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Cocoa bean shell (CBS), a by-product of the chocolate industry, was employed as substrate for the sustainable recovery of bioactive compounds using ohmic heating (OH). Total phenolic content and antioxidant activity of the treated CBS were optimized by experimental design. Maximum extraction of antioxidant phenolic compounds (23mg GAE/g CBS) was obtained at 67°C, 50min and 44% ethanol (v/v). The antioxidant activity of the extracts obtained under of the central point conditions was 284.5M Fe2+/g extract (FRAP) and 36.4MTE/g extract (DPPH). The use of OH increased the extraction of bioactive phenolic compounds when compared to the conventional process (CH) (approximately 40%). An increased on the chemical antioxidant activity was also observed, ranging from 4 to 20%. The metabolic activity of the extracts obtained by the two methods (OH and CH) was evaluated in non-tumoral (HEK293T and L929) and tumoral cell lines (Caco-2, HT-29, and HeLa). The CBS extracts presented low toxicity in non-tumoral cells and ROS preventive effects. These characteristics make them ideal to be used in food processing and formulation, as well as nutraceutical products due to their antioxidant protection. The use of OH results in an extract with higher phenolic content and higher antioxidant activity and low environmental impact., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of both the strategic funding of UIDB/04469/2020 unit and LA/P/0029/2020 (LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems), and by the Project SV-PA-21-AYUD/2021/51041 from the Economy and Employment Office of Principality of Asturias (Spain). J. S. G.-D. acknowledges the PhD scholarship 2021.06136.BD provided by FCT. Pedro Ferreira-Santos would like to express gratitude to the Spanish Ministry of Science, Innovation and Universities for financial support (FJC2021-046978-I)., info:eu-repo/semantics/publishedVersion

The development of more efficient and sustainable technologies for the recovery of valuable compounds holds the ability to change the paradigm of the use of resources. Electric field-based technologies have been regarded as highly promising towards an efficient extraction of biometabolites while addressing sustainability. Algal biomass is a potential valuable resource. Besides its nutritional value, it provides a wide range of bio compounds with a variety of industrial applications in different sectors (e.g., food, feed, cosmetics). Implementing a cascade biorefinery approach based on electric-fields application would boost the production of high-value-added products from algal biomass, opening up new opportunities for the development of a flourishing blue bioeconomy. This review paper provides an overview of the use of alternative extraction technologies in algae biomass, focusing mainly on electric field-based methodologies, suggesting cascade biorefinery approaches considering different alternative technologies depending on the target fraction, and envisaging their environmental sustainability and economic feasibility., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020, LABBELS - Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, and the PhD grant reference 2021.07623.BD (Sara G. Pereira). Ricardo N. Pereira acknowledge FCT for its Assistant Research program under the scope of Scientific Stimulus Employment with reference CEECIND/02903/2017., info:eu-repo/semantics/publishedVersion

Emerging processing technologies are gaining importance due to ecological benefits and high extraction efficiency. Agarophytes are seaweeds highly recognized for their agar content. This work aimed at evaluating the potential of combining subcritical water extraction and moderate electric fields (MEF) for agar extraction. Different temperature and time binomials were applied, keeping the same severity factor, under subcritical water conditions with and without MEF. The influence in the agar extraction yields, its composition and technological properties was evaluated. Moderate electric fields proved to be a valuable tool in agar recovery, particularly for G. sesquipedale, improving agar's extraction yield up to 40% at 140°C in the presence of MEF. Applied treatments and extraction technologies did not affect gelling ability, rheological behaviour, structure, and molecular weight distribution of the obtained agars. Therefore, the feasibility of alternative processing technologies was demonstrated, with gains in efficiency and processing time., This work was supported by the Portuguese Foundation for Science and Technology (FCT), by the European Regional Development Fund (FEDER) and by COMPETE 2020 – Competitiveness and Internationalization Operational Program under the scope of OH2O project (POCI-01-0145-FEDER-029145; PTDC/EQU-EQU/029145/2017). The work was also supported by FCT under the scope of the strategic funding of UID/BIO/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Sara G. Pereira acknowledges FCT for the scholarship 2021.07623.BD. Joana S. Gomes-Dias also acknowledges FCT for the PhD grant with reference 2021.06136.BD., info:eu-repo/semantics/publishedVersion

This study examined the influence of ohmic heating (OH), compared to the conventional heating (Conven) and Control (solvent) methods, on the extraction of olive mill leaves. The main extraction parameters were: (i) solvent ratio (aqueous ethanol; 40%, 60%, and 80%, v/v), and (ii) extraction temperature; 45 °C, 55 °C, and 75 °C (for OH and Conven), and room temperature (for Control). The selected response variables were extraction yield (%), total phenolic content (TPC), and antioxidant activity (ABTS and DPPH). The ohmic system, compared to Conven and Control, exhibited the greatest effects (p < 0.001) on increasing (i) extraction yield (34.53%) at 75 °C with 80% ethanol, (ii) TPC at 55 °C (42.53, 34.35, 31.63 mg GAE/g extract, with 60%, 40%, and 80% ethanol, respectively), and (iii) antioxidant potency at 75 °C detected by DPPH and ABTS, in the range of 1.21–1.04 mM TE/g, and 0.62–0.48 mM TE/g extract, respectively. Further, there were relatively similar trends in TPC and antioxidant activity (both methods), regardless of solvent ratios, p < 0.001. These findings demonstrate the potential of ohmic heating, as a green processing tool, for efficient extraction (15 min) of olive leaves. To date, no literature has described ohmic application for olive leave extraction., This work was supported by the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UID/BIO/04469/2020 unit., info:eu-repo/semantics/publishedVersion

Microalgae biomass is a promising raw material for several bioproducts suitable for food, energy and pharmaceutical industries. The aim of the present work was to optimize the extraction of bioactive compounds from Chlorella vulgaris using Ohmic Heating (OH). A rotatable central composite design for two factors was used to assess the effects of temperature and solvent (% ethanol in water) on lipid extraction yields and fatty acid profile. OH extraction experiments were powered by low-frequency (50 Hz) and high-frequency (25 kHz) to identify the presence of non-thermal effects and its influence on composition and bioactive properties of the generated extracts. Lipid extraction using OH was successfully optimized with the best extraction conditions found at 70 ºC for 5 min using 88 % ethanol as a solvent. No effect on the application of the electrical frequencies was observed. These conditions allowed to recover up to 87 % of lipids from biomass, polyunsaturated fatty acids (PUFAs) accounting for 43 % of the extracted lipids against 26 % of saturated fatty acids (SFAs). The fatty acid profile reveals that C16:2, C16:3, C18:2 and C18:3 correspond to the PUFAs extracted from Chlorella vulgaris. Regarding lipid extraction yields OH was statistically equivalent to conventional heating (COV); however, in terms of heating kinetics, OH reaches the required extraction temperature 5 times faster than COV. Results showed that OH has potential to be applied as a treatment for the production of Chlorella vulgaris PUFAs richextracts providing high recovery yields with reduced treatment times and less energy consumption., This study was supported by: the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and project OH2O – PTDC/EQU-EQU/029145/2017; by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) under the scope of Project Algavalor (POCI-01-0247-FEDER-035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234) and OH2O (POCI-01-0145-FEDER-029145); AgriFood XXI R & D & I project, operation number NORTE-01-0145-FEDER-000041, co-financed by the European Regional Development Fund (FEDER) through NORTE 2020 (Northern Regional Operational Program 2014/2020). Ricardo N. Pereira acknowledge FCT for its Assistant Research contract obtained under CEEC Individual 2017. Leandro Madureira acknowledges FCT for its PhD fellowship (SFRH/BD/151474/2021) obtained under MIT Portugal Program, info:eu-repo/semantics/publishedVersion

Rotondas Red Eggplant belongs to the family of Solanum aethiopicum species, cultivated in a specific area of Potenza (Basilicata, Italy) and has gained the Protected Designation of Origin certification. Recently, Red Eggplant was associated with different biological properties, related to health benefits, as antioxidant, hypoglycemic, antihypertensive and hypolipidemic, probably due to its chemical composition. The search for "greener" technologies has been one of the focus of research, having in mind industrial applications. In this sense, electrotechnologies, such as ohmic heating (OH), have been explored, showing to have potentially lower energy costs, higher extraction yields, being less time consuming and increasing the biocompounds chemical stability. The aim of this study was to determine the chemical/nutritional composition of Red Eggplant and to assess conventional and OH methods in the extraction and recovery of the main bio-compounds present in this material. Red Eggplant was characterized for the nutritional profile (AOAC procedures): proteins, carbohydrates, total extractives, lipids, ash and minerals. The selected conditions for the extraction of the main biocompounds, for conventional and OH methods, were: solid/liquid ratio 1:10 (w:v), 80 °C, 25 min and water or mixture of 50% (v/v) ethanol/water as a solvent. Subsequently, assays were carried out to determine the content of proteins (Bradford), total carbohydrates (phenolsulfuric acid) and phenolics (Folin-Ciocalteu), and antioxidant activity (FRAP and DPPH) of the obtained extracts. Monosaccharides profile after hydrolysis and phenolics were also quantified by HPLC. Results showed that carbohydrates were the major macronutrient, followed by protein, lipids and ash. K, Ca and Mg were the major minerals. Regarding the monosaccharide composition, glucose, fructose, xylose and arabinose were the most abundant sugars. Ethanolic extractives represented approximately 46 % of the Red Eggplant composition. Independently of the solvent, higher amounts of polyphenols, proteins and carbohydrates were extracted when OH was applied. Moreover, OH extracts showed greater antioxidant activity compared to extracts obtained by the conventional extraction method. According to the polyphenol profile of the extracts the main compounds found were taxifolin, rutin and rosmarinic acid. These results demonstrate the potential of this vegetable to be used for nutraceuticals or functional food., The authors wish to thank for the financial support to funding program Marie Skłodowska-Curie grant (MSCA-RISE; FODIAC; 778388). Pedro Santos is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15), funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte (NORTE-08-5369-FSE-000036). Zlatina Genisheva is supported by the project OH2O (POCI-01-0145-FEDER-029145) funded by FCT and Fundo Europeu de Desenvolvimento Regional (FEDER) under the scope of Programa Operacional de Competividade e Internacionalizaçao (POCI)-COMPETE 2020 and Portugal 2020., info:eu-repo/semantics/publishedVersion

The development of new green and sustainable extraction technologies with low environmental impact and providing high nutritional functionalities, safety and applicability, has become relevant. Ohmic heating (OH) has emerged as a novel electric-based technology which allows high heating rates, higher selectivity of interest compounds, less energy and solvent consumption and energetically more efficient.[1,2] Ulva rigida is a green seaweed with high exploitation potential for commercial application. Its major compound of interest is ulvan, a sulfated polysaccharide present in the cell wall that has a wide range of possible applications in nutraceuticals, functional foods, agriculture and biomaterials.[3] The aim of this work was to access the feasibility of OH assisted extraction as an alternative extraction technology in the recovery of biocompounds (e.g.: polysaccharides, proteins, phenolic compounds and pigments) from Ulva rigida using different ratios of water/ethanol in different times of extraction. Extractions were made using a solid:solvent ratio of 1:30 and water/ethanol mixtures ranging from 0 to 75 % ethanol to extract different seaweeds fractions. The extractions were performed at 82 °C during 1 h, 2 h and 3 h, and the frequency was set at 25 kHz. Control extractions were made using a water bath (conventional extraction) with the same conditions, but without the presence of an electric field. Different parameters were assessed including extraction yield, lipids, ashes, moisture, total polysaccharides, protein, total phenolic compounds and pigments (chlorophylls and carotenoids) content, antioxidant activity and gelling ability. Higher extraction and ulvan-fraction yields, as well as total polysaccharides were obtained after 3 h of extraction for aqueous extracts. Total phenolic compounds, proteins and pigments content and antioxidant activity were higher for mixtures with higher amounts of ethanol. For antioxidant activity and total phenolic compounds, the 50:50 H2O/EtOH mixture under OH proved to be more efficient, with an increase up to 10 %, when compared to conventional extraction. Also, for pigments and protein content, the 25:75 H2O/EtOH mixture allowed promising results for 1 h of extraction. Gelling ability was identical in all produced extracts. As conclusion, these results showed that OH is a good alternative for a low cost and environmental-friendly extraction technology, for the recovery of bioactive compounds from green seaweeds., info:eu-repo/semantics/publishedVersion

Pulsed electric fields (PEF) is attracting great attention as nonthermal extraction technology on downstream processing of emergent marine bioresources such as micro- and macroalgae. These uni- and multicellular algae enclose a diverse and interesting biochemical composition and are recognized as important sources of valuable bioactive compounds and functional food nutrients. This chapter intends to give an overview about the role of electric field processing toward the extraction of proteins and carbohydrates given particular emphasis on PEF technology, addressing the latest scientific developments in the field. Electric field-based technologies have a great potential as a green tool, being an energy efficient and environmentally friendly processing technology, while reducing the operational time by allowing a strong electro permeation, that is electroporation of cellular membranes, that enhances the extraction yields while reducing the use of solvents. The possibility of combining PEF with other processing variables (i.e., ohmic heating) can bring more flexibility to the extraction process and will be highlighted., (undefined), info:eu-repo/semantics/publishedVersion

Tese de Doutoramento em Ciência e Tecnologia Alimentar e Nutrição, Atualmente, o esgotamento de recursos, o acúmulo de resíduos e as mudanças climáticas são os principais fatores que impulsionam a necessidade de práticas sustentáveis. Algumas das estratégias para a redução do impacto ambiental gerado são o reaproveitamento de biorresíduos industriais, bem como o aumento do uso de recursos subexplorados por processos efetivos/intensificados para obtenção de produtos sustentáveis, ecológicos, seguros e de alta qualidade. A presente tese teve como objetivo desenvolver metodologias eficientes e ecologicamente corretas envolvendo a aplicação de campos elétricos e enzimas, além de solventes de baixo custo e não tóxicos para a valorização de biorecursos subvalorizados, como casca de Pinus pinaster e microalga Spirulina platensis. Os extratos de casca de pinheiro (PBE) apresentaram alta atividade antioxidante, antibacteriana e anti-hiperglicêmica, com baixa citotoxicidade e atividade seletiva contra células cancerosas, pois foram afetados negativamente. Essas propriedades foram atribuídas ao seu perfil fenólico e flavonóide. Além disso, o Aquecimento Óhmico (OH) evidenciou um aumento na extração de compostos fenólicos antioxidantes sem degradação. Os resultados também indicam extração preferencial sob o solvente apropriado, presumivelmente devido à ação do campo elétrico agindo em sinergia com o calor e solventes, promovendo efeitos de eletroporação da membrana. A microencapsulação por spray-drying de PBE utilizando maltodextrina foi eficaz na proteção de compostos fenólicos durante as condições gastrointestinais, controlando sua liberação e aumentando os seus benefícios para a saúde, diminuindo a produção de espécies reativas de oxigênio implicadas no processo de estresse oxidativo associado a algumas patologias. A Spirulina platensis é interessante para a indústria alimentar devido à sua composição geral e alto teor em C-ficocianina (corante azul natural). A presente tese mostrou que campos elétricos moderados associados à temperatura promovem efeitos de desintegração celular e eletroporação melhorando a extração e estabilidade de compostos intracelulares da Spirulina, tendo também um efeito positivo no rendimento de extração de C-ficocianina com tempos de tratamento mais curtos. Uma extração sequencial em múltiplos estágios avaliada por extração térmica convencional, OH, tratamento enzimático (EAE), e suas combinações, evidenciou que a extração combinada de OH e EAE permitiu a recuperação seletiva de ficobiliproteínas na etapa de extração aquosa. Pigmentos, como clorofila e carotenóides, foram extraídos seletivamente com etanol (95%), bem como a fração lipídica da microalga. De modo geral, a tecnologia OH tem potencial para ser uma alternativa "verde", melhorando a extração de compostos intracelulares, além de reduzir o tempo de processo, solventes e consumo de energia, oferecendo a possibilidade de ajustar a seletividade e estabilidade dos compostos extraídos. Além disso, processos integrados que abordem simultaneamente diferentes frações, em direção a uma economia de “desperdício zero”, são desejáveis e podem até ser benéficos para a eficácia de cada fração individual. Os biocompostos obtidos podem ser usados como aditivo tecnológico natural e/ou ingredientes funcionais para aplicações alimentares, cosméticas e farmacêuticas., Nowadays, resources depletion, waste accumulation and climate change are key factors driving the need for sustainable practices. Some of the strategies for the reduction of generated environmental impact are the reuse of industrial biowastes, as well as increasing the use of underexploited resources by effective/intensified processes to obtain sustainable, ecological, safe and high quality products. The present thesis aimed to develop efficient and environmentally friendly methodologies involving the application of electric fields and enzymes, as well as low cost and non-toxic solvents for the valorization of undervalued bioresources, as Pinus pinaster bark and Spirulina platensis microalgae. Pine bark extracts (PBE) presented high antioxidant, antibacterial and antihyperglycemic activities, with low cytotoxicity and a selective activity against cancer cells as these were negatively affected. These properties were ascribed to their phenolic and flavonoid profile. Moreover, Ohmic Heating (OH) evidenced an increased in the extraction of antioxidant phenolic compounds with no degradation. Results also indicate preferential extraction under the appropriate solvent, presumably due to the action of electric field effects acting in synergy with heat and solvents, promoting membrane electroporation effects. Spray-drying microencapsulation of PBE using maltodextrin was effective in protecting phenolic compounds during gastrointestinal conditions, controlling their delivery and enhancing its health benefits, decreasing the production of reactive oxygen species (ROS) implicated in the process of oxidative stress associated with some pathologies. Spirulina platensis is interesting for the food industry due to its overall composition and high content in C-phycocyanin (natural blue colorant). The present thesis showed that moderated electric fields associated with temperature promotes cell structure disintegration and electroporation effects improving the extraction and stability of intracellular compounds from Spirulina, having also a positive effect on the extraction yield of C-phycocyanin at shorter treatment times. A sequential multi-stage extraction evaluated by conventional thermal extraction, OH, enzymatic treatment (EAE), and their combinations evidenced that the combined OH and EAE extraction allowed selective recovery of phycobiliproteins in the aqueous extraction step. Pigments, such as chlorophyll and carotenoids were selectively extracted with 95% ethanol, as well as the lipid fraction of the microalgae. Overall, OH technology holds the potential to be a "green" alternative, improving intracellular compounds extraction, in addition to reducing the process time, solvents and energy consumption, offering the possibility of tuning the selectivity and stability of the extracted compounds. Moreover, integrated processes simultaneously addressing different fractions, towards a “zero waste” economy, are desirable and may be even beneficial to the effectiveness of each individual fraction. The obtained biocompounds can be used as natural technological additive and/or functional ingredient for food, cosmetic and pharmaceutical applications.