Protein is an important fraction within bovine milk. This milk protein is not only vital for calf growth and development, but also includes bioactive proteins and peptides that have been shown to enhance the health of animals and humans. Research efforts are focusing on factors, such as nutrition, that can influence the quantity and profile of proteins within the bovine milk proteome. The research outlined herein investigated the impact of diet on the bovine milk proteome. The first experiment examined whether dietary inclusion of grape marc (GM), a condensed tannin (CT) containing by-product from the viticulture industry, could alter the bovine milk proteome through altered nitrogen (N) metabolism. In this experiment, 10 lactating Holstein cows were fed either 2.0 kg dry matter (DM)/ cow/ day of beet pulp: soy hulls in a 50% mixture (control), or 1.5 kg DM/ cow/ day of GM as part of a balanced dairy cow ration for a 28-d trial. Milk samples were obtained for analysis of the high- and low-abundance protein fractions. Skimmed milk samples collected for high-abundance protein analysis were measured using high performance liquid chromatography (HPLC), and liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to identify proteins in the low-abundance protein enriched fraction. Skimmed milk samples collected for low-abundance milk protein analysis were fractionated and enriched to remove higher abundance proteins. Enriched milk samples were then digested and labeled with isobaric tandem mass tags (TMT) prior to protein identification using LC-MS/MS analysis. There were no changes in the high-abundance protein fraction in response to diet; however, 16 of 127 low-abundance proteins were identified at different relative-abundances due to diet (P ≤ 0.05). While there were no alterations in the metabolic or N status of animals due to GM supplementation, the 12% change in the low-abundance milk protein fraction highlighted the potential for dietary alteration of the bovine milk proteome. A second experiment evaluated the inclusion of alternative forage crops (AFC) as a means to alter the bovine milk proteome. In this experiment, both the skimmed milk and milk fat globule membrane (MFGM) protein fractions were included in analysis. Milk samples were collected from 16 lactating Jersey cattle included in a 21-d grazing experiment, where cows were offered one of two diets. The control group (CON, n=8) grazed a grass-legume pasture mixture containing orchardgrass (Dactylis glomerata), timothy (Phleum pratense), Kentucky bluegrass (Poa pratensis), and white clover (Trifolium repens). The treatment group (AFC, n=8) grazed a similar base pasture that was strip-tilled with oat (Avena sativa), buckwheat (Fagopyrum esculentum), and chickling vetch (Lathyrus sativus) so that the AFC species comprised 10% of the AFC group’s pasture DM intake (DMI). Milk samples were collected for HPLC analysis of the high abundance milk proteins, and LC-MS/MS analysis of the low abundance protein enriched skim milk fraction and MFGM-associated protein fraction. Cows that grazed pastures containing AFC had higher αs1-CAS content (P = 0.005), and higher relative-abundances of 7 low-abundance proteins within the skim milk and MFGM fractions (P ≤ 0.05). While it is plausible that the inclusion of AFC in pasture increased nutrient availability to the mammary gland, the specific mechanisms that could have caused the shifts observed remain unclear. Further investigation is necessary to fully understand the role of diet and the milk protein profile.

Vanderghem Caroline. (2009). La membrane du globule gras du lait : études physico-chimiques et sa valorisation technofonctionnelle dans les babeurres (Thèse de doctorat en Anglais). Gembloux, Belgique, Faculté Universitaire des Sciences Agronomiques de Gembloux. 198p., 14 tabl., 59 fig. Résumé La membrane du globule gras du lait (MGGL) est une structure complexe qui enveloppe, protège et délivre des composants bioactifs et des nutriments dune façon efficace au nouveau-né. Sa structure est unique par rapport à dautres systèmes biologiques de transport de lipides. Lobjectif général de ce travail a été de contribuer à augmenter les connaissances et la compréhension de cet émulsifiant naturel. La première partie de cette thèse concerne létude de la structure de la MGGL. Limportance des protéines membranaires concernant la stabilité et leur disposition dans la MGGL ont été étudiés. Les protéines de la MGGL sont présentes en petite quantité dans le lait et une technique de pré-fractionnement simpose afin de les isoler par rapport aux autres protéines du lait (caséines et protéines du lactosérum). Une technique dextraction qui a été développée dans notre laboratoire a été testée afin disoler la MGGL de la crème laitière. Une analyse détaillée a révélé que cette procédure est très bien adaptée pour lélimination dun maximum de protéines du lait écrémé. Une approche protéomique a été établie et a permis lidentification de protéines majeures de la MFGM ainsi que dautres protéines mineures (GTP-binding proteins, annexins, actin) afin de compléter le protéome. Par la suite, différentes protéases ont été testées en vue dobtenir différents degrés et/ou sélectivité dhydrolyse des protéines de la MGGL. La distribution asymétrique des protéines de la MGGL a été étudiée par une approche basée sur lattaque protéolytique du globule gras natif. Sur base de nos résultats et des données bibliographiques récentes, un nouveau modèle de la structure de la MGGL est proposé. La deuxième partie de cette thèse est consacrée à évaluer les propriétés technofonctionnelles de la MGGL dans les babeurres. Les propriétés interfaciales, moussantes et émulsifiantes du babeurre sont comparées à dautres ingrédients laitiers comme le lait écrémé et le concentré protéique de lactosérum. Nos résultats montrent le bon pouvoir émulsifiant du babeurre en comparaison des autres ingrédients laitiers. Dans les émulsions recombinées, la stabilité envers le crémage a été améliorée et les membranes ont présenté une meilleure résistance face à la coalescence. Les résultats sur la tension interfaciale et les propriétés rhéologiques interfaciales ont été mis en relation avec certaines propriétés des mousses et des émulsions. Vanderghem Caroline. (2009). The milk fat globule membrane: physico-chemical studies and its techno-functional valorisation in buttermilk (Thèse de doctorat). Gembloux, Belgium, Gembloux Agricultural University, 198p., 14 tabl., 59 fig. Summary The milk fat globule membrane (MFGM) is a complex structure that surrounds, protects and delivers bioactive compounds and nutrients in an efficient manner to the neonate. Its structure is unique in relation with any other biological lipid transport system. The overall aim of this work was to contribute to increase the knowledge and comprehension of this natural emulsifier. The first part of this thesis concerned the study of the structure of the MFGM. Membrane proteins were targeted and their importance regarding stability and disposition in the MFGM was studied. MFGM proteins are present at low concentrations in milk and a pre-fractionation of the sample is required in order of MFGM proteins to be visible among other milk proteins (caseins and whey proteins). A mild procedure developed in our laboratory was tested in order to isolate MFGM from cream. Detailed analysis revealed that this procedure is very well suited for the elimination of a maximum of skim milk proteins. A proteomic approach was established and allowed the identification of the most important MFGM proteins and additional minor MFGM proteins for additional completion of the proteome (GTP-binding proteins, annexins, actin). Subsequently, different proteases were screened in an attempt to obtain different degrees and/or selective proteolysis of MFGM proteins. Asymmetric arrangement of MFGM proteins was studied with an approach based on the proteolytic attack on the native fat globule. Based on our results and recent bibliographic data, an updated model of the MFGM structure was proposed. The second part of this thesis was devoted to assess the techno-functional properties of the MFGM in buttermilks. Interfacial, foaming and emulsifying properties of buttermilk were assessed and compared to classic milk ingredients such as skim milk and whey protein concentrate. Our results highlighted that buttermilk possesses good emulsifying power compared to other milk ingredients. In buttermilk recombined emulsions stability towards creaming was improved and the recombined membrane presented a great resistance to coalescence. Results of the interfacial pressure and the interfacial rheological properties were related to some foams and emulsions properties.