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- Enzymatic soy protein hydrolysis: a tool for biofunctional food ingredient productionPublication . Coscueta, Ezequiel R.; Campos, Débora A.; Osório, Hugo; Nerli, Bibiana B.; Pintado, ManuelaThis work aimed to evaluate the digestive stability of the peptides previously identified from a Corolase PP soy protein hydrolysate (SPH) and to respond to the uncertainty about the merit of controlled hydrolysis. For this purpose, we applied an empirical and theoretical analysis, determining peptide sequences, oxygen radical scavenging (ORAC) and ACE inhibitory (iACE) activities, and the effect of hydrolysis on solubility. Results showed that during digestion most of SPH peptides were degraded as smaller ones. However, both SPH bioactivities improved significantly after digestion (3.9 ± 0.1 μmol TE/mg protein for ORAC and IC50 = 52 ± 4μg protein/mL for iACE) with similar values for soy protein isolate (SPI). With respect to solubility, the controlled hydrolysis considerably increased this functional property. In conclusion, the results indicated that controlled enzymatic hydrolysis of SPI with Corolase PP produced an ingredient more apt to be incorporated in certain nutritional or nutraceutical formulations.
- Chitosan-olive oil microparticles for phenylethyl isothiocyanate delivery: optimal formulationPublication . Coscueta, Ezequiel R.; Sousa, Ana Sofia; Reis, Celso A.; Pintado, ManuelaPhenylethyl isothiocyanate (PEITC), a chemopreventive compound, is highly reactive due to its considerably electrophilic nature. Furthermore, it is hydrophobic and has low stability, bioavailability and bioaccessibility. This restricts its use in biomedical and nutraceutical or food applications. Thus, the encapsulation of this agent has the function of overcoming these limitations, promoting its solubility in water, and stabilizing it, preserving its bioactivity. So, polymeric microparticles were developed using chitosan-olive oil-PEITC systems. For this, an optimisation process (factors: olive oil: chitosan ratio and PEITC: chitosan ratio) was implemented through a 3-level factorial experimental design. The responses were: the particle size, zeta-potential, polydisperse index, and entrapment efficiency. The optimal formulation was further characterised by FTIR and biocompatibility in Caco-2 cells. Optimal conditions were olive oil: chitosan and PEITC: chitosan ratios of 1.46 and 0.25, respectively. These microparticles had a size of 629 nm, a zeta-potential of 32.3 mV, a polydispersity index of 0.329, and entrapment efficiency of 98.49%. We found that the inclusion process affected the optical behaviour of the PEITC, as well as the microparticles themselves and their interaction with the medium. Furthermore, the microparticles did not show cytotoxicity within the therapeutic values of PEITC. Thus, PEITC was microencapsulated with characteristics suitable for potential biomedical, nutraceutical and food applications.
- Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysisPublication . Coscueta, Ezequiel R.; Amorim, Maria M.; Voss, Glenise B.; Nerli, Bibiana B.; Picó, Guillermo A.; Pintado, M. E.Enzymatic hydrolysis of soybean meal protein isolate (SPI) obtained under two temperature conditions with Corolase PP was studied, assessing the impact of hydrolysis on potential antioxidant and antihypertensive activities. The protein was isolated from soybean meal under controlled conditions of time and temperature (70 C, 1 h; 90 C, 30 min). Degree of hydrolysis assessed the progress of hydrolysis at different sampling times. For hydrolysates the antioxidant and angiotensin-converting-enzyme (ACE) inhibitory activities were measured. As observed, the DH was increasing until reaching 20% at 10 h with disappearance of globular proteins and generation of low molecular weight peptides (less than 3 kDa). A significant increase in antioxidant and ACE inhibitory capacities was observed. Five main peptides were identified, which may explain through their sequences the bioactive properties analyzed. Through this study was possible to obtain for the first time with Corolase PP soy hydrolysates with potential antioxidant and ACE inhibitory activities, which can be used to obtain new added value functional ingredients from soy meal.
- Hydrolysate from mussel mytilus galloprovincialis meat: enzymatic hydrolysis, optimization and bioactive propertiesPublication . Cunha, Sara A.; Castro, Rita de; Coscueta, Ezequiel R.; Pintado, ManuelaMussel production generates losses and waste since their commercialisation must be aligned with target market criteria. Since mussels are rich in proteins, their meat can be explored as a source of bioactive hydrolysates. Thus, the main objective of this study was to establish the optimal production conditions through two Box–Behnken designs to produce, by enzymatic hydrolysis (using subtilisin and corolase), hydrolysates rich in proteins and with bioactive properties. The factorial design allowed for the evaluation of the effects of three factors (hydrolysis temperature, enzyme ratio, and hydrolysis time) on protein/peptides release as well as antioxidant and anti-hypertensive properties of the hydrolysates. The hydrolysates produced using the optimised conditions using the subtilisin protease showed 45.0 ± 0.38% of protein, antioxidant activity via ORAC method of 485.63 ± 60.65 µmol TE/g of hydrolysate, and an IC50 for the inhibition of ACE of 1.0 ± 0.56 mg of protein/mL. The hydrolysates produced using corolase showed 46.35 ± 1.12% of protein, antioxidant activity of 389.48 ± 0.21 µmol TE/g of hydrolysate, and an IC50 for the inhibition of ACE of 3.7 ± 0.33 mg of protein/mL. Mussel meat losses and waste can be used as a source of hydrolysates rich in peptides with relevant bioactive properties, and showing potential for use as ingredients in different industries, such as food and cosmetics, contributing to a circular economy and reducing world waste.
- Optimization of bromelain isolation from pineapple byproducts by polysaccharide complex formationPublication . Campos, Débora A.; Coscueta, Ezequiel R.; Valetti, Nadia Woitovich; Pastrana-Castro, Lorenzo M.; Teixeira, José A.; Picó, Guillermo A.; Pintado, Maria ManuelaA simple method for bromelain extraction from industrial pineapple residues (stems and peels) was developed and optimized through factorial experimental design. The developed methodology, based on precipitation with carrageenan, represents an alternative to the use of organic solvents and inorganic salts (common industrial precipitation) and allows achieving extracts with high bromelain purity. High recovery yield – 80–90% - of active bromelain was obtained for both crude juices (stems and peels) making possible to obtain ca. 0.3 g of bromelain from 100 g of pineapple byproducts using a low polysaccharide concentration (0.2–0.3% w/v).
- Continuous method to determine the trypsin inhibitor activity in soybean flourPublication . Coscueta, Ezequiel R.; Pintado, Manuela E.; Picó, Guillermo A.; Knobel, Gastón; Boschetti, Carlos E.; Malpiedi, Luciana Pellegrini; Nerli, Bibiana B.The determination of trypsin inhibitor (TI) activity is of importance to evaluate the nutritional value of soybean flours. An analytical method, which involves a continuous spectrophotometric rate determination for trypsin activity against the substrate N-benzoyl-DL-arginine p-nitroanilide, is proposed as an alternative to the standard discontinuous assay. Stopping the reaction with acetic acid and a centrifugation/filtration step to decrease turbidity are not required, thus reducing costs and sample preparation time. The TI activity of different flour samples, determined by both assays, demonstrated to be statistically comparable, irrespective of the TI concentration level. The coefficients of variation of the novel method did not exceed 8% at any concentration level. The curves of progress reaction showed a non-linear behavior in samples without TI. A reduction of incubation time from 10min to 2min increased the method sensitivity and extended its linear range. A more economical, faster and simpler assay was developed.
- Phenylethyl isothiocyanate extracted from watercress by-products with aqueous micellar systems: development and optimisationPublication . Coscueta, Ezequiel R.; Reis, Celso A.; Pintado, ManuelaPhenylethyl isothiocyanate (PEITC) was reported as a useful antioxidant, anti-inflammatory, and chemopreventive agent. Due to technological and stability issues, it is necessary to be able to extract PEITC from its natural matrix (watercress) through sustainable and scalable methodologies. In this article, we explored, for the first time, the extractive capacity of aqueous micellar systems (AMSs) of two non-ionic surfactants. For this, we compared the AMSs with conventional organic solvents. Furthermore, we developed and optimised a new integral PEITC production and extraction process by a multifactorial experimental design. Finally, we analysed the antioxidant capacity by the oxygen radical absorbance capacity (ORAC) and ABTS methods. As results, the AMSs were able to extract PEITC at the same level as the tested conventional solvents. In addition, we optimised by response surface methodology the integrated process (2.0% m/m, 25.0 C, pH 9.0), which was equally efective (ca. 2900 g PEITC/g watercress), regardless of the surfactant used. The optimal extracts showed greater antioxidant capacity than pure PEITC, due to other antioxidant compounds extracte in the process. In conclusion, by the present work, we developed an innovative cost-effective and low environmental impact process for obtaining PEITC extracts from watercress by-products.
- Collagen-based bioactive bromelain hydrolysate from salt-cured cod skinPublication . Coscueta, Ezequiel R.; Brassesco, Maria Emilia; Pintado, ManuelaConsiderable amounts of fish processing by-products are discarded each year. About 30% of this material may be skin and bone. Fish skin has more than 80% of its total protein content as collagen. Furthermore, in recent years, there has been a growing demand for collagen-based peptides due to their beneficial health effects. So, the objective of the present study was to optimise the obtaining bioactive hydrolysates from salt-cured cod skin using the protease Bromelain at 0.5% (w/w) concentration. This study developed a sustainable process that consumes less time and energy and uses an alternative source as raw material. In addition, bromelain allows hydrolysates with important antioxidant (ORAC, 514 μmol Trolox Equivalent/g protein) and antihypertensive activities (inhibition of ACE, IC50 of 166 μg protein/mL) as well as excellent biocompatibility with dermal and subcutaneous cells.
- Cod skin collagen extraction and characterizationPublication . Coscueta, Ezequiel; Brassesco, María; Pintado, Manuela
- Phenethyl isothiocyanate, a bioactive agent for gastrointestinal healthPublication . Coscueta, Ezequiel; Sousa, Ana Sofia; Reis, Celso; Pintado, ManuelaThe incidence of gastrointestinal diseases (cancer in particular) has increased progressively with considerable morbidity, mortality, and a high economic impact on the healthcare system. Dietary intake of natural bioactive phytochemicals showed to have cancer-preventing and therapeutic effects. This includes the cruciferous vegetable derivative phenethyl isothiocyanate (PEITC), a bioactive compound from watercress. PEITC antioxidant, anti-inflammatory and anti-cancer properties are of particular importance. This review summarizes the current knowledge on the role of PEITC as a potential natural nutraceutical or an adjuvant against oxidative/inflammatory-related disorders in the gastrointestinal tract. We also discuss the safe and recommended dose of PEITC. Besides, we establish a framework to guide the research and development of sustainable methodologies for obtaining and stabilizing this natural nutraceutical for industrial use. This is a topic that still needs more scientific development, but with the potential to lead to a viable strategy in the prevention of cancer and other associated diseases of the gastrointestinal tract.