Browsing by Author "Coscueta, E. R."
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- Bioactive peptides produced from the mussel mytilus galloprovincialis by enzymatic hydrolysis with corolasePublication . Cunha, S. A.; Castro, R. de; Coscueta, E. R.; Pintado, M. E.Bioactive peptides have been produced from several marine sources since they can exhibit positive effects for humans and animals. Mytilus galloprovincialis is highly consumed in several countries and has a meat rich in proteins, which has been described as a source of bioactive peptides with relevant properties such as antioxidant, anti-hypertensive and antimicrobial activities. Mussel commercialization generates waste, since the small or broken mussels are discarded. Thus, in this work discarded mussels were used with the goal of producing water soluble extracts rich in proteins and bioactive peptides. The mussel meat was firstly minced, and then submitted to different conditions of enzymatic hydrolysis, using the protease corolase to generate peptides. The variable factors evaluated were temperature, incubation time and enzyme concentration. To achieve the optimal extraction conditions, a Box-Behnken experimental design was performed using statgraphic centurion software. Factorial design allowed the evaluation of the effects of the three factors on protein release, antioxidant and anti-hypertensive properties of the extracts. The protein content of each extract was determined by Kjeldahl, the antioxidant activity was determined by oxygen-radical absorbance-capacity (ORAC) assay and anti-hypertensive property was determined by the inhibition method of Angiotensin-I converting enzyme (ACE). The incubation of the minced mussel meat with 3% of enzyme, at 40 ºC for 3 hours, appears to be the best conditions to obtain the best results of protein extraction, and antioxidant and anti-hypertensive properties. With these conditions, the obtained extracts showed 48% of protein content, an antioxidant activity of 821 μmol TE / g of extract, and an ability of inhibiting the activity of ACE in 61% (using a concentration of 10 mg / mL). Thus, the factorial design allowed to confirm the combination of experimental factors that leads to the most efficient extraction of antioxidant and anti-hypertensive peptides of the mussel Mytilus galloprovincialis. In conclusion, the use of discarded mussels to produce functional ingredients for food, cosmetic and pharmaceutic industries contribute to valorise world waste in a circular economy context.
- Bioactive potential and chromatographic characterization of body mucus from Portugal coastal fish halobatrachus didactylusPublication . Cunha, M. Fernandez; Coscueta, E. R.; Brassesco, M. E.; Almada, F.; Gonçalves, D.; Pintado, M.The body mucus in fish provides a stable physical or chemical barrier against invading pathogens. In mucus, antimicrobial peptides are secreted as a response to immune stimulation. Studies have shown antimicrobial activity against multidrug-resistant pathogens and low toxicity to eukaryotic cells. Previously, body mucus from five captive Halobatrachus didactylus individuals was collected. We aim to explore the mucus studying molecules with bioactive potential. Size exclusion highperformance liquid chromatography (SEHPLC) analyses were performed on the five body mucus samples showing a similar molecular size distribution with a maximum peak of ca. 800 Da. These five mucus samples were pooled to assess the following bioactivities: antioxidant (ABTS and ORAC), antimicrobial (minimal inhibitory concentration), and cytotoxicity (Caco2 and HaCaT human cell lines). The protein content in the mucus, determined by the bicinchoninic acid methodology, was 16836 ± 1020 µg BSA/mL. The antioxidant activity resulted in 268 ± 11 µmol TE/g mucus protein for ABTS and 306 ± 11 µmol TE/g mucus protein for ORAC. The antibacterial activity was assessed against five pathogenic bacteria: Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, within minimal inhibitory concentrations of 421 to 105 µg mucus protein/mL. Moreover, the mucus showed noncytotoxic for Caco2 cells in concentrations between 196 to 25 µg mucus protein/mL, while it showed cytotoxicity for HaCaT cells. In the future, liquid chromatographytandem mass spectrometry (LCMS/MS) analysis will be performed to determine the molecules behind these bioactivities, namely antimicrobial peptides.
- Collagen-based bioactive hydrolysates production from Blue Shark skinPublication . Brassesco, M. E.; Coscueta, E. R.; Pintado, Maria ManuelaPortugal is the country with the highest levels of fish consumption in the world with an average of 59 kg per capita year. As a result, a huge amount of fish residues is produced, of which up to 30% may be skin and bone. Is well-known that fish skin has more than 80% of its total protein content as collagen. This structural protein is present in the extracellular matrices of animals and has widely applications in food, cosmetic, biomedical, and pharmaceutical industries. For decades, the main industrial exploitations of collagen have been bovine and porcine origins. However, due to religious beliefs and infectious diseases, other collagen sources are being debated. In this regard, the use of collagen with marine origin is being considered highly attractive by the industry as an important alternative source. Blue shark skin, which is an abundant by-product of Portugal processing industry rich in collagen, represents a potential source of this protein. Thus, a collagen hydrolysate was obtained from Blue Shark skin with bromelain, a plant enzyme, for the first time. As a result, low molecular weight hydrolysates were obtained with a protein concentration of 0.5%(p/p). The antioxidant activity was analysed in vitro, obtaining a result of 4.077 µmol Trolox Equivalent/mg protein. The ACE inhibition activity was also analysed obtaining an IC50 of 14.3 ug protein/mL at 1h hydrolysis. Therefore, it was possible to obtain an ingredient (hydrolysed collagen) that is currently required by industries such as cosmetic, food and nutraceutical, with high potential antioxidant and antihypertensive activities. For this, it was possible to apply a sustainable process that consumes less time and uses an alternative and abundant raw material.
- Collagen-based bioactive hydrolysates production from blue shark skinPublication . Brassesco, M. E.; Coscueta, E. R.; Pintado, M.Portugal is the country with the highest levels of fish consumption in the world with an average of 59 kg per capita year. As a result, a huge amount of fish residues is produced, of which up to 30% may be skin and bone. Is well-known that fish skin has more than 80% of its total protein content as collagen. This structural protein is present in the extracellular matrices of animals and has widely applications in food, cosmetic, biomedical, and pharmaceutical industries. For decades, the main industrial exploitations of collagen have been bovine and porcine origins. However, due to religious beliefs and infectious diseases, other collagen sources are being debated. In this regard, the use of collagen with marine origin is being considered highly attractive by the industry as an important alternative source. Blue shark skin, which is an abundant by-product of Portugal processing industry rich in collagen, represents a potential source of this protein. Thus, a collagen hydrolysate was obtained from Blue Shark skin with bromelain, a plant enzyme, for the first time. As a result, low molecular weight hydrolysates were obtained with a protein concentration of 0.5%(p/p). The antioxidant activity was analysed in vitro, obtaining a result of 4.077 µmol Trolox Equivalent/mg protein. The ACE inhibition activity was also analysed obtaining an IC50 of 14.3 ug protein/mL at 1h hydrolysis. Therefore, it was possible to obtain an ingredient (hydrolysed collagen) that is currently required by industries such as cosmetic, food and nutraceutical, with high potential antioxidant and antihypertensive activities. For this, it was possible to apply a sustainable process that consumes less time and uses an alternative and abundant raw material.
- Exploring the role of human gut microbiota in the phenolic compound's metabolism: an in vitro approachPublication . Pais, A. C. S.; Ribeiro, T. B.; Coscueta, E. R.; Pintado, M. M.; Silvestre, A. J. D.; Santos, S. A. O.
- gBIOT - Nutraceutical biopolymeric-biocatalytic microbot against gut inflammatory disordersPublication . Sousa, A. S.; Pintado, M. M.; Matos, R. D.; Sousa, C. C.; Machado, M. F.; Coelho, M.; Rodrigues, P. M.; Magalhães, A. M.; Coscueta, E. R.gBiOT is a novel project that proposes micro-robotic technology for nutraceuticals and therapeutic foods for gastrointestinal tract disorders. Gastrointestinal disorders have high prevalence and morbidity, leading to neoplasia and cancer. Recent advances in nanotechnology have opened up new opportunities in the food sector, allowing for the development of health-food products containing nanosized ingredients that can benefit the entire food chain. However, colon-targeted drug delivery systems have gained the most attention as potential carriers for the local treatment of colonic disorders. While current delivery systems have some benefits, they must be improved to be more than passive delivery systems. Advanced steps would include developing biocompatible devices that can actively identify inflamed regions in the gastrointestinal tract, counteract proinflammatory metabolites, and release drugs to alleviate abnormality. Such devices could propel themselves in the gastrointestinal tract environment, making them active in various tasks. gBiOT aims to address these unmet needs by developing a modular microbot prototype that executes intelligent functions under gastrointestinal disorders. The project is developing functionalized microbots that will test to assess the microbiota interaction and preclinical tests. This would lead to the development of practical solutions for the prophylaxis and control of gastrointestinal diseases with minimal impact on patient quality of life and via sustainable methodologies.
- P82 Peptide fraction identification by SE-HPLC and LC-MS/MS analysis of the body mucus from Portugal coastal fish Halobatrachus didactylusPublication . Cunha, M.; Coscueta, E. R.; Brassesco, M. E.; Almada, F.; Gonçalves, D.; Pintado, M. ManuelaThe mucus covers the fish's body, working as a protective barrier. Besides physical protection, mucus provides molecules that protect the fish from pathogens damaging 1,2. These include antimicrobial peptides secreted in the mucus, which play an essential role in defense against microbial pathogens since these belong to the innate immune system2,3. In this study, two adult Halobatrachus didactylus individuals were captured from the wild in Sesimbra. Then, mucus collection was performed by scraping the dorsal-lateral body of the fish with a sponge. Our objective was the identification of new peptides with bioactive potential in mucus samples by chromatography analysis. Size exclusion highperformance liquid chromatography (SE-HPLC) analysis performed on mucus samples from the two individuals revealed a similar profile with an intense highlight peak which resulted in a distribution of about 775 Dalton. With interest in that peak, the two mucus samples were pooled for fractionation by SEC. The resulting fraction was analyzed by liquid chromatography-tandem mass spectrometry (LCMS/MS) to identify the most probable peptide sequences. Identification from databases did not provide reliable results, indicating a lack of information on the matrix analyzed. We resorted to de novo sequencing with good results using PEAKS Studio software. Five identified peptides were selected according to their bioactivities predicted in silico. Furthermore, the five identified peptides were synthesized, and the molecular size was validated by SE-HPLC analysis. Overall, this chromatographic approach enabled the identification of promising peptides, which bioactivities will be evaluated in vitro in future work.
- Production, characterization and ultrafiltration of bioactive extracts derived from the microalgae Senedesmus obliquusPublication . Cunha, S. A.; Castro, R. de; Coscueta, E. R.; Silva, J. L. da; Pintado, M. E.Algae are very abundant worldwide and contains compounds with high biological value, such as proteins, amino acids, antioxidants. It is noteworthy that some algae species contain protein levels similar to meat, soybean and milk. Micro and macroalgae have been studied as a source of bioactive compounds, such as bioactive peptides, with properties of interest for humans and animals. In vitro, enzymatic hydrolysis is one of the most described methods for producing peptides. Thus, in this work the microalgae Senedesmus obliquus was used with the goal of producing water soluble extracts rich in proteins and bioactive peptides. The Senedesmus obliquus used showed 45.7% of protein, 9.1% of lipids, 15.6% of carbohydrates and 15.8% 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 a cellulase to weaken the cellular wall and help releasing proteins. Secondly, the mixture was incubated with a protease at 40ºC for 2h, to hydrolyse the proteins into smaller peptides. One half of the produced extract was fractionated by ultrafiltration in a Tangential Flow Filtration System (Cogent μScale) using a cut-off of 3KDa. The protein content was determined by Kjeldahl, the antioxidant activity was determined by ORAC and ABTS assays and anti-hypertensive property was determined by the inhibition method of Angiotensin-I converting enzyme (iACE). The hydrolysis strategy resulted in high levels of hydrolysis with a yield of 52% of soluble protein. The biological activity of non-fractionated extract showed ORAC and iACE results of 530 μmol TE/g of extract and an IC of 200 μg protein/mL, respectively. The showed ORAC results of 597 μmol TE/g of extract. So, due to its protein richness and interesting bioactivities, Senedesmus obliquus extracts may have potential to be used in food and cosmetic industries.
- UHPLC-DAD-MSn analysis of phenolic compounds bioavailability throughout in vitro simulated gastrointestinal digestionPublication . Pais, A. C. S.; Coscueta, E. R.; Pintado, M. M.; Silvestre, A. J. D.; Santos, S. A. O.Phenolic compounds, one of the most widely distributed and structural diverse plant secondary metabolites families, have been the focus of several studies due to their vast range of biological activities (such as antioxidant, anti-inflammatory and/or antiproliferative). Since they are commonly present in human diet, phenolic compounds could be responsible for human health beneficial effects, preventing some disorders. Notwithstanding, these health effects are strictly dependent on their bioavailability, which consists in the amount of each ingested compound that reaches the target tissue where it can have a promising biological effect. Therefore, compound’s structure, human enzymatic activity and gut microbiota are some of the numerous factors that influenced phenolic compounds bioavailability, and consequently their human health beneficial effects.In this vein, the bioavailability of phenolic compounds from different classes, particularly, flavonols (rutin), flavanones (naringenin and naringin), dihydrochalcones (phloretin) and tannin monomeric units (phloroglucinol), were evaluated in an in vitro simulated gastrointestinal digestion and further analyzed and quantified through ultra-high performance liquid chromatography with diode-array detection and coupled to electrospray ionization tandem mass spectrometry (UHPLC-DAD-MSn). Most of them showed a bioavailability >70% in intestinal digestion phase and seemed to be absorbed, reaching the systemic circulation. Thus, these results could be a future remark to evaluate the human health effects of promising phenolic compounds combination, or of plant-based extracts with a similar composition or even extracts enriched with them.