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Research conducted for this dissertation had three goals; 1) determine if CLA can induce milk fat depression immediately postpartum, 2) determine if CLA can alter energy availability, 3) determine the mechanism behind the mammary gland's decreased sensitivity to CLA immediately postpartum. The first study provides strong evidence indicating CLA can decrease milk fat synthesis immediately postpartum, but the dose required is approximately 3x greater than in established lactation. This trial also provided evidence that CLA can alter energy status, as CLA decreased days to EBAL nadir by nearly 5 days. This is relevant as recovery of EBAL from its lowest point provides an important signal for initiating ovarian activity and days to nadir is highly correlated with days to first ovulation. Study two was designed to determine if CLA induced milk fat depression could improve energy status during heat stress. Rumen-inert CLA reduced milk fat synthesis, and was able to improve energy availability, but did not increase milk yield or yield of other milk components. Although production was unchanged in this study, the study did provide further evidence that rumen-inert CLA can alter energy availability. Study three utilized intravenous infusion of CLA in cows in mid and early lactation to determine the mechanism for the mammary gland's decreased sensitivity in early lactation. It is postulated that increased fatty acid oxidation and subsequent enhanced levels of circulating NEFA present during the transition period competitively prevent adequate CLA uptake by the mammary gland. In the current study, trans-10, cis-12 CLA concentration in milk was not different between early and established lactation, while milk fat yield was drastically reduced on d 4 and 5 of trans-10, cis-12 CLA infusion in mid lactation cows, but unaltered in early lactation. Further, NEFA levels were nearly 3 fold higher in early lactation than in mid lactation, providing further evidence that increased circulating NEFAs in early lactation are unlikely to be the source of the mammary gland's decreased sensitivity during this time. Do to the variation in gene expression observed in this trial, we were unable to make any definitive conclusions as to the sensitivity of the expression of genes involved in milk lipid synthesis to CLA in early vs. mid lactation.

The objective of these studies was to investigate the milk fat synthesis regulation by transcription factors. In the first study, bovine mammary epithelial (MAC-T) cells were treated with sterol regulatory element binding protein-1 (SREBP-1) specific siRNA. The mRNA and protein expression of SREBP-1 were decreased by more than 90% by siRNA. Fatty acid (FA) synthesis, uptake, and selected lipogenic enzyme expression were reduced in cells treated with SREBP-1 siRNA. Therefore, SREBP-1 plays an important role in integrated regulation of lipid synthesis in MAC-T cells through regulation of key enzymes. In the second study, MAC-T cells treated with hormones or FA were transfected with luciferase reporter constructs containing response elements for SREBP-1, peroxisome proliferator-activated receptor γ (PPARγ), or liver X receptor (LXR). The activation of PPARγ and SREBP-1 were stimulated by insulin and insulin combined with leptin, respectively. Trans-10, cis-12 conjugated linoleic acid (CLA) inhibited SREBP-1 activation, and this inhibition was not attenuated by insulin and leptin. Neither trans-10 nor cis-12 double bond inhibited SREBP-1 activation. Taken together, trans-10 and cis-12 double bonds need to be conjugated in CLA to reduce SREBP-1 activation and this inhibition cannot be overcome by insulin and leptin combination in MAC-T cells. In the third study, lactating dairy cows were intravenously infused with 0.625 g/h trans-10, cis-12 CLA for 14 h. We confirmed the appearance of trans-10, cis-12 CLA in the milk of CLA treated cows. Milk and component yield were not affected by the CLA treatment. The desaturation of stearic acid was reduced by CLA. The mRNA and protein expression of transcription factors or lipogenic enzymes were not affected by trans-10, cis-12 CLA. DNA-binding activities for PPARγ and LXR and the activation of SREBP-1 to its mature form were not changed by the treatment. The infusion time in this study was probably too short to induce any changes in transcription factors and lipogenic enzymes. We confirmed DNA-binding activities of PPARγ and LXR in bovine mammary gland. Overall, a prominent role for SREBP-1 in mammary epithelial cell lipid synthetic pathways was described and regulation of transcription factor activation by trans-10, cis-12 CLA was specific to SREBP-1. / Ph. D.

The milk fat globule membrane (MFGM) obtained from bovine milk is a source of bioactive compounds. In this research, the hypothesis that the MFGM possesses anticarcinogenic capacity and immune modulatory properties was tested, along with the hypothesis that processing history of the fat globules will affect the bio-functionality of the MFGM. Throughout all experiments, lipopolysaccharide (LPS) was minimized by extracting the milk using a catheter, working under aseptic conditions and avoiding contamination via reagents. To study the anticarcinogenic capacity, native MFGM was tested in vitro using two colon adenocarcinoma cell lines (HT-29, Caco-2). Stimulated cells showed a dose-dependent decrease in cell proliferation, starting at very low concentrations (1 μg/mL based on protein) and its efficiency was comparable or even superior to the commercial anticarcinogenic drugs melphalan and C2-ceramide. Cytotoxicity and apoptosis were found to be mechanisms responsible for this bioactivity, as shown by the lactate dehydrogenase enzyme activity and the increase in caspase-3 production. MFGM- immune modulatory capacity was also tested by studying its effects on splenocyte proliferation, apoptosis and cytokine production. While splenocyte proliferation was not affected when the MFGM isolate was used alone, it suppressed cell division in the presence of polyclonal activators (LPS, Concanavalin A) used to simulate inflammatory conditions. Cytokine production suggests inhibition of the splenocytes’ activation process as the mechanism behind its bioactivity. Milk heating caused a significant decrease in bioactivity, indicating that protein interactions and denaturation as well as the structural changes caused by this treatment, directly affect the MFGM bio-functionality. Partial hydrolysis (by trypsin and phospholipase-A2) caused a similar effect, suggesting that not only the phospholipids are involved in the anticarcinogenic capacity, but also, the complex mixture of proteins which form part of the MFGM complete system. In summary, the bioactivity of the MFGM extracted from milk is not limited to its anticarcinogenic but also to its immune regulatory ability, by helping control the response of the immune system when inflammatory conditions arise. However, pre-treatment of the raw material greatly affects this bioactivity, suggesting that greater care may be needed in processing of fat globules to maintain such important characteristics of their components. / Canadian Dairy Council (CDC), Ontario Dairy Council (ODC), Natural Sciences and Engineering Research Council of Canada (NSERC).

Orientadores: Mirna Lúcia Gigante, Renato Grimaldi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T07:11:54Z (GMT). No. of bitstreams: 1 Queiros_MayaradeSouza_M.pdf: 10790431 bytes, checksum: 9180a58c6d003d9587519df936a3423e (MD5) Previous issue date: 2015 / Resumo: Embora muito valorizada por suas características de sabor e aroma, a manteiga tem como desvantagem a difícil espalhabilidade a baixas temperaturas. A modificação das características funcionais da manteiga pode ser alcançada modificando-se sua composição e estrutura física. O objetivo deste trabalho foi avaliar o efeito da adição de oleína sobre as características de composição e estrutura da manteiga. A fração oleína adicionada ao creme de leite antes da fabricação da manteiga foi obtida através do fracionamento a seco duplo da gordura anidra do leite (GAL). O fracionamento duplo da GAL para obtenção da oleína foi realizado em reator encamisado com controle de agitação e temperatura. As frações obtidas foram caracterizadas quanto à composição em ácidos graxos, conteúdo de gordura sólida, isoterma de cristalização, e comportamento térmico. A manteiga foi fabricada utilizando-se creme de leite ou creme de leite adicionado de 50% de oleína. As manteigas foram caracterizadas quanto à composição físico-química e de ácidos graxos, cor instrumental, conteúdo de gordura sólida, ponto de fusão, isoterma de cristalização e comportamento térmico. Foi ainda avaliado o efeito do tempo de exposição à temperatura ambiente sobre a firmeza das manteigas. O delineamento experimental utilizado foi um esquema fatorial 2 x 2 em blocos inteiramente casualizados. O experimento completo foi repetido três vezes e os dados foram avaliados por Análise de Variância (ANOVA) e teste de Tukey para comparação de médias ao nível de significância de 5%. A oleína obtida após duplo fracionamento apresentou características físicas que evidenciaram seu potencial para ser adicionada ao creme de leite, a fim de modificar a funcionalidade da manteiga. A manteiga adicionada de oleína diferiu significativamente da manteiga controle e apresentou menor conteúdo de gordura sólida, maior tempo para cristalizar, menor ponto de fusão, menor gasto de energia para fundir e consequentemente menor firmeza. Estas características sugerem que a menor firmeza observada para a manteiga adicionada de oleína deveu-se, possivelmente, à forma de organização da rede de cristais de gordura sólida na gordura líquida. Quando exposta à temperatura ambiente, a firmeza das manteigas diminuiu ao longo do tempo, no entanto, a manteiga adicionada de oleína apresentou significativamente menor firmeza, tanto após 01 dia de armazenamento a 5 °C, como após 07 dias. O conjunto dos resultados indicou que a manteiga com adição de oleína apresentou menor firmeza sob refrigeração e quando exposta ao ambiente, possivelmente devido à organização da rede de cristais de gordura sólida na maior fração de gordura líquida na manteiga adicionada de oleína / Abstract: Although butter is highly valued for its flavor and aroma characteristics, it has the disadvantage of unsatisfactory spreadability at low temperatures. The functional characteristics of butter can be modified by changing its composition or physical structure. The objective of this study was to evaluate the effect of olein addition on the composition and structure of butter. The olein fraction added to the cream before butter manufacture was obtained by double dry fractionation of anhydrous milk fat (AMF), which was carried out in jacketed reactor with agitation and temperature control. The obtained fractions were characterized according to the fatty acid composition, solid fat content, isothermal crystallization and thermal behavior. Butter was manufactured using commercial cream or cream containing 50% olein. Butter samples were characterized for physicochemical composition and fatty acids, instrumental color, solid fat content, melting point, isothermal crystallization and thermal behavior. The effect of exposure time at room temperature on the firmness of butter was also assessed. The experimental design was a 2 x 2 factorial arrangement in completely randomized blocks with three repetitions. Data were evaluated by analysis of variance (ANOVA) and Tukey's test for means comparison at 5% significance level. The olein fractions obtained by double fractionation presented physical characteristics with potential to be added to the cream to modify butter functionality. Butter containing olein was significantly different from the control, and presented lower solid fat content, higher crystallization time, lower melting point, lower energy required to melt and consequently lower firmness. These results suggest that the lower firmness observed for butter containing olein was possibly due to the structural organization of the solid fat crystals network in the liquid fat. Despite the firmness of butter decreased over time when exposed to room temperature, butter containing olein exhibited significantly lower firmness, both after 01 and 07 days of storage. The results indicated that butter containing olein presented less firmness under refrigeration conditions and when exposed to the environment, possibly due to the organization of solid fat crystals network in the higher liquid fat fraction of butter containing olein / Mestrado / Tecnologia de Alimentos / Mestra em Tecnologia de Alimentos

Cheddar cheese was made by the traditional 4.5-h method from three experimental lots of milk, each standardized to casein/fat ratios of approximately 0.64, 0.67 and 0.70. The effect of casein/fat ratio on milk fat recovery was determined. The effects of milk composition on curd firmness at cutting, cheese composition and resulting yield of cheese were evaluated. Correlations between milk constituents and various cheese components were obtained. Milk fat recovery was unaffected by casein/fat ratios within the limits of 0.64 and 0.71. Average milk fat recovery was 91.58 ± 1.73%. Cheese yield was a function of milk protein, milk fat and cheese moisture; and a modified Van Slyke equation predicted cheese yield better than the original equation within the limits of casein/fat ratio studied. Strong negative correlations were observed between casein/fat ratio and cheese fat and cheese fat in the dry matter whereas positive correlations were observed between casein/fat ratio and cheese protein. At constant protein levels curd firmness increased directly with the amount of fat in cheese milk.

Durante anos, a manteiga foi muito apreciada pelo consumidor, mas por conter grande quantidade de ácidos graxos saturados e colesterol, seu consumo tem diminuído. Recentemente, a descoberta dos malefícios causados pelos ácidos graxos trans presentes nas margarinas produzidas por hidrogenação parcial aumentou o interesse por métodos alternativos de modificação de lipídios. Assim, o objetivo deste trabalho foi desenvolver um lipídio estruturado à base de gordura do leite que apresentasse maiores teores de ácidos graxos insaturados que a manteiga original e, consequentemente, melhor espalhabilidade, além de conter fitosteróis, constituindo a base de um alimento funcional com capacidade de reduzir os níveis de colesterol no sangue. Foram realizadas misturas da gordura do leite (GL) com óleo de girassol (OG) e ésteres de fitosteróis (EF) e foi efetuada interesterificação química e enzimática. As misturas e os lipídios estruturados obtidos foram analisados quanto a composição em ácidos graxos, consistência, conteúdo de gordura sólida (CGS), cinética de cristalização e estrutura cristalina, entre outras propriedades físico-químicas. Foram obtidos produtos contendo cerca de 60% de ácidos graxos insaturados pela adição de até 40% de OG e 25% de EF à gordura do leite, que originalmente possuía cerca de 33% de ácidos graxos insaturados. A interesterificação química melhorou a plasticidade das misturas, enquanto que a modificação enzimática não modificou de maneira expressiva sua consistência. A adição de pequenas quantidades de OG à GL provocou grande diminuição de sua consistência, enquanto que a diminuição do CGS ocorreu em proporções semelhantes às esperadas por interpolação linear. A edição de vídeos que permitam a observação da formação da estrutura cristalina de gorduras mostrou ser uma ferramenta de grande utilidade no estudo da cristalização de óleos e gorduras. A emulsão preparada com a mistura contendo 25,0% de EF, 7,5% de OG e 67,5% de GL apresentou diminuição expressiva na consistência quando comparada à manteiga, representando um efeito benéfico. / For many years, butter has been held in the highest esteem by consumer but its world consumption has steadily declined due to its high contents of saturated fatty acids and cholesterol. More recently, trans fatty acids, which are commonly resent in hydrogenated argarines, have been found to cause detrimental effects when consumed in high amounts. This observation has increased the interest in alternative methods of lipid modification. Therefore, the objective of this work was to develop a milkfat based structured lipid, presenting higher contents of unsaturated fatty acids than original butter and consequently better spreadability, besides containing phytosterols and serving as a base of a functional food with a cholesterol lowering health claim. Blends containing milkfat (MF), sunflower oil (SO) and phytosterol esters (PE) were prepared and then chemically and enzimatically interesterified. The fatty acid composition, consistency, solid fat content (SFC), crystallization kinetics and crystal structure, among other physical and chemical properties were analyzed. The blends presented about 60% of unsaturated fatty acids with the addition of up to 40% of SO and 25% of PE to MF, which originally presented close to 33% of unsaturated fatty acids. Chemical interesterification improved the plasticity of the blends, while the enzymatic reaction did not substantially alter it. The addition of small amounts of SO and PE to MF caused a great decrease in its consistency, while SFC decreased almost proportionally to the addition of these components. The edition of videos made possible the observation of crystal structure formation and it represented a tool of great importance in the fats and oils crystallization research. The emulsion prepared with the blend containing 25.0% of PE, 7.5% of SO, and 67.5% of MF presented a substantial improvement in consistency when compared to butter commercially available.

O presente trabalho objetivou avaliar o efeito da gordura do leite bovino na resistência térmica do Mycobacterium fortuitum. Amostras de leite bovino integral e desnatado foram contaminadas com inóculo padronizado de M. fortuitum, atingindo a concentração de aproximadamente 107 UFC/mL de leite, e submetidas à pasteurização lenta (65ºC/30mim.). Foi realizada a contagem do agente nos tempos 0, 5, 10, 15, 20, 25 e 30 minutos de pasteurização (em meio Lowenstein-Jensen, incubação a 37ºC/5 dias), e os resultados, em logarítmo, foram plotados em diagrama de dispersão com posterior regressão linear para construção da curva de sobreviventes (ou curva de morte térmica). Foram obtidas 3 curvas para o leite integral e 3 para o leite desnatado, porém, para o cálculo do valor D65ºC, utilizou-se a melhor reta obtida para cada tipo de leite. Encontraram-se valores D65ºC do Mycobacterium fortuitum iguais a 18,02 minutos para o leite integral e 7,82 minutos para o desnatado; a gordura do leite influenciou no padrão da curva de morte térmica e teve efeito protetor sobre o M. fortuitum. Conclui-se que a pasteurização lenta é capaz de reduzir 3,85 log de M. fortuitum em leite desnatado e 1,67 log em leite integral, resultando em diferentes níveis de segurança ao consumidor. / This study aimed to evaluate the effect of cow milk fat on the thermal resistance of Mycobacterium fortuitum. Samples of whole and skim cow milk were contaminated with a standardized inoculum of M. fortuitum, until 107 cfu/mL, and heated at 65ºC/30 minutes (Holder Pasteurization). It was repeated tree times, with milk (whole and skim) from the same animal. Survivors were enumerated by plate count method, after heating for 0, 5, 10, 15, 20, 25 and 30 minutes (in Lowenstein-Jensen medium, incubation at 37ºC/5 days) and the logarithmic results were plotted on dispersion diagram with posterior linear regression for the construction of the survivors? curve (or thermal death curve). Those data resulted on 3 curves for the whole milk and 3 for the skim milk, although, for the D65ºC value calculation, it was selected the best-fit line for each kind of milk. The D65ºC value for Mycobacterium fortuitum achieved was 18,02 minutes for the whole milk and 7,82 minutes for the skim milk; the milk fat influenced the pattern of thermal death curve and had a protective effect on the M. fortuitum. It was inferred that the holder pasteurization (65ºC/30 min) is capable to reduce 3,85 log of the M. fortuitum at skim milk and 1,67 log at whole milk, resulting in different security levels for the customers.