Browsing by Author "Cunha, Sara A."
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- Anti-hypertensive activity of horse mackerel pulverized with three different extracts from marine originPublication . Nova, Paulo; Cunha, Sara A.; Barros, Diana; Ramos, Carlos; Castro, Inês; Vaz-Velho, Manuela; Pintado, Manuela; Gomes, AnaHypertension is a serious risk factor and the most prevalent trigger to fatal cardiovascular diseases such as stroke and myocardial infarction. The marine environment is a unique source of molecules with biological activity such as antioxidant, anti-coagulant and anti-hypertensive activities. In this study, the anti-hypertensive activity of horse mackerel fillets (HMF) pulverized with each of the following marine extracts (derived from enzymatic hydrolysis) were determined: microalgae (Tetraselmis sp. incubated with subtilisin and cellulase), Mussel_Sub (Mytilus galloprovincialis minced and incubated with subtilisin protease) and Mussel_ Pro+Alc (Mytilus galloprovincialis boiled and incubated with Protamex and Alcalase). Horse mackerel fillets were evenly sprayed with each extract and frozen until subsequent analyses. Non-sprayed HMF were used as control. Aqueous extracts were prepared from HMF by sonication. Anti-hypertensive activity was determined by the angiotensin I-converting enzyme (ACE) inhibitory activity method. Data are presented in Table.1 as average±standard deviation of two replicates. Table 1. Anti-hypertensive activity of Horse Mackerel fillets sprayed with Extracts from Marine Origin. Horse mackerel fillet showed by itself an interesting anti-hypertensive profile. Notwithstanding, the tested marine-derived extracts effectively increased its biological potential(up to four-fold), with the best anti-hypertensive profile being achieved with microalgae and Mussel_Pro+Alc extracts. These extracts have an enormous potential to be used in the development of innovative food products that emphasize functionality, convenience, nutrition and health – goal of the project VALORMAR (POCI-024517-FEDER)(PPS1).
- Anti-hypertensive activity of horse mackerel pulverized with three different extracts from marine originPublication . Nova, Paulo; Cunha, Sara A.; Barros, Diana; Ramos, Carlos; Castro, Inês; Vaz-Velho, Manuela; Pintado, Manuela; Gomes, Ana
- Antioxidant peptides produced from unexplored marine resourcesPublication . Cunha, Sara A.; Castro, Rita; Pintado, ManuelaThe sea contains about one half of the global biodiversity, thereby being a good source for novel compounds of interest, such as bioactive peptides. Mussels are consumed and appreciated over the world, although commercialized mussels are pre-selected before being delivered for sale, so small or broken mussels are discarded. Microalgae are photosynthetic organisms, easy to cultivate since they do not require cultivable land. The microalgae used in this work are produced in a company in which the CO2 generated in the production of cement is injected to microalgae growth tubes transforming it in oxygen, resulting in a decrease of CO2 emission. The chosen species, Chlorella vulgaris and Nannochloropsis oceanica, have interesting protein content, making them a promising sustainable protein source. Discarded mussels from the specie Mytilus galloprovincialis, supplied by a national mussel trading company, and the microalgae grown up with CO2 generated from cement production, were used to produce water soluble extracts rich in proteins and bioactive peptides, showing potential to the development of sustainable industrial alternatives, promoting new perspectives for a circular economy. Four extracts were produced and analysed: Mussel_Sub, hydrolysed with subtilisin (1.5%,3h); Mussel_Cor, hydrolysed with corolase (3%,3h); Chlorella, hydrolysed with acetic acid (0.5%,1h), cellulase (5%,2h) and subtilisin (3.9%;2h); and Nannochloropsis, hydrolysed with cellulase (5%,2h) and subtilisin (1.7%,5h). The protein content was determined by Kjeldahl and the antioxidant activity by ORAC and ABTS assays. The Chlorella extract showed 44.71±1.75% protein, 462.83±39.37 (ORAC) and 76.12±7.53 (ABTS) μmol TE/g. Nannochloropsis showed 31.01±0.27% protein, 361.32±49.29 (ORAC) and 68.07±6.97 (ABTS) μmol TE/g. Mussel_Cor showed 48.00±0.15% protein, 389.50±0.29 (ORAC) and 62.76±8.88 (ABTS) μmol TE/g; Mussel_Sub showed 45.23±0.14% protein, 485.62±60.65 (ORAC) and 66.11±2.35 (ABTS) μmol TE/g. The enzymatic hydrolysis of marine species may allow to produce extracts rich in proteins and antioxidant peptides useful for industrial applications, contributing to the valorisation of these species and to a circular economy, since mussel commercialization generates waste and microalgae can transform CO2 from other industries to O2, reducing the environmental impact.
- Assessment of the bioaccessibility and bioavailability prediction of omega 3 and conjugated fatty acids by in vitro standardized digestion model (INFOGEST) and cell modelPublication . Salsinha, Ana Sofia; Cunha, Sara A.; Machado, Manuela; Rodriguez-Alcalá, Luís Miguel; Relvas, João B.; Pintado, ManuelaOmega 3 EPA and DHA are polyunsaturated fatty acids with relevant health benefits. Conjugated linoleic and linolenic acids are known for their anti-carcinogenic effect, anti-inflammatory properties and body weight reduction. To achieve therapeutical doses, high amounts of these fatty acids’ food sources must be consumed. Thus, the intake of enriched oils with a high concentration of these fatty acids is often used. But several factors influence their bioavailability. Here, by using the INFOGEST static in vitro protocol of gastrointestinal tract digestion it was studied the bioaccessibility of these fatty acids in different matrixes: Pomegranate and Fish oil and omega 3, CLA and CLNA soft-gel enriched capsules. After digestion, the Recovery Index for the major bioactive PUFAs are very low: Pomegranate oil is 2%, Fish oil 11–13%, CLNA 17%, CLA 6% and Omega 3 capsules 3%. Higher initial concentrations of these PUFAs seem to be related to higher degrees of oxidation. In Pomegranate oil, CLNA and Omega 3 capsules, the digestion process negatively influenced the antioxidant potential. The opposite was verified for the Fish oil and CLA capsules. Importantly, bioaccessibility studies of similar matrixes are very scarce and intestinal permeability is absent in most of the studies. Intestinal permeability studies were performed using a Caco-2/HT29-MTX co-culture: there is significative incorporation of the bioactive fatty acids into the intestinal cells, which may affect their permeability performance. Interestingly, most fatty acids remain in the non-bioaccessible fraction which may be relevant when designing oral routes of administration and in gut microbiota modulation.
- Bioactive hydrolysates from chlorella vulgaris: optimal process and bioactive propertiesPublication . Cunha, Sara A.; Coscueta, Ezequiel R.; Nova, Paulo; Silva, Joana Laranjeira; Pintado, Maria ManuelaMicroalgae have been described as a source of bioactive compounds, such as peptides. Microalgae are easy to produce, making them a sustainable resource for extracting active ingredients for industrial applications. Several microalgae species have interesting protein content, such as Chlorella vulgaris with around 52.2% of protein, making it promising for peptide hydrolysate production. Therefore, this work focused on the production of water-soluble hydrolysates rich in proteins/peptides from the microalgae C. vulgaris and studied bioactive properties. For that, a design of experiments (DOE) was performed to establish the optimal conditions to produce hydrolysates with higher levels of protein, as well as antioxidant and antihypertensive properties. Four experimental factors were considered (cellulase percentage, protease percentage, hydrolysis temperature, and hydrolysis duration) for three responses (protein content, antioxidant activity, and antihypertensive activity). The optimal conditions determined by the DOE allowed producing a scaled-up hydrolysate with 45% protein, with antioxidant activity, measured by oxygen radical absorbance capacity assay, of 1035 µmol TE/g protein, IC50 for angiotensin-converting enzyme inhibition activity of 286 µg protein/mL, and α-glucosidase inhibition of 31% (30 mg hydrolysate/mL). The obtained hydrolysates can be used as functional ingredients for food and nutraceuticals due to their antioxidant, antihypertensive, and antidiabetic potential. Moreover, the antioxidant potential of the extracts may be relevant for the cosmetic industry, especially in antiaging formulations.
- Chemical and antioxidant properties of solvent and enzyme-assisted extracts of Fucus vesiculosus and Porphyra dioicaPublication . Nova, Paulo; Cunha, Sara A.; Costa-Pinto, Ana R.; Gomes, Ana MariaExtraction strategies impact the efficiency and nature of extracted compounds. This work assessed the chemical composition and antioxidant capacity of ethanolic, hydroethanolic, and aqueous versus enzyme-assisted extracts (isolated or with the sequential use of alcalase®, cellulase®, and viscozyme®) of the macroalgae Fucus vesiculosus (brown, Phaeophyceae) and Porphyra dioica (red, Rhodophyta. For both macroalgae, enzyme-assisted extraction (EAE) was the most efficient process compared to solvent-assisted extraction (SAE), independent of solvent. Fucus vesiculosus extraction yields were higher for EAE than for SAE (27.4% to 32.2% and 8.2% to 30.0%, respectively). Total phenolics content (TPC) was at least 10-fold higher in EAE extracts (229.2 to 311.3 GAE/gextract) than in SAE (4.34 to 19.6 GAE/gextract) counterparts and correlated well with antioxidant capacity (ABTS and ORAC methods), with EAE achieving values up to 8- and 2.6-fold higher than those achieved by SAE, respectively. Porphyra dioica followed F. vesiculosus’s trend for extraction yields (37.5% to 51.6% for EAE and 5.7% to 35.1% for SAE), TPC, although of a lower magnitude, (0.77 to 8.95 GAE/gextract for SE and 9.37 to 14.73 GAE/gextract for EAE), and antioxidant capacity. Aqueous extracts registered the highest DPPH values for both macroalgae, with 2.3 µmol TE/gextract and 13.3 µmol TE/gextract for F. vesiculosus and P. dioica, respectively. EAE was a more efficient process in the extraction of soluble protein and reducing sugars in comparison to SAE. Furthermore, an improved effect of enzyme-assisted combinations was observed for almost all analyzed parameters. This study shows the promising application of enzyme-assisted extraction for the extraction of valuable compounds from F. vesiculosus and P.dioica, making them excellent functional ingredients for a wide range of health and food industrial applications.
- Chlorella vulgaris extracts as modulators of the health status and the inflammatory response of gilthead seabream juveniles (Sparus aurata)Publication . Reis, Bruno; Ramos-Pinto, Lourenço; Cunha, Sara A.; Pintado, Manuela; Silva, Joana Laranjeira da; Dias, Jorge; Conceição, Luís; Matos, Elisabete; Costas, BenjamínThis study aimed to evaluate the effects of short-term supplementation, with 2% Chlorella vulgaris (C. vulgaris) biomass and two 0.1% C. vulgaris extracts, on the health status (experiment one) and on the inflammatory response (experiment two) of gilthead seabream (Sparus aurata). The trial comprised four isoproteic (50% crude protein) and isolipidic (17% crude fat) diets. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of C. vulgaris biomass (Diet D1); 0.1% inclusion of C. vulgaris peptide-enriched extract (Diet D2) and finally a 0.1% inclusion of C. vulgaris insoluble fraction (Diet D3). Diets were randomly assigned to quadruplicate groups of 97 fish/tank (IBW: 33.4 ± 4.1 g), fed to satiation three times a day in a recirculation seawater system. In experiment one, seabream juveniles were fed for 2 weeks and sampled for tissues at 1 week and at the end of the feeding period. Afterwards, randomly selected fish from each group were subjected to an inflammatory insult (experiment two) by intraperitoneal injection of inactivated gram-negative bacteria, following 24 and 48 h fish were sampled for tissues. Blood was withdrawn for haematological pro-cedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters. The anterior gut was also collected for gene expression measurements. After 1 and 2 weeks of feed-ing, fish fed D2 showed higher circulating neutrophils than seabream fed CTR. In contrast, dietary treatments induced mild effects on the innate immune and antioxidant functions of gilthead sea-bream juveniles fed for 2 weeks. In the inflammatory response following the inflammatory insult, mild effects could be attributed to C. vulgaris supplementation either in biomass form or extract. However, the C. vulgaris soluble peptide-enriched extract seems to confer a protective, anti-stress effect in the gut at the molecular level, which should be further explored in future studies.
- Enzymatic approach for the extraction of bioactive fractions from red, green and brown seaweedsPublication . Teixeira-Guedes, Catarina; Gomes-Dias, Joana S.; Cunha, Sara A.; Pintado, Manuela E.; Pereira, Ricardo N.; Teixeira, José A.; Rocha, Cristina M. R.Although several enzymatic approaches have been applied with this intent, the sequential use of enzymes covering both cellulolytic and proteolytic activity has never been performed in seaweeds. Sequential use of these enzymes improved the overall extraction yield by up to 160%, 30% and 80% in the different seaweeds when compared to the control condition (water extraction), use of a carbohydrases’ cocktail alone and use of proteases alone, respectively. Regarding the proximate composition of extracts, it proved to be an efficient approach for the solubilization of carbohydrates (up to 28% in G. vermiculophylla, 66% in P. dioica, 77% in U. rigida and 35% in F. vesiculosus) and protein (up to 55% in G. vermiculophylla, 47% in P. dioica, 52% in U. rigida and 42% in F. vesiculosus). For all biomasses, the combination of enzymes induced a significant increase in antioxidant activity, not only by the increase of phenolic compounds but also by the hydrolysis of protein to peptides. Moreover, extracts from red seaweeds displayed prebiotic activity which can be ascribed to their increased content in oligosaccharides and protein/peptides. Overall, the sequential use of enzymes with different activities demonstrated to be an efficient approach for extracting functional fractions to be used as functional ingredients to improve the nutritional value (e.g. in proteins) and/or to include antioxidant and prebiotic features in the food product.
- Enzymatic hydrolysis allows an integral valorization of nannochloropsis oceanica resulting in the production of bioactive peptide extracts and an eicosapentaenoic acid enriched fractionPublication . Cunha, Sara A.; Coscueta, Ezequiel R.; Alexandre, Agostinho M. R. C.; Partidário, Ana Maria Carvalho; Fernández, Naiara; Paiva, Alexandre; Silva, Joana Laranjeira; Pintado, Manuela E.Nannochloropsis oceanica is a microalga with relevant protein content, making it a potential source of bioactive peptides. Furthermore, it is also rich in fatty acids, with a special focus on eicosapentaenoic acid, an omega-3 fatty acid mainly obtained from marine animal sources, with high importance for human health. N. oceanica has a rigid cell wall constraining protein extraction, thus hydrolyzing it may help increase its components' extractability. Therefore, a Box-Behnken experimental design was carried out to optimize the hydrolysis. The hydrolysate A showed 67±0.7% of protein, antioxidant activity of 1166±63.7 μmol TE/g of protein and an ACE inhibition with an IC50 of 379 μg protein/mL. The hydrolysate B showed 60±1.8% of protein, antioxidant activity of 775±13.0 μmol TE/g of protein and an ACE inhibition with an IC50 of 239 μg protein/mL. The by-product showed higher yields of total fatty acids when compared to “raw” microalgae, being 5.22 and 1%, respectively. The sustainable developed methodology led to the production of one fraction rich in bioactive peptides and another with interesting EPA content, both with value-added properties with potential to be commercialized as ingredients for different industrial applications, such as functional food, supplements or cosmetic formulations.
- Enzymatic hydrolysis of the microalgae tetraselmis sp. production, characterization and ultrafiltration of antimicrobial extractsPublication . Cunha, Sara A.; Castro, Rita de; Pintado, Manuela E.Microalgae have been studied for their bioactive compounds, such as peptides, with enzymatic hydrolysis appearing as the most frequently used method for producing bioactive peptides. Microalgae are easy to produce, making them a sustainable resource for the extraction of active ingredients for industrial applications. Therefore, this work focused on the production of water-soluble extracts rich in proteins and bioactive peptides with molecular weight lower than 3 KDa. The intact Tetraselmis sp. used showed 23.0% of protein, 6.2% of lipids, 18.9% of carbohydrates and 17.0% of fibers. Extracts were produced from the microalgae by enzymatic hydrolysis, using ultrapure water as the solvent. First, the mixture was incubated at 50ºC for 2h with 4.7% of cellulase. Secondly, the mixture was incubated with 5% of a subtilisin protease at 40ºC for 2h, to hydrolyze the proteins into smaller peptides. The produced extract was then fractionated by ultrafiltration in a Tangential Flow Filtration System (Cogent® μScale) using a cut-off of 3KDa. The resulting extract