FROM’MIR : Développer des outils de prédiction et de conseil pour maîtriser la fromageabilité des laits destinés à la fabrication des fromages traditionnels franc-comtois

Ce volume regroupe les textes issus du programme Casdar "Innovation et Partenariat" et "Recherche finalisée et innovation" de 2014. Il a été réalisé sous l’égide du GIS Relance Agronomique.Mid-infrared spectroscopy prediction equations of the cheese-making properties of milk, established inthe Franche-Comté PDO/PGI context, exist for the first time in France. Laboratory curd yield in DryMatter was consistent with the yields observed in mini-manufactures of soft and pressed cookedcheeses and it is the best predicted parameter. Under our conditions, some coagulation properties suchas curd firmness could be estimated. The acidification properties, which heavily depend on themicrobiological component of milk, are poorly estimated. The best prediction performances wereobtained on individual cow milks. The performances were poorer on the scale of bulk milks, herd tankmilk but especially dairy vat milk. The study of variation factors made it possible to highlight theimportant weight of genetics with a high level of heritability and strong effects of the genome regionsinvolved. The quality and quantity of fodder and the distribution of calves were influential in the contextstudied. In this same context, few factors of variation have been identified at the scale of dairy vat milks,as the practices were very much governed by the PDO specifications. At the end of this project, anobservatory, from the quality of the milk to the quality of the cheese, will be set up in Franche Comté.Studies will also be carried out at the national level to consolidate and improve the equations in othercontexts.Des équations MIR (spectrométrie moyen infrarouge) d'estimation de la fromageabilité des laits,établies en contexte AOP/IGP franc-comtois, existent pour la première fois en France. Le rendementlaboratoire extrait sec (ES), cohérent avec les rendements observés en mini-fabrications de fromages àpâte molle et à pâte pressée cuite, est le paramètre le mieux prédit. Dans nos conditions, certainsaspects de l'aptitude à la coagulation enzymatique, comme la fermeté des gels, peuvent être estimés.L’aptitude à l’acidification, dépendant fortement de la composante microbiologique des laits, est quant àelle mal estimée. Les meilleures performances de prédiction sont obtenues sur les laits individuels devaches. Les performances sont moins bonnes à l’échelle des laits de mélange, des laits de troupeauxmais surtout des laits de cuves de fromagerie. L'étude des facteurs de variation a permis de mettre enévidence le poids important de la génétique avec un niveau d’héritabilité élevé et des effets forts desrégions du génome impliquées. La qualité et la quantité de fourrages ainsi que la répartition desvêlages sont influents dans le contexte étudié. Dans ce même contexte, peu de facteurs de variationont été mis en évidence à l’échelle des laits de cuves, les pratiques étant très encadrées par le cahierdes charges AOP. A l’issue de ce projet, un observatoire, depuis la qualité des laits jusqu’à celle desfromages, va être mis en place en Franche Comté. Des études seront aussi mises en œuvre au niveaunational pour permettre notamment une consolidation et une amélioration des équations dans d'autrescontextes

ELISA to detect proteolysis of ultrahight-temperature milk upon storage

International audienceCasein proteolysis can occur in milk during storage leading to its gelation. The two main proteolytic systems suspected to be involved are the plasmin and the proteases produced by psychrotrophic bacteria. The latter have been shown to cleave κ-casein at the Phe105−Met106 bond. Although several techniques allow the determination of plasmin in milk, few rapid and easy-to-perform analytical techniques are available to check for bacterial proteolytic activity. This study presents the development of an inhibition ELISA allowing for the quantification of the κ-casein intact at the Phe105−Met106 bond. It uses a monoclonal antibody specifically directed against this peptide bond that binds to the protein as long as the molecule's cleavage site is intact but not when it is cleaved. This simple technique allows for the rapid analysis of more than 20 samples within 3 h. Applied to commercial milks, this assay allowed for the detection of unstable milk

An ELISA to detect proteolysis of UHT milk upon storage

International audienc

An ELISA to detect proteolysis of UHT milk upon storage

International audienc

Quantification of pepsin in rennet using a monoclonal antibody-based inhibition ELISA

6 graph.Pepsin and chymosin are milk-clotting enzymes found in rennets. They differ in their pH and temperature sensitivities and their milk-clotting activity (MCA)/proteolytic activity ratio which impact cheese technology. Therefore, characterization of rennet should not be limited to its total MCA, but also its enzymatic composition. Monoclonal antibodies against pepsin, obtained from mice immunized with purified pepsin, were characterized. A specific inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of pepsin in rennets. The limit of quantification was 143 ng pepsin/ mL. The precision within runs ranged from 7.0 to 9.4%, for rennets with low and high pepsin concentrations, respectively. The precision among runs also ranged from 8.8 to 11.4%. Satisfying recovery, from 84.7 to 100.3%, was found with spiked samples. The applicability of the developed inhibition ELISA for pepsin quantification was assessed by analyzing commercial rennets, and compared to the standard chromatographic method 110A of International Dairy Federation. The relation and agreement between methods were evaluated using Deming regression analysis and Bland-Altman plot. A good agreement was found with a fixed bias. By means of bias subtraction, inhibition ELISA could be a reliable alternative for rapid and sensitive determination of pepsin in rennet

Development and evaluation of a monoclonal antibody-based inhibition ELISA for the quantification of chymosin in solution

Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clottingactivity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987)

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies

Contact: [email protected] theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (Rs1, Rs2, β, and k) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of Rs1-, Rs2-, β-, and k-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of k-casein was highly accessible and located at the periphery of the structure.When casein micelles were submitted to proteolysis, the C-terminal extremity of k-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of Rs1- and Rs2-casei