Browsing by Author "Fontes, Ana L."
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- 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.
- In situ CLA and CLNA production: a potential strategy to elaborate food products enriched in bioactive fatty acidsPublication . Pimentel, Lígia L.; Fontes, Ana L.; Rodríguez-Alcalá, Luis M.; Gomes, Ana M.
- In situ milk enrichment with microbial CLA/CLNA using vegetable oils as substrate sourcePublication . Fontes, Ana L.; Pimentel, Lígia L.; Domingues, M. Rosário; Rodríguez-Alcalá, Luis M.; Gomes, Ana M.
- Influence of shaking and viable cell numbers on microbial conjugated linoleic acid (CLA) productionPublication . Fontes, Ana L.; Pimentel, Lígia L.; Salsinha, Ana S.; Rodríguez, Juan M.; Domingues, M. Rosário; Rodríguez-Alcalá, Luis M.; Gomes, Ana M.
- 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.
- Modified cellulose nanocrystals encapsulating cannabigerol: a step forward in controlling intestinal inflammatory disordersPublication . Casanova, Francisca; Pereira, Carla F.; Ribeiro, Alessandra B.; Castro, Pedro M.; Martins, Eva; Freixo, Ricardo; Tavares-Valente, Diana; Pimentel, Lígia L.; Fontes, Ana L.; Rodríguez-Alcalá, Luís M.; Fernandes, João C.; Pintado, Manuela E.; Ramos, Óscar L.Cannabigerol (CBG) from Cannabis sativa L. is known for its anti-inflammatory, antibacterial, and antioxidant properties, showing potential against intestinal inflammation. However, its lipophilic nature limits its absorption and stability. Researchers have explored cellulose nanocrystals (CNCs) to deliver lipophilic compounds and enhance their biological outcomes. This study investigated the capability of modified CNC with cetyltrimethylammonium bromide (CTAB) to effectively deliver CBG. The encapsulation process’s impact on cytotoxicity, biological activity, and controlled release during digestion was assessed. Results indicated that CNC-CTAB encapsulation significantly reduced CBG’s cytotoxicity on intestinal cells, allowing safer administration of higher doses. The antioxidant and anti-inflammatory properties of the encapsulated CBG were retained, resulting in a decrease in reactive oxygen species and cytokine levels in intestinal cells. Additionally, the system inhibited the growth of the intestinal pathogen Campylobacter jejuni. The study supports using CNC-CTAB as an efficient delivery system to enhance CBG’s potential against intestinal inflammation. Incorporating this system into food matrices could lead to novel functional foods for managing intestinal inflammation.
- Stability of a fermented milk enriched with microbial CLA/CLNAPublication . Fontes, Ana L.; Pimentel, Lígia L.; Rodríguez-Alcalá, Luis M.; Gomes, Ana M.There is an increasing interest towards the development of innovative value-added food products with a potential to prevent or counteract disease conditions, many times impelled by unbalanced diets, such as cardiovascular diseases and cancer. Several promising food-derived lipids with potential bioactive properties have been identified over the last decade, and these include conjugated linoleic (CLA) and conjugated linolenic (CLNA) acids [1], [2]. Due to concentration and availability limitations in their natural sources (e.g. ruminants’ milk and meat or vegetable oils) [3], [4], the in situ microbial production may reveal itself to be a good strategy to increment CLA/CLNA daily intake. Several probiotic strains have demonstrated the ability to produce CLA/CLNA isomers using linoleic (LA) and alpha-linolenic (α -LNA) acids as precursor substrates, respectively [5], [6]. This research team has previously assayed a combination of screening tools from a wide collection of probiotic strains and selecting the best producer of CLA and CLNA isomers – Bifidobacterium breve DSM 20091 – to proceed afterwards with studies on CLA/CLNA-enriched milk. Seeking to explore industrial viability, commercial edible vegetables oils were applied as precursor substrate sources instead, being previously hydrolysed with lipases to enhance the amounts of free LA/α- LNA. After a series of optimization assays, the flaxseed oil (FSO), which is rich in α-LNA, provided the highest yield of microbial conjugated FA (~1 mg/g) at 2 mg/mL α-LNA. After elaborating the new functional milk, the next required step is evaluating its compositional stability throughout storage. Therefore, the aim of the present work was to investigate i f a microbial CLA/CLNA-enriched fermented milk is stable, in terms of probiotic viable counts, pH and fatty acid (FA) profile, at conditions mimicking shelf-life. Pasteurized cow milk was inoculated with B. breve DSM 20091 and 2 mg/mL of α-LNA (from hydrolysed FSO) or not (control) and distributed by 100 mL containers for each sampling point and in triplicate. The containers were fermented for 22 h at 37 ºC under anaerobic conditions. After fermentation, three containers from each substrate condition were separated for further analysis (T0d) and the rest was stored at 4 ºC, being well sealed with parafilm and protected from light with aluminium foil. Samples were taken from storage each 7 days until the end of the assay (T28d). At each sampling point, it was performed viable cell counting in cys-MRS agar plates and total microbial count in PCA plates, measure of pH and FA analysis content through GC-FID. According to the obtained results, after 7 days of storage, viable cell counts of B. breve DSM 20091 decreased significantly, especially in the enriched milk (from 8.09 to 4.51 log10), and kept decreasing, reaching insignificant counts by the end of the assay. Concerning to total microbial count in PCA, insignificant numbers were detected throughout all storage period at both conditions. In terms of pH, it maintained constant overall (Control: 5.05-5.14; Enriched milk: 4.86-4.99). As for FA profile, in the non-esterified fraction, it was detected higher levels of CLA and CLNA isomers in the enriched milk, and consequently of total polyunsaturated FA (PUFA) as well, at T28d, comparing to all other sampling points, with values of 0.17 mg/g CLA, 1.11 mg/g CLNA and 2.04 mg/g PUFA. In the esterified fraction, was observed variations throughout storage in total saturated FA of control (7.85-9.18 mg/g), and in total monounsaturated FA of both conditions (Control: 2.23- 2.81 mg/g; Enriched milk: 2.19-2.66 mg/g). In conclusion, a microbial CLA/CLNA-enriched fermented milk is not entirely stable during storage, including its CLA/CLNA content, which was in fact increased.
- Stability of a lyophilized milk enriched with microbial CLA/CLNAPublication . Fontes, Ana L.; Pimentel, Lígia L.; Rodríguez-Alcalá, Luis M.; Gomes, Ana M.