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- Platform design for extraction and isolation of Bromelain: complex formation and precipitation with carrageenanPublication . Campos, Débora A.; Valetti, Nadia Woitovich; Oliveira, Ana; Pastrana-Castro, Lorenzo M.; Teixeira, José A.; Pintado, Maria Manuela; Picó, GuillermoThe main objective of this work was to investigate for the first time the molecular mechanism of com-plex formation between bromelain (a positively charged enzyme) and carrageenan (a natural strongpolyelectrolyte, negatively charged) using spectroscopy techniques and thermodynamic approaches. TheBromelain-Carrageenan complex showed a maximal non-solubility at pH around 5.1. The solubility wasdependent on pH and ionic strength of the medium. To re-dissolve the formed complex, the pH waschanged and 500 mM of NaCl was added to the initial solution, proving the columbic mechanism forthe formation of non-soluble complex. The formation of the carrageenan-bromelain complex increasedin 8◦C the enzyme thermal stability, while its biological activity was not modified. The amount of totalenzyme recovered in solution after precipitation with around 0.08% w/v of carrageenan was 85–90%.
- Anthocyanin recovery from grape by-products by combining ohmic heating with food-grade solvents: phenolic composition, antioxidant, and antimicrobial propertiesPublication . Coelho, Marta; Silva, Sara; Costa, Eduardo; Pereira, Ricardo N.; Rodrigues, António Sebastião; Teixeira, José António; Pintado, ManuelaUsually, wine-making by-products are discarded, presenting a significant environmental impact. However, they can be used as a source of bioactive compounds. Moreover, consumers’ increasing demand for naturally nutritious and healthy products requires new formulations and food product improvement, together with sustainable, environmentally friendly extraction methods. Thus, this work aimed to compare ohmic heating (OH) with conventional methodology (CONV), using food-grade solvents, mainly water, compared to standard methanol extraction of anthocyanins. No significant differences were found between the CONV and OH for total phenolic compounds, which were 2.84 ± 0.037 and 3.28 ± 0.46 mg/g DW gallic acid equivalent, respectively. The same tendency was found for antioxidant capacity, where CONV and OH presented values of 2.02 ± 0.007 g/100 g and 2.34 ± 0.066 g/100 g ascorbic acid equivalent, respectively. The major anthocyanins identified were malvidin-3-O-acetylglucoside, delphinidin-3-O-glucoside, petunidine-3-O-glucoside, cyanidin-3-O-glucoside, and peonidine-3-O-glucoside. These extracts displayed antimicrobial potential against microorganisms such as Yersinia enterocolitica, Pseudomonas aeruginosa, Salmonella enteritidis, methicillin-sensitive Staphylococcus aureus, a methicillin-resistant Staph. aureus (MRSA), and Bacillus cereus. In conclusion, OH provides similar recovery yields with reduced treatment times, less energy consumption, and no need for organic solvents (green extraction routes). Thus, OH combined with water and citric acid allows a safe anthocyanin extraction from grape by-products, thus avoiding the use of toxic solvents such as methanol, and with high biological potential, including antimicrobial and antioxidant activity.
- Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applicationsPublication . Balcão, Victor M.; Costa, Carla I.; Matos, Carla M.; Moutinho, Carla G.; Amorim, Manuela; Pintado, Manuela E.; Gomes, Ana M. P.; Vila, Marta M.; Teixeira, Jose A.Lactoferrin has for long captured the interest of many researchers as a natural compound with a wide variety of uses. Lactoferrin is a monomeric, iron-binding 80 kDa glycoprotein, and appears to be the subfraction of whey with the best documented antiviral, antimicrobial, anticancer and immune modulating/enhancing effects. It belongs to the family of transferrin proteins, and serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. In the present research effort, production of lactoferrin derivatives (starting from a purified commercial extract), encompassing full stabilization of its three-dimensional structure, has been attempted via nanoencapsulation within lipid nanovesicles, integrating a multiple water-in-oil-in-water emulsion. Long-term storage of the multiple nanoemulsions produced did not lead to leaching of protein, thus proving the effectiveness of the encapsulation procedure. Furthermore, lactoferrin nanovesicle derivatives prepared under optimal conditions were successfully employed at lab-scale antimicrobial trials.
- 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).
- In vitro digestibility and fermentability of fructo-oligosaccharides produced by Aspergillus ibericusPublication . Nobre, C.; Sousa, S.C.; Silva, S.P.; Pinheiro, A.C.; Coelho, E.; Vicente, A.A.; Gomes, A.M.P.; Coimbra, M.A.; Teixeira, J.A.; Rodrigues, L.R.The bifidogenic potential of fructo-oligosaccharides (FOS) produced by a newly isolated strain – Aspergillus ibericus was studied. Their activity was compared to FOS produced by Aureobasidium pullulans and to a nonmicrobial commercial FOS sample (Raftilose® P95). FOS fermentability by a number of probiotic bacteria and their hydrolytic resistance to the simulated harsh conditions of the digestive system was evaluated. Aspergillus ibericus FOS sample effectively promoted probiotic bacteria growth. Overall, microbial-derived FOS promoted greater cellular growth compared to the commercial sample. FOS fermentation was both substrate and strain specific. The FOS structural differences identified may explain their distinct assimilation by the probiotics. [Fru (2→6)Glc] (possibly blastose) and a reducing trisaccharide (possibly [Fru(β2→6)Glc(α1↔β2)Fru], neokestose) were only found in microbial-derived FOS samples, while Raftilose® P95 was richer in inulobiose/inulotriose. 1- Kestose and nystose were only slightly hydrolyzed in the presence of gastric and intestinal fluid. FOS synthesized by Aspergillus exhibited great potential as food ingredients with likely prebiotic features.
- Emergent technologies for the extraction of antioxidants from prickly pear peel and their antimicrobial activityPublication . Alexandre, Elisabete M. C.; Coelho, Marta C.; Ozcan, Kardelen; Pinto, Carlos A.; Teixeira, José A.; Saraiva, Jorge A.; Pintado, ManuelaPhenolic compounds are important bioactive compounds identified in prickly pear peel that have important antioxidant and antimicrobial properties. However, conventional thermal extraction methods may reduce their bioactivity, and technologies such as high pressure (HP) and ohmic heating (OH) may help preserve them. In this study, both technologies were analyzed, individually and combined (250/500 MPa; 40/70◦C; ethanol concentration 30/70%), and compared with Soxhlet with regard to total phenolics, flavonoids, and carotenoids as well as antioxidant (ABTS, DPPH, ORAC), DNA pro-oxidant, and antimicrobial (inhibition halos, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), growth curves, and viable cells) activities of prickly pear peel extracts. Total phenolics extracted by each technology increased 103% (OH) and 98% (HP) with regard to Soxhlet, but the contents of total flavonoids and carotenoids were similar. Antioxidant activity increased with HP and OH (between 35% and 63%), and OH (70◦C) did not induce DNA degradation. The phenolic compound present in higher amounts was piscidic acid, followed by eucomic acid and citrate. In general, their extraction was significantly favored by HP and OH. Antimicrobial activity against 7 types of bacteria showed effective results only against S. aureus, S. enteritidis, and B. cereus. No synergetic or additive effect was observed for HP/OH.
- Advances in extraction methods to recover added-value compounds from seaweeds: sustainability and functionalityPublication . Matos, Gabriela S.; Pereira, Sara G.; Genisheva, Zlatina A.; Gomes, Ana Maria; Teixeira, José A.; Rocha, Cristina M. R.Seaweeds are a renewable natural source of valuable macro and micronutrients that have attracted the attention of the scientists in the last years. Their medicinal properties were already recognized in the ancient traditional Chinese medicine, but only recently there has been a considerable increase in the study of these organisms in attempts to demonstrate their health benefits. The extraction process and conditions to be used for the obtention of value-added compounds from seaweeds depends mainly on the desired final product. Thermochemical conversion of seaweeds, using high temperatures and solvents (including water), to obtain high-value products with more potential applications continues to be an industrial practice, frequently with adverse impact on the environment and products’ functionality. However more recently, alternative methods and approaches have been suggested, searching not only to improve the process performance, but also to be less harmful for the environment. A biorefinery approach display a valuable idea of solving economic and environmental drawbacks, enabling less residues production close to the much recommended zero waste system. The aim of this work is to report about the new developed methods of seaweeds extractions and the potential application of the components extracted.
- Nanostructures of whey proteins for encapsulation of food ingredientsPublication . Ramos, Oscar L.; Pereira, Ricardo N.; Simões, Lívia S.; Madalena, Daniel A.; Rodrigues, Rui M.; Teixeira, José A.; Vicente, António A.The most current and high-level research is being taken on the use of nanoscience and nanotechnology due to its varied application in numerous fields of science. Food nanotechnology, and in particular, the development and application of bio-based nanostructures are an emerging area having a high potential to engender new products and processes in the food industry. This chapter intends to discuss whey protein-based nanostructured systems (i.e., whey protein isolate, whey protein concentrate, β-lactoglobulin, and α-lactalbumin) for encapsulation of food ingredients. These protein nanostructures have unique properties, such as a high nutritional value, GRAS nature, gelling capability, and can be easily prepared and controlled. They have also the ability to conjugate a large variety of food ingredients (e.g., antioxidants, vitamins, minerals, flavors, and odors) via amino groups or ionic and hydrophobic interactions. This behavior will prevent the degradation of sensitive bioactives, while permitting a site-specific action and controlled delivery rate due to the swelling behavior of the gel in reaction to external and physical stimuli such as temperature, enzymes, pH, or ionic strength), thus contributing to an improved bioavailability of such ingredients. The potential of whey protein nanostructures for encapsulation and controlled delivery of food ingredients will be addressed in a critical manner in this chapter. Moreover, various techniques used for their nanoencapsulation and evaluation of their stability during storage will also be discussed. The behavior and bioavailability of whey nanostructures and their associated/encapsulated food ingredients will be discussed using insights from in vitro and in vivo gastrointestinal systems together with potential cytotoxicity, cellular uptake, and allergenicity via in vitro cell lines. Finally, examples of such nanostructures applied in food matrices will be described, as well as the main challenges for their commercial use.
- Saccharomyces cerevisiae oxidative response evaluation by cyclic voltammetry and gas chromatography−mass spectrometryPublication . Castro, Cristiana C.; Gunning, Caitriona; Oliveira, Carla M.; Couto, José A.; Teixeira, José A.; Martins, Rui C.; Ferreira, António C. SilvaThis study is focused on the evaluation of the impact of Saccharomyces cerevisiae metabolism in the profile of compounds with antioxidant capacity in a synthetic wine during fermentation. A bioanalytical pipeline, which allows for biological systems fingerprinting and sample classification by combining electrochemical features with biochemical background, is proposed. To achieve this objective, alcoholic fermentations of a minimal medium supplemented with phenolic acids were evaluated daily during 11 days, for electrochemical profile, phenolic acids, and the volatile fermentation fraction, using cyclic voltametry, high-performance liquid chromatography−diode array detection, and headspace/solid-phase microextraction/gas chromatography−mass spectrometry (target and nontarget approaches), respectively. It was found that acetic acid, 2- phenylethanol, and isoamyl acetate are compounds with a significative contribution for samples metabolic variability, and the electrochemical features demonstrated redox-potential changes throughout the alcoholic fermentations, showing at the end a similar pattern to normal wines. Moreover, S. cerevisiae had the capacity of producing chlorogenic acid in the supplemented medium fermentation from simple precursors present in the minimal medium.
- Integral valorization of pineapple (ananas comosus l.) by-products through a green chemistry approach towards added value ingredientsPublication . Campos, Débora A.; Ribeiro, Tânia; Teixeira, José A.; Pastrana, Lorenzo; Pintado, Maria ManuelaIndustrial by-products are produced every day through fruit processing industries. Pineapple is not an exception; when processed, around 60% (w/w) of its weight are peels, stem, trimmings, and crown, the only used fruit part for human consumption. Due to high concerns of sustainability in the food system and negative high impact of human practice in the environment, a strategy has to be developed. Therefore, a green chemistry approach was applied to pineapple by-products to make an integrated valorization by the extraction of bioactive molecules. Two pineapple by-products (peels and stems) were studied, applying a green chemistry approach, which means the non-use of organic solvents or extreme methodologies. A subdivision of each by-product was done by the application of a juice machine. The peels and stems in the fresh state were ground separately, creating two fractions for each by-product—a juice and a wet pulp (press cake). The press cake was characterized, dried, and ground to create a fine powder flour. To the juice, a precipitation methodology with polysaccharides was applied, which allowed the bromelain separation (developing of an enzymatic fraction) from the fruit juice. The enzymatic extract was freeze-dried, and the juice was spray-dried, developing two more fine powders. Thus, three new ingredients were produced from each by-product, creating a total of six new ingredients. Overall, the enzymatic fractions represented around 0.26% (w/w) of pineapple weight. Pineapple stem juice represented 4.8% (w/w), and peel juice represented 17.3% (w/w). Pineapple stem flour represented 3.1% (w/w), and peel flour represented 11.4% (w/w) of the total pineapple weight. To valorize the by-products juices, a full characterization was performed of bioactive molecules and biological activities. When comparing the two juices, the peel juice showed lower content of total phenolic compounds, lower antioxidant capacity, and lower content of vitamin C. The different phenolic compounds were identified by HPLC analysis in the two pineapple by-products juices. However, the same compounds in both juices were quantified (chlorogenic, caffeic, and ferulic acids). On the other hand, the by-products flours had a high content of insoluble dietary fiber (IDF), mainly cellulose and hemicellulose. Therefore, the approach applied in this work opens the door to the production of green products, as a result of by-products valorization. This could be applied not only in the food industry but also in the nutraceutical and cosmetic industries.
