Bioactive peptides produced from the mussel mytilus galloprovincialis by enzymatic hydrolysis with corolase

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.info:eu-repo/semantics/publishedVersio

Bioactive potential and chromatographic characterization of body mucus from Portugal coastal fish halobatrachus didactylus

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.info:eu-repo/semantics/publishedVersio

Flow analysis method for hydroxyproline determination to assess collagen content in fish skin

Collagen is a protein with several applications, with weak antigenicity, low toxicity, and high nutritional value. Usually, it is extracted from bovine skin, but a project was designed proposing an alternative to extract collagen from fish skin. This alternative was tested by assessing the collagen content in fish skin based on the determination of hydroxyproline (HYP), one of the most abundant amino acid present in collagen (Sotelo et al., 2016). Therefore, the determination of HYP requires the hydrolysis of the fish skin, to break collagen in its amino acids, and the HYP value quantified relates to the collagen content. This was previously assessed to be 38 μg of HYP per mg of pure collagen. In this context, the aim of the described work was to develop an automatic method based on flow injection analysis approach to determine HYP. The determination was based on the redox reaction with permanganate (Farokhi et al., 2002) and the consequent decrease of colour intensity registered. The best conditions for the determination were studied, namely, reagent concentration, sample volume, flow rate and reaction time. The developed method enables the determination of the HYP in a faster, simpler and accurate way, with less toxic solutions.info:eu-repo/semantics/publishedVersio

Collagen determination in fish skin: development of a flow analysis system for quantification of hydroxyproline

Collagen is a protein with various applications, namely in the food area. It has valuable properties, since it is a polymer with weak antigenicity, low toxicity, and high nutritional value, among other features [1]. Its extraction from mammalian sources, i.e., bovines, is decreasing due to health and environmental problems and, therefore, fish have become a good alternative for collagen resources [2].One way to quantify the collagen present in fish skin, in order to obtain high-value fractions, is the determination of hydroxyproline (HYP), an amino acid highly present in collagen [1]. The determination of HYP from fish skin requires the hydrolysis of a skin section, to break collagen in its amino acids and the HYP value quantified is compared to the amount present in pure collagen, studied previously (38 μg of HYP per mg of pure collagen). The quantification of HYP is based on its oxidation combined with the reaction with DAB (dimethylaminobenzaldehyde) that forms a chromophore-coloured product. The HYP can then be correlated with the spectrophotometric measurement of this coloured product. A batchwise approach was performed to study the best reaction conditions, namely different reagents, heating times and proportions.The main goal of this work is to develop an automated flow injection analysis (FIA) method, to miniaturize the determination of HYP. Several operation parameters like flow rates, number of channels, tube diameters and lengths of reactors will be studied to optimize the developed FIA method.info:eu-repo/semantics/publishedVersio

UHPLC-DAD-MSn analysis of phenolic compounds bioavailability throughout in vitro simulated gastrointestinal digestion

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.info:eu-repo/semantics/publishedVersio

gBIOT - Nutraceutical biopolymeric-biocatalytic microbot against gut inflammatory disorders

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.info:eu-repo/semantics/publishedVersio

The health-promoting potential of Salix spp. bark: from the phenolic composition to the in vitro bioactivity

info:eu-repo/semantics/publishedVersio

P82 Peptide fraction identification by SE-HPLC and LC-MS/MS analysis of the body mucus from Portugal coastal fish Halobatrachus didactylus

The 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.info:eu-repo/semantics/publishedVersio

Production of enzymatic hydrolysates from blue shark skin by-product using a biorefinery approach

Portugal is the third country in the world with the highest fish consumption, with an average of 59 kg per capita per year. That results in a considerable amount of fish waste, up to 30% of which can be skin and bones. Fish skin mainly comprises collagen (more than 70% of its total protein content). Collagen is a structural protein found in animal extracellular matrices and has a wide range of commercial applications s. In the past, bovine and porcine sources have been the primary sources of collagen, but due to religious beliefs and some infectious diseases, other sources are in debate. Marine-based collagen is an attractive alternative for the industry. Blue shark skin is a by-product of the fishing industry that is rich in collagen. This study used a biorefinery approach to valorize blue shark skin waste. We used a process we reported for another type of marine matrix (cod skin) to obtain collagenic peptide hydrolysates and non-collagenic protein hydrolysates. We evaluated two proteases: alcalase (of microbial origin) and bromelain (of vegetable origin). We performed a multifactorial optimization study for both matrices and both enzymes, considering as responses the degree of hydrolysis and antioxidant activity for all models and, additionally, protein solubilization for the case of collagen hydrolysates. In all cases, alcalase was the enzyme with the best performance. Once we validated the predictive models, we chose the most viable conditions, and the production of these hydrolysates was scaled up to pilot production. The optimized hydrolysates present molecular profiles, bioactivities, and organoleptic characteristics of interest for the nutraceutical and cosmetic industries (in the case of collagen hydrolysates) and aquaculture (in the case of non-collagen hydrolysates). In conclusion, this study has demonstrated that blue shark skin waste can be valorized to produce collagenic peptide hydrolysates and non-collagenic protein hydrolysates, which have potential applications in the nutraceutical, cosmetic, and aquaculture industries.info:eu-repo/semantics/publishedVersio

Integrated ultrafiltration, nanofiltration, and reverse osmosis pilot process to produce bioactive protein/peptide fractions from sardine cooking effluent

Sardine cooking effluents contain a high level of organic matter, such as proteins and lipids, which allows them to be forward into a chain exploiting high added-value compounds attained from these effluents, increasing their economic value while reducing their environmental effect. Thus, the purpose of this work was to develop an innovative pilot-scale integrated membrane process, with or without enzymatic hydrolysis, to obtain fractions with high protein/peptide and low NaCl contents, as well as optimized bioactive properties. The research strategy followed involved the use of ultrafiltration (UF) and nanofiltration (NF) technologies of the pretreated sardine cooking effluent followed by reverse osmosis (RO) at a pilot scale levels. Moreover, it allowed for the attainment of fractions rich in protein/peptides that might be used in the food, pharmaceutical, or cosmetic industries, particularly after RO, as they present a lower NaCl content. The RO retentate (hydrolyzed sample) coupled with UF and NF resulted in the fractions with the best bioactive properties (higher antioxidant capacity and antimicrobial activity) of all the analyzed samples. Overall, the current work demonstrated the feasibility of exploiting liquid by-products as a source of functional components as well as reinforcing this strategy’s potential relevance in future effective management strategies for this type of effluents.info:eu-repo/semantics/acceptedVersio