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- Suitable simple and fast methods for selective isolation of phospholipids as a tool for their analysisPublication . Pimentel, Lígia; Fontes, Ana Luiza; Salsinha, Sofia; Machado, Manuela; Correia, Inês; Gomes, Ana Maria; Pintado, Manuela; Rodríguez-Alcalá, Luis MiguelLipids are gaining relevance over the last 20 years, as our knowledge about their role has changed from merely energy/structural molecules to compounds also involved in several biological processes. This led to the creation in 2003 of a new emerging research field: lipidomics. In particular the phospholipids have pharmacological/food applications, participate in cell signalling/homeostatic pathways while their analysis faces some challenges. Their fractionation/purification is, in fact, especially difficult, as they are amphiphilic compounds. Moreover, it usually involves SPE or TLC procedures requiring specific materials hampering their suitableness for routine analysis. Finally, they can interfere with the ionization of other molecules during mass spectrometry analysis. Thus, simple high‐throughput reliable methods to selectively isolate these compounds based on the difference between chemical characteristics of lipids would represent valuable tools for their study besides that of other compounds. The current review work aims to describe the state‐of‐the‐art related to the extraction of phospholipids using liquid‐liquid methods for their targeted isolation. The technological and biological importance of these compounds and ion suppression phenomena are also reviewed. Methods by precipitation with acetone or isolation using methanol seem to be suitable for selective isolation of phospholipids in both biological and food samples.
- Impact of sterilization on phytosterols in canned tuna-based productsPublication . Machado, Manuela; Pimentel, Lígia L.; Rodríguez-Alcalá, Luis M.; Pisco, Ana; Lopes, Margarida; Gomes, Ana M; Pintado, ManuelaPhytosterols (PS) are characterized by a tetracyclic ring and a side chain linked to carbon 17. They can be classified according to the presence or absence of a double bond at .5 position (sterols or stanols respectively)(1,2). These compounds occur naturally in all foods of plant origin, such as vegetable oils, nuts, seeds, grain products, fruits and vegetables. Normal daily intake from natural sources is 200–400 mg/day (3). PS enriched foods are considered a valuable option as part of a healthy diet (1). The beneficial effects of these compounds have been demonstrated by several studies and consequently the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA) approved their use in functional foods. EFSA recommends a continuous daily intake of 3 g of PS during two to three weeks to obtain their claimed effects. In this context, the main objectives of this study were the characterization of the sterols profile of a commercial functional PS-rich ingredient as well as the evaluation of its stability after sterilization (119°C; 45min) of a canned tuna-based product enriched in this ingredient. Accordingly, the sterol profile was assayed by GC-FID (4). The results showed that the sterilization process did not affect the sterol profile, with ß-sitosterol being the predominant sterol in all the tested samples. PS content in the novel tuna-based product complies with EFSA recommendations regarding cardiovascular health.
- Functional lipid enriched dairy products geared towards obesity management : technological feasibility and nutraceutical potentialPublication . Machado, Manuela de Fátima Ferraz; Pintado, Maria Manuela Estevez; Gomes, Ana Maria Pereira; Alcalá, Luís Miguel RodríguezThe number of people afflicted by excess weight has been growing each year thus making obesity a worldwide public health problem. Furthermore, conventional treatment regiments (surgery or drug based) are of difficult access to the public and their results more often than note fall short of the desired effects. Thus, alteration of eating behaviours has become the main recommendation for obesity management as it is easier to implement, easier to access and has a wider reach for a larger number of citizens. Nowadays, one of the main dietary drawbacks is the high saturated fat content of some foodstuffs which creates various health problems. However, there are also some health benefits related with fat ingestion, as polyunsaturated ones are responsible for reducing cholesterol and triglycerides levels in blood. Considering this dichotomy this thesis main goal was to understand if it was possible to develop bioactive lipids rich dairy products capable of modulating or exerting a positive effect upon obesity related metabolism. To that end, the work was divided in 4 distinct stages. In stage one, three different vegetable oils (pomegranate, coconut, and avocado) were selected due to the differences in their fatty acids profile and posteriorly their chemical composition and biological potential was characterized. The data obtained showed that of the three oils pomegranate was richer in conjugated fatty acids (73% of the total fatty acids), coconut richer in medium chain fatty acids (60% of the total fatty acids) and, the avocado oil was an excellent source of monounsaturated fatty acids (80% of the total fatty acids). From a biological perspective, all three oils significantly reduced adipocytes’ lipid accumulation in (79% for pomegranate, 76% for avocado and 49% for coconut oil, respectively) and were capable of modulating gut cells’ inflammatory response, reducing IL-6 pro-inflammatory cytokine’ secretion (33% pomegranate oil, 22% coconut oil and 11% avocado oil). The addition of vegetable oil into foods has several challenges with one of them being their susceptibility to oxidation. For this reason, the second step of the work consisted in the development of a carboxymethyl cellulose (hydrophilic component) based bigel in conjunction with either Tween 80 or Geleol to protect the oil during processing and the gastrointestinal tract passage. It was found that the solution developed allowed to reduce the degradation of the main fatty acids of each oil throughout the gastrointestinal tract. On stage three, vegetable oils were incorporated into dairy products. The first approach consisted in their incorporation into yogurts either in its free or bigel form. The data obtained showed that oils addition did not negatively affect the yogurts rheological characteristics, however, the addition of coconut oil bigel impairs the yogurts visual appearance. In terms of nutritional profile, the oil’s addition led to a significant increase (over 50%) in their essential fatty acids content. Following the simulated digestion of the yogurts the data obtained showed that the digested samples reduced lipidic accumulation in hepatocytes (8 to 17% for the non-induced model and 30 to 40% for the steatosis induced model) and in adipocytes (23 to 25%), with the best performer being for the avocado oil yogurt (13% and 25%, respectively). The incorporation in bigel was not a valid alternative for pomegranate oil, as during the digestive process it showed a high fatty acids loss (64%). Thus, a chitosan-based microparticle (2% w/v) solution for incorporation of the pomegranate oil in yogurts was developed. This approach allowed for an increase in the amount of conjugated fatty acids that reached the intestine and, consequently, increase the yogurts bioactive potential. This was reflected in the yogurts capacity to modulate adipocytes metabolism, as seen by the reduction of 77% in leptin secretion while simultaneously increasing by 50% adiponectin levels, and in the reduction of pro-inflammatory cytokines secretion (IL-6 and TNF-α by 50% and 12%, respectively) in colon cells. As dairy products are among the most relevant and consumed products worldwide, a second development avenue, in the form of fresh cheese, was explored. In this approach in addition to the oil’s incorporation the produced products were supplemented with probiotic microorganisms (Lactiplantibacillus plantarum 299v e Bifidobacterium animalis Bo), as these have been shown to possess an important anti-obesogenic role. The data obtained showed that the fresh cheese was a better matrix than yogurt, as it presented a higher oil retention percentage and consequently a higher nutritional potential, due to the higher fatty acid content. Additionally, fresh cheeses showed a better bioactive potential than yogurts, particularly due to their higher capacity to reduce leptin secretion (87% for cheese vs 80% for yogurts). The fourth and final stage consisted in using the dairy product prototype (avocado oil bigel fresh cheese), which showed the highest bioactive potential, and perform in-depth studies using more complex cellular models. To do so, the amount of fatty acids (oleic and palmitic) capable of permeating through a simulated intestinal barrier (Caco-2/HT29-MTX co-culture) was evaluated and the permeated fatty acids’ modulation of adipocytes metabolism and macrophages inflammatory response was studied. The data showed that permeated fatty acids reduced leptin (8.7%) and the pro-inflammatory cytokines MPC-1 (96%) and IL-1β (78%) in adipocytes. Similarly, for macrophages a significant reduction of inflammatory response modulation was observed (60% in IL-6 secretion). In general, all prototypes developed could reduce lipidic accumulation in both hepatocytes and adipocytes, with the different fatty acids’ profiles leading to distinct modulation of adipokines secretion. Pomegranate yogurts showed an elevated capacity to reduce leptin secretion when in comparison with the coconut and avocado ones. However, this did not reflect itself in the adiponectin secretion as the increase registered was similar for the three oils. Similarly, to the adipocyte’s metabolism modulation the samples impact upon the macrophages inflammatory response also appeared to be lipidic profile dependent, with only the pomegranate oil yogurts being capable of reducing TNF-α secretion in LPS stimulated macrophages. On the other hand, all yogurts were capable of reducing IL-6 secretion. Considering the results obtained, the produced bioactive lipids functionalized dairy products showed promising anti-obesity results and potential to be used as viable food alternatives in obesity management and prevention.