Video files: Some images may require stereoscopic glasses / Fat in milk and cream is present as tiny droplets, which are each enveloped in a thin membrane, called the milk fat globule membrane (MFGM). The MFGM can easily be damaged by factors such as pumping the milk and applying other forms of agitation. MFGM damage is believed to reduce processing efficiency and compromise the quality of manufactured products. A comprehensive review of the literature showed that our understanding of changes occurring in the MFGM post secretion of the fat globule by the mammary secretory cell is still rudimentary. Furthermore, it was found that a fundamental understanding of MFGM damage in raw milk is lacking. Hence, this study sought to develop analytical techniques for studying the MFGM. Fluorescent probes were identified that associated with the MFGM (bovine, ovine, human) in one of two ways: either by embedding in the phospholipid bilayer (lipophilic probe) or by binding to carbohydrate moieties of glycosylated chains in the glycocalyx (lectin probes). The use of these probes, in combination with either conventional fluorescence microscopy or confocal laser scanning microscopy, allowed 2-D images and 3-D images of fat globules to be made. Application of water-soluble lipophilic probes and the lectin wheat germ agglutinin (WGA) directly to milk allowed the staining of the MFGM in its native environment. Variable distribution patterns of the probes in the MFGM were observed, which suggests that the MFGM of fat globules in harvested milk is structurally and chemically heterogeneous both within and among globules from the same species and between species, and even among fat globules within the milk of an individual animal. Furthermore, the binding behaviour of WGA to the MFGM of native fat globules (in bovine milk) and washed fat globules (in model systems) following heat treatment implicated β-lactoglobulin, α-lactalbumin, immunoglobulin M and/or the glycosylated proteins Periodic acid Schiff 6/7 in the disappearance of fat globule aggregation upon elevated heat treatment of milk. The results of the current study showed that the use of membrane-specific fluorescent probes, particularly in combination with confocal laser scanning microscopy, has significant potential for providing real time structural and chemical information about the MFGM in matrices such as harvested milk and milk products. In addition to the fluorescence microscopy techniques, development of other techniques was also conducted. Flow cytometry was shown to have significant potential for the quantitative determination of various properties of fat globules and their membranes. Although no suitable sample preparation technique could be developed in this study, atomic force microscopy is believed to have significant potential for studying structural and physical properties of the MFGM. Selective harvesting of individual fat globules was shown to be possible by using a micromanipulator. In future work, this technique is expected to be used in combination with fluorescence microscopy, or atomic force microscopy. The present study has shown that the development and application of novel analytical techniques has advanced, and in the future will further advance, understanding of the MFGM.

Orientadores: Mirna Lucia Gigante, Rafael Jimenez-Flores / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-11T21:12:34Z (GMT). No. of bitstreams: 1 Costa_MarceladeRezende_D.pdf: 1269005 bytes, checksum: be9b972dd7766eba5cc6917dd666c5f0 (MD5) Previous issue date: 2008 / Resumo: O leitelho de soro é um subproduto do processamento de manteiga a partir do creme de soro. Esse subproduto lácteo contém fragmentos da membrana do glóbulo de gordura do leite (MGGL), material rico em componentes com funções nutricionais e efeitos benéficos à saúde, destacando-se os fosfolipídeos. O objetivo desse trabalho foi obter um ingrediente lácteo enriquecido em fosfolipídeos da MGGL a partir do leitelho de soro utilizando a associação de duas tecnologias de fracionamento: ultrafiltração e extração com fluido supercritico (EFS). O leitelho de soro foi submetido à ultrafiltração e a cinco diafiltrações a 25 °C e o retentado obtido foi seco em spray-dryer, obtendo-se o leitelho de soro em pó (LSP), o qual foi submetido a três ciclos de extração a 50 °C com dióxido de carb ono supercrítico. As matériasprimas, produtos finais e uma amostra comercial de leitelho tradicional em pó (LTP) foram analisados quanto a sua composição centesimal, teor de fosfolipídeos, perfis lipídico e protéico. Os leitelhos de soro em pó, submetidos ou não à EFS, e o LTP foram avaliados quanto ao tamanho de partículas e suas propriedades funcionais (solubilidade protéica e capacidade emulsificante) em pH 5 e 7. A filtração em membrana reduziu em 74 e 96% os teores de lactose e cinzas no retentado e aumentou os de proteínas, lipídeos e fosfolipídeos em 91, 190 e 300%, respectivamente. A EFS reduziu em 55,4% o teor de lipídeos, removendo exclusivamente lipídeos apolares, com isso aumentou em 71% o teor de fosfolipídeos no leitelho de soro em pó, resultando em um produto com 73% de proteínas, 21% de lipídeos, 3% de lactose, 3% de cinzas e 12% de fosfolipídeos, em base seca. As propriedades funcionais dos leitelhos de soro em pó, submetidos à extração supercrítica (LSP-EFS) ou não (LSP), foram pouco ou não afetadas pelo pH, enquanto o abaixamento de pH prejudicou as características do LTP, devido ao alto teor de caseínas nesse tipo de pó em relação aos de leitelhos de soro. Os pós em soluções (5% de proteína) apresentaram de 69 a 84% de suas partículas, em % volumétrica, entre 10 e 100 µm. A solução de LTP em pH 5 foi a que apresentou a maior quantidade de partículas acima de 100 µm. LSP e LSP-EFS apresentaram solubilidades protéicas em torno de 86 e 84%, respectivamente, independente do pH. O LTP teve a solubilidade reduzida de 86 para 73% quando o pH foi reduzido de 7 para 5. Em pH 7, as emulsões (20% óleo de canola, 1% proteína) de LSP e de LSPEFS apresentaram melhor estabilidade (IC de 0,2 e 0,4%, respectivamente) do a emulsão de LTP (IC de 3,0%). Em pH 5, a emulsão de LSP-EFS foi a que mostrou melhor estabilidade, apresentando um IC de 7%, valor cerca de 82% menor do que os das emulsões de LSP e LTP. Os leitelhos de soro em pó obtidos podem ser considerados ingredientes com características promissoras, associando propriedades tecnológicas, especialmente para uso em alimentos de baixo pH, e conteúdo de compostos potencialmente benéficos à saúde, principalmente após o tratamento com extração supercrítica / Abstract: OBTENTION OF DAIRY INGREDIENT ENRICHED IN POLAR LIPIDS FROM WHEY BUTTERMILK. Whey buttermilk is a by-product from the whey cream processing into butter. This dairy by-product contains milk fat globule membrane (MFGM) fragments, a material rich in components with nutritional functions and beneficial health effects, especially phospholipids. The objective of this research was obtaining a dairy ingredient enriched in MFGM phospholipids from whey buttermilk using the association of two fractionation technologies: ultrafiltration and supercritical fluid extraction (SFE). Whey buttermilk was submitted to ultrafiltration and five diafiltrations at 25 °C. The retentate was spray-dried and later s ubmitted to three cycles of extraction at 50 °C with supercritical carbon dioxi de. Raw materials, final products and a commercial traditional buttermilk powder sample (BMP) were analyzed for gross composition, lipid and protein profiles, and phospholipids content. Whey buttermilk powders, before and after the SFE, and the BMP had particle size and some functional properties (protein solubility and emulsifying capacity) evaluated in pH 5 and 7. Membrane filtration reduced in 74 and 96% lactose and ash contents in the retentate and increased proteins, lipids and phospholipids in 91, 190 and 300%, respectively. SFE reduced in 55.4% the lipids content, removing exclusively non-polar lipids, while increased in 71% the phospholipids content in whey buttermilk powder, resulting in a product with 73% of proteins, 21% of lipids, 3% of lactose, 3% of ash and 12% of phospholipids, in dry matter basis. Functional properties of the whey buttermilk powders, treated (WBP-SFE) or not through SFE (WBP), were little or not affected by pH, while dropping the pH impaired the BMP features, due the high casein content in this type of powder in relation to the ones from whey buttermilk. The powders in solutions (5% protein) presented from 69 to 84% of the particles, in volume%, between 10 and 100 µm. The BMP solution in pH 5 was the one with the biggest amount of particles above 100 µm. WBP and WBP-SFE showed protein solubilities around 86 and 84%, respectively, independent of pH. BMP had the solubility reduced from 86 to 73% when pH was reduced from 7 to 5. In pH 7, the emulsions (20% canola oil, 1% protein) of the WBP and the WBP showed better stability (CI of 0.2 and 0.4%, respectively) than the BMP emulsion (CI of 3.0%). When in pH 5, WBP-SFE emulsion had the best stability, presenting CI of 7%, value about 82% smaller than the ones of WBP and BMP emulsions. Whey buttermilk powders obtained in this work can be considered ingredients with promising features, combining technological properties, especially for use in low pH foods, and content of components with potential health benefits, mainly after the supercritical extraction treatment / Doutorado / Doutor em Tecnologia de Alimentos

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.

La lipolyse est une réaction enzymatique qui influence négativement les qualités organoleptiques et technologiques du lait. La lipolyse spontanée (LS) correspond à la part de la lipolyse qui dépend de l’animal et du système d’élevage. La LS résulte de l’action de la lipoprotéine lipase (LPL) et de ses cofacteurs sur les globules gras (GG). L’objectif de la thèse a été de comprendre les variations de LS à l’échelle zootechnique et à l’échelle du lait. Les vaches (VL) ont pu être classées en 2 groupes selon leur phénotype : « susceptible » et « non susceptible » à la LS, confirmant l’importance de l’effet individu. Chez les VL susceptibles, nous avons confirmé un effet de la race/génétique, de la parité, du stade physiologique, du moment de la traite, de la fréquence de traite et de l’alimentation.La LS était plus élevée dans les laits issus de la traite du soir, chez les VL Holstein, génotypées KK au locus de DGAT-1. Elle l’était également en fin de lactation et en début de lactation uniquement chez les multipares hautes productrices. Un bilan énergétique négatif pourrait expliquer ces variations. L’augmentation de la fréquence de traite, la restriction alimentaire, l’alimentation à base d’ensilage de maïs comparé à l’herbe conservée/fraîche et la supplémentation lipidique ont également augmenté la LS. Le mécanisme d’action implique probablement une inhibition de la LS par la protéose peptone 5. La membrane des GG semblerait avoir un rôle crucial dans le maintien de l’intégrité du GG, l’interaction avec la LPL et l’équilibre des cofacteurs. L’impact des facteurs zootechniq / Lipolysis is an enzymatic reaction which leads to off-flavor in milk and impairs technological properties of milk. Spontaneous lipolysis (SL) is the fraction of lipolysis which depends on cows and breeding systems. SP corresponds to the hydrolysis of milk fat in milk fat globules (MFG) by the lipoprotein lipase (LPL) and its cofactors. The aim of the PhD was to understand SL variations at both zootechnical and biochemical levels. Cows were sorted in two groups according their phenotype: “susceptible” and “non-susceptible” to SL, confirming the strong impact of the individual effect. Among cows “susceptible” to SL, we confirmed the effects of breed/genetics, parity, physiological stage, milking moment, milking frequency and feeding systems. SL was higher in evening milks of Holstein cows and of cows having the KK genotype at the DGAT-1 locus. SL was higher in late lactation and, in early lactation only for high merit multiparous cows, probably in relation to negative energy balanceAn increase in milking frequency, feeding restriction, maize silage based diets compared to fresh grass and conserved grass based diets and lipid supplementation enhanced SL. At a biochemical level, LS might be inhibited by proteose peptone 5. The MFG membrane might play an important role on MFG integrity, LPL and MFG interactions, and cofactors balance. The impact of zootechnical and biochemical factors on SL is still difficult to hierarchize

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