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- Utilization of omega-3 and conjugated fatty acids to develop new antiobesity strategiesPublication . Salsinha, Ana Sofia Nobre; Pintado, Maria Manuela Estevez; Relvas, João Bettencourt; Alcalá, Luis Miguel RodriguesAccording to the World Health Organization's latest data, overweight and obesity prevalence is increasing mostly in urban settings. Diet, particularly the rise of the Western diet, characterized by high-sugar and high-saturated fat consumption, has been pointed out as one important contributor to this trend. Western diet has been shown to detrimentally affect brain function by inducing chronic low-grade inflammation. This neuroinflammation is in part mediated by microglia, so the first part of this study intended to determine how different bioactive fatty acids, namely omega-3 and conjugated fatty acids isomers (conjugated linoleic (CLA) and linolenic acid (CLNA) isomers) modulate microglia activity and if they can suppress the negative effect of obesogenic nutrients (palmitic acid and fructose), intended to mimic in this study the Western diet main ingredients. Importantly, short-time exposure to omega-3, CLA, and CLNA was demonstrated, for the first time, to be sufficient to abolish NF-κB pathway activation, reactive oxygen species production, and the activation of LynSrc in a human microglia cell line, suggesting a potential for neuroprotection. Interestingly, the NF-κB pathway activation was demonstrated for the first time for CLA and CLNA, to be mediated by the GPR120 receptor. Nevertheless, despite this promising potential one of the most straightforward routes of administration is through oral supplementation and the use of enriched oils has been used as a strategy to achieve therapeutical doses of the mentioned bioactive fatty acids. So, to explore the antiobesity potential of omega-3 and conjugated fatty acids, through oral supplementation, bioaccessibility and bioavailability are important parameters to consider. So, in the second stage of this work, the bioaccessibility of these fatty acids in different matrixes was determined using the INFOGEST static in vitro protocol of gastrointestinal tract simulation. After digestion the recovery index for most of the major bioactive fatty acids – punicic acid, the omega-3 EPA, DPA, and DHA fatty acids, and the CLA isomers, rumenic acid, and C18:2 t10c12 - was very low between 1.90-17.03%, demonstrating the negative effect of gastrointestinal digestion upon these fatty acids in this type of matrix. Besides, using a Caco-2/HT29-MTX co-culture it was demonstrated that a significative percentage of these fatty acids is incorporated in the intestinal cells (8.44-26.71%), which may affect their permeability performance and explain the null apparent permeability values obtained. Moreover, due to this observation, it was demonstrated that in fact, most of them remain in the non-bioaccessible/ colon-available fraction. Despite the drawbacks of this observation in terms of brain bioavailability, the potential of gut microbiota modulation was considered. Western diet has been considered one important player in the gut microbiota alterations in industrialized societies and a causal role between gut microbiota changes and obesity has been demonstrated. So, the third stage of this work intended to assess the effects of omega-3, CLA, and punicic acid on human gut microbiota modulation using in vitro fecal fermentations. Indeed, all the samples demonstrated a positive impact, demonstrated by positive relative difference indexes concerning the negative control (RD>0), in the growth of key bacteria that have been associated with a positive effect on obesity treatment namely Akkermansia spp., Bifidobacterium spp., and Roseburia spp. In addition, most samples (Fish oil, Omega-3, CLA, and CLNA capsules content) were able to increase butyrate concentration, a known anti-inflammatory short-chain fatty acid. Moreover, demonstrating the potential of gut-brain axis modulation and its importance in brain function restoration, the sources of omega-3 (Fish oil and Omega-3 capsules content) were able to increase the concentration of GABA and several amino acids that are known precursors of neurotransmitters. Importantly, this first gut microbiota study demonstrated the potential role of these fatty acids in gut microbiota modulation and a possible prediction of their preventive and therapeutical role in obesity. So, a second study intended to assess if the selected oils enriched in omega-3 (Fish oil) and punicic acid (Pomegranate oil) could restore the gut microbiota changes associated with the Western diet that are frequently linked to conditions like obesity and other metabolic diseases, such as type 2 diabetes mellitus. So, Fish oil (as the selected source of omega-3), Pomegranate oil (as a source of punicic acid), and a mixture of both oils (1:1 w/w) were subjected to in vitro fermentations using cecal samples from rats subjected either to a control standard chow diet or to a western diet (high-fat and high-sugar diet). Fish oil+Pomegranate oil was able to increase α-diversity, the relative abundance of the Firmicutes and Bacteroidetes phylum as well as Akkermansia and Blautia, which were affected by the western diet consumption. All samples were able to increase butyrate and acetate concentration in the western diet group. Once again, demonstrating the great potential of these samples to modulate the gut-brain axis, especially in a metabolic syndrome context, tyrosine, a precursor for dopamine and norepinephrine, and GABA concentrations increase in the Fish oil+Pomegranate oil sample from the western diet group. Finally, to tentatively uncover some of the molecular mechanisms behind these bioactive fatty acids' effects on gut permeability and adipogenesis, the role of CB1 in both these processes and GPR120 in adipogenesis was studied. Interestingly, it was demonstrated that CB1 may have a role in intestinal permeability and different bioactive polyunsaturated fatty acids may respond differently to this receptor. Moreover, there seems to be an important role of both GPR120 and CB1 (studied separately) in Fish and Pomegranate oil inhibitory adipogenesis effects in the studied cell model (3T3-L1 adipocytes). In this thesis, it was demonstrated, for the first time, the great potential of using bioactive fatty acids, namely omega-3 and punicic acid to promote a systemic effect in obesity prevention and therapy. Moreover, it was demonstrated a synergistic effect of these bioactive fatty acids, by using a mixture of Fish and Pomegranate oil, to restore the gut microbiota changes associated with western diet consumption. Despite these promising results, further studies using as a first approach animal models (such as rodent models) and afterward a clinical trial using human patients, are required to fully elucidate the bioavailability of such bioactive fatty acids in a dietary context and consequently, their possible beneficial role in brain inflammatory processes either directly or via gut-brain axis modulation.
- Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii groupPublication . Rocha, Joana; Botelho, João; Ksiezarek, Magdalena; Ugarcina Perovic, Svetlana; Machado, Miguel; Carrico, João André; Pimentel, Lígia L.; Salsinha, Sofia; Rodríguez-Alcalá, Luis M.; Pintado, Manuela; Ribeiro, Teresa G.; Peixe, LuísaOne Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus . Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii , respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus , for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).
- Influence of shaking and viable cell numbers on microbial conjugated linoleic acid (CLA) productionPublication . Fontes, Ana Luiza; Pimentel, Lígia; Salsinha, Ana Sofia; Rodriguez, Juan Miguel; Domingues, MR; Rodríguez-Alcalá, Luís M.; Gomes, Ana Maria
- 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.
- Microbial production of conjugated linoleic acid and conjugated linolenic acid relies on a multienzymatic systemPublication . Salsinha, Ana S.; Pimentel, Lígia L.; Fontes, Ana L.; Gomes, Ana M.; Rodríguez-Alcalá, Luis M.Conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have gained significant attention due to their anticarcinogenic and lipid/energy metabolism-modulatory effects. However, their concentration in foodstuffs is insufficient for any therapeutic application to be implemented. From a biotechnological standpoint, microbial production of these conjugated fatty acids (CFAs) has been explored as an alternative, and strains of the genera Propionibacterium, Lactobacillus, and Bifidobacterium have shown promising producing capacities. Current screening research works are generally based on direct analytical determination of production capacity (e.g., trial and error), representing an important bottleneck in these studies. This review aims to summarize the available information regarding identified genes and proteins involved in CLA/CLNA production by these groups of bacteria and, consequently, the possible enzymatic reactions behind such metabolic processes. Linoleate isomerase (LAI) was the first enzyme to be described to be involved in the microbiological transformation of linoleic acids (LAs) and linolenic acids (LNAs) into CFA isomers. Thus, the availability of lai gene sequences has allowed the development of genetic screening tools. Nevertheless, several studies have reported that LAIs have significant homology with myosin-cross-reactive antigen (MCRA) proteins, which are involved in the synthesis of hydroxy fatty acids, as shown by hydratase activity. Furthermore, it has been suggested that CLA and/or CLNA production results from a stress response performed by the activation of more than one gene in a multiple-step reaction. Studies on CFA biochemical pathways are essential to understand and characterize the metabolic mechanism behind this process, unraveling all the gene products that may be involved. As some of these bacteria have shown modulation of lipid metabolism in vivo, further research to be focused on this topic may help us to understand the role of the gut microbiota in human health.
- Characterization of a concentrated second cheese wheyPublication . Faustino, Margarida; Salsinha, Ana Sofia; Pimentel, Lígia; Alcalá, Luis; Freitas, Ana Cristina; Gomes, Ana Maria; Pereira, Carlos Dias; Pintado, Manuela
- Lactobacillus femina sp. nov., a new species of lactobacillus delbrueckii groupPublication . Rocha, Joana; Botelho, João Francisco Tomaz Santos; Ksiezarek, Magdalena; Ribeiro, Teresa Goncalves; Machado, Miguel Paulo; Carrico, Joao Andre; Peixe, Luisa Maria Vieira; Pimentel, Lígia; Salsinha, Ana Sofia; Rodríguez-Alcalá, Luís M.; Pintado, Manuela