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- 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.
- Microbiological in vivo production of CLNA as a tool in the regulation of host microbiota in obesity controlPublication . Pimentel, Lígia Leão; Fontes, Ana Luiza; Salsinha, Ana Sofia; Cardoso, Beatriz Batista; Gomes, Ana Maria; Rodríguez-Alcalá, Luís MiguelMetabolic disorders associated with dietary patterns have become a social and economic problem. Obesity is indeed considered a central feature that increases the risks associated with a vast array of diseases (insulin resistance, type 2 diabetes, fatty liver disease, atherosclerosis, hypertension, stroke, cancer, and asthma), with significant morbidity and mortality. Although lipids may be involved in the development of these illnesses, recent studies have stated their role as cellular mediators and they have been assayed as bioactive compounds in possible treatments. Clear examples are the conjugated isomers of linoleic acid (CLA), which have been associated with antiatherogenic, antioxidative, immunostimulation, and body fat reduction activities. Recently, increased interest in other conjugated PUFAs has emerged, linked to the health-promoting properties of conjugated linolenic acid isomers (CLnA) like C18:3 c9t11c15 (rumelenic acid, RLA). These fatty acids combine in the same molecule a double conjugated bond system with the n3 structure of linolenic acid (ALA; C18:3 c9c12c15) resulting in a high bioactive potential. Animal studies have revealed that CLnA regulates leptin production and increases β-oxidation in the liver thus reducing perirenal and epididymal adipose tissue. Elsewhere it was reported that RLA increased PPARα levels in adipocytes, which suggests that it is a candidate functional ingredient for use in the prevention of obesity, diabetes, and dyslipidemia. CLA and CLnA are natural fatty acids (FAs) mainly found in dairy products and beef, because they are produced as intermediates of the biohydrogenation pathway of PUFAs by ruminal bacteria. Therefore it is hypothesized that other microorganisms may also be able to produce CLA and conjugated FAs. Indeed, recent investigations demonstrated that it is possible to elaborate fermented dairy products containing CLA and CLnA produced by these bacteria. Other authors have identified Bifidobacterium strains with a high rate of substrate conversion to yield CLA, CLnA, and stearidonic acids producing these FAs in vivo, increasing the RA concentration in murine and pigs livers as well as DHA and EPA in mice adipose tissue. An important issue arises from these results: host fatty acid composition can be manipulated by oral administration of CLA-producing microorganisms. In the last 5 years an increasing number of studies have found strong evidence of the role not only of diet but also of human gut microbiota in the development of diabetes and obesity. Indeed, host microbiota regulates the content of triglycerides, cholesterol esters, sphingomyelin, and phosphatidylcholine in the plasma, liver, and adipose tissue as well as energy metabolites. This chapter will review the current state-of-the-art microbiological in vivo production of CLA and CLnA and its effect on host microbiota and health.
- 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.
- Considerations about the in situ derivatization and fractionation of EFA and NEFA in biological and food samplesPublication . Pimentel, Lígia L.; Fontes, Ana L.; Gomes, Ana M.; Rodríguez-Alcala, Luis M.Despite their important role in tissues, fluids and foods, the analysis of non-esterified fatty acids (NEFA) asmethyl esters (NEFAME) is performed using expensive, cumbersome and time-consuming procedures that needs of isolation, fractionation and derivatization steps. However, Yi et al. [1] proposed a promising in situ, single-step procedure to analyze esterified fatty acids (EFA) and NEFA from a same sample on the basis that acylglycerols and free fatty acids can be derivatized using specific reactions. However, according to the data presented in this research work, some modifications need to be performed to increase the reliability of the method: Increment of the transesterification performance by adding hexane to the reaction mixture, decreasing the time for the derivatization of acylglycerols from 10min to 3–4min and stopping the reaction with sulfuric acid. Avoid cross-contamination of the NEFAME extract by adding 500 mu L of water after collection of EFA methyl esters (EFAME). Samples are spiked with three internal standards: a triacylglycerol (to calculate the concentration of EFA), a free fatty acid (to calculate NEFA) and a FAME (to control isolation of FAME and cross-contamination).
- Evidences and perspectives in the utilization of CLNA isomers as bioactive compound in foodsPublication . Fontes, Ana L.; Pimentel, Lígia; Simões, Catarina D.; Gomes, Ana M. P.; Rodríguez-Alcalá, Luís M.Conjugated linolenic acid (CLNA) isomers are promising lipids due to their similarities with CLA but exerting their bioactivity at lower doses; some isomers also belong to the omega 3 family. This review aims to summarize the state of the art about the utilization of CLNA as a functional ingredient. Indeed, in vitro and in vivo studies reported that CLNA exerted anti-cancer, anti-inflammatory, anti-obese and antioxidant activities. However, CLNA has not been tested in humans yet. These compounds are naturally present in meat and milk fat from ruminants but the highest concentrations are found in vegetable oils. Their incorporation in foodstuffs is one of the most effective strategies to elaborate CLNA-enriched products together with the microbiological production. Lactobacilli, propionibacteria and bifidobacteria strains have been assayed to produce CLNA isomers but at the current moment there are not high CLNA concentration products elaborated using these strains. Furthermore, it is known that CLNA are highly prone to oxidation when compared with linoleic acid and CLA but it is unknown the possible effects of elaboration and storage on high CLNA products. The utilization of CLNA as a functional compound remains still a challenge that requires more research to address all the technological and bioactivity aspects about it.