C at the end of ripening. Polymorphic phase transitions of milk fat are also suggested during ripening of Emmental Cheese.
Julien Dherbecourt - One of the best experts on this subject based on the ideXlab platform.
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Identification of a secreted lipolytic esterase in Propionibacterium freudenreichii
2010Co-Authors: Julien Dherbecourt, Julien Jardin, Frédérique Barloy-hubler, Hélène Falentin, Anne ThierryAbstract:Free fatty acids are important flavour compounds in Cheese, where they bring “pungent”, “rancid”, “Cheese”, and “fruity” notes. They mainly result from the lipolytic activity of Cheese micro-organisms. In Emmental Cheese, the main agent of lipolysis is Propionibacterium freudenreichii, a species used as a ripening culture. By combining in silico prediction of subcellular localisation, zymography, and identification by mass spectrometry, we recently demonstrated that P. freudenreichii CIRM1T possess one secreted lipolytic esterase, PF279. PF279 possesses an atypical motif TXSXG instead of the classical GXSXG of esterases, and shares only weak homology with the proteins of NCBI nr database. It is constitutively expressed during Pf growth and is active on milk fat. Transformed P. freudenreichii CIRM1T clones over-expressing PF279 showed ~5 times more lipolytic activity on milk fat than the wild strain. PF279 is likely a key component in Emmental Cheese lipolysis.
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Identification of a secreted lipolytic esterase in Propionibacterium freudenreichii, a ripening process bacterium involved in Emmental Cheese lipolysis.
Applied and Environmental Microbiology, 2010Co-Authors: Julien Dherbecourt, Julien Jardin, Frédérique Barloy-hubler, M.-b. Maillard, F. Baglinière, Anne ThierryAbstract:Lipolysis plays an important role in the formation of Cheese flavor. In Emmental Cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii, a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in Cheese lipolysis by P. freudenreichii. Since Cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico. The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in Cheese lipolysis by P. freudenreichii.
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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental Cheese
Microbial Cell Factories, 2008Co-Authors: Julien Dherbecourt, Hélène Falentin, Stéphane Canaan, Anne ThierryAbstract:Background Lipolysis is an important process of Cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental Cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027^T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental Cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. Results The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027^T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. Conclusion We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental Cheese.
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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental Cheese
Microbial cell factories, 2008Co-Authors: Julien Dherbecourt, Hélène Falentin, Stéphane Canaan, Anne ThierryAbstract:Lipolysis is an important process of Cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental Cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental Cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental Cheese.
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A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental Cheese
Microbial Cell Factories, 2008Co-Authors: Julien Dherbecourt, Hélène Falentin, Stéphane Canaan, Anne ThierryAbstract:Background: Lipolysis is an important process of Cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental Cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental Cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study their activity and specificity of hydrolysed substrates.