R - Teses de Doutoramento / Doctoral Theses
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- Acorn starch extraction and modification by emerging technologiesPublication . Castro, Luís Manuel Gomes de; Pintado, Maria Manuela Estevez; Saraiva, Jorge Manuel Alexandre; Alexandre, Elisabete Maria da CruzNative to the Northern Hemisphere, the trees of the genus Quercus spp. are found between the temperate and tropical latitudes of the Americas, Asia, Europe, and North Africa. These oaks are evergreen, semi-deciduous, or deciduous trees and produce small fruits called acorns. According to the National Institute of Statistics, these trees represent 35% of the forest area in mainland Portugal, where 85% of cork oaks and 92% of holm oaks are found in the Alentejo, while 94% of the remaining species are concentrated in the North and Center regions. For this reason, large quantities of acorns are produced, and it is estimated that the annual production is 401,585 tons. Despite being a fruit rich in polyphenols and gluten-free (resistant) starch, about 55% of the acorns produced are underused. Currently, as starch has multiple applications in the food and non-food industry, the exploitation of acorns as a source of starch would be advantageous. According to the literature, alkaline extraction is the methodology that allows obtaining the highest extraction yields in starch with the highest purity and with the most relevant properties. However, these starches cannot be used as food additives in the European Union due to a lack of approval. Thus, the main objective of this thesis was to value acorns within the scope of the principle of sustainable development through the creation of high-added value and zero-waste ingredients. Different species of acorns (Q. pyrenaica, Q. robur, and Q. ilex) were collected to study the effect of different dehulling methods on their nutritional and phytochemical composition. Each specie was dehulled separately by three different methods: manual, thermal (by thermal shock), and drying. It was found that the carbohydrate content was not significantly affected, but there was a loss of resistant starch of 76, 42 and 62% after thermal dehulling and a loss of 35, 27 and 25% after dehulling by drying compared to manual dehulling for Q. pyrenaica, Q. robur, and Q. ilex, respectively. As Q. robur was the specie with the highest resistant starch/total starch ratio (0.89), as well as the highest phenolic contents (19.3±1.3 mg GA Eq./g DF), hydrolyzable and condensable tannins (73.2±4.6 mg TA Eq./g DF and 0.103±0.01 mg TA Eq./g DF, respectively) comparatively to Q. pyrenaica and Q. ilex, the manually dehulled acorn of the Q. robur specie was selected in the following tests. Then, the optimization of the starch extraction was conducted at room temperature using water as extraction solvent by high hydrostatic pressure (HP) (pressure levels between 0.1 and 500 MPa and extraction times between 5 and 20 min) and pulsed electric field (PEF) (field strengths between 0.1 and 20 kV/cm and times between 35 and 85 μs) technologies. Due to the presence of large tannin content, which are anti-nutritional compounds of great importance in leather production, their extraction was also optimized, as well as other phenolic compounds, to obtain antioxidant extracts with high added value. Regarding the HP extraction, the optimal extraction condition was found at 333 MPa for 17.4 min with a desirability of 79.5%. The starch yield was 35.4±1.1% (w/w DF) and the extracts had a phenolic and hydrolyzable tannin content of 23.1±0.3 mg GA Eq./g DF and 54.1±0.1 mg TA Eq./g DF, respectively, and an antioxidant activity measured by ABTS of 53.6±0.6 mg TX Eq./g DF. As for the PEF extraction, the optimal extraction condition was obtained at 0.1 kV/cm for 63.3 μs with a desirability of 86.5%. The starch yield was 34.5±0.4% (m/m DF) and the extracts had a phenolic and hydrolyzable tannin content of 27.0±1.2 mg GA Eq./g DF and 56.7±0.6 mg TA Eq./g DF, respectively with antioxidant activity of 59.1±1.9 mg TX Eq./g DF. Condensed tannins were not detected in any of the extracts. Compared to alkaline extraction, the results observed under optimal conditions represent an increase in phenolic yields, tannins, and antioxidant activity by 2, 900, and 4% by HP and an increase by 24, 1012, and 15% by PEF, respectively. Thus, these technologies make it possible to obtain aqueous extracts with a higher tannin content than alkaline extraction, thus valuing these compounds. However, it was the PEF extraction technology that made it possible to obtain extracts with a higher content of polyphenols (phenolics and tannins) and with greater antioxidant activity than HP. Concerning starch, although the extraction yields obtained by HP and PEF under optimal conditions were lower than those verified in the respective alkaline extractions (49.7±0.5 and 48.9±1.2% w/w DF for HP and PEF, respectively), these starches are clean-label and can be used more safely as an additive and/or ingredient in human food than alkaline starches. As the extraction yields under optimal conditions were like each other, the impact of technologies on starch properties was studied and the properties were compared to commercial corn starch to try to identify the most advantageous extraction technology. Regarding the starch obtained by HP under optimal conditions, pressurization preserved the type of polymorphism of the acorn starch and did not significantly alter the relative crystallinity and the onset gelatinization temperatures (i.e., it preserved the hydrogen bonds). However, the significant increase in the amylose/amylopectin ratio by 97%, motivated by the cleavage of the amylopectin chains, led to changes in the properties of starches. Pressurization led to a decrease in the solubility and swelling power of the starches relative to the control and led to an increase in the resistance of the gels to deformation. However, no differences were observed in the in-vitro digestibility, pseudo-plastic behavior, or resistance to flow after pressurization. Regarding the starch obtained by PEF under optimal conditions, the amylose and amylopectin contents were not altered, as well as the onset gelatinization temperatures (hydrogen bonds). Although the type of polymorphism was not changed, there was a decrease in relative crystallinity of 17%. PEF treatment did not alter the starch properties concerning the control starch (solubility, swelling power, in-vitro digestibility, and rheology). Compared to commercial corn starch, acorn starches showed lower gelatinization temperatures and enthalpies, better pseudo-plastic behavior, lower in-vitro digestibility, greater resistance to complex flow, and lower resistance to deformation. Overall, acorn starches also showed higher solubility and swelling power up to 80 °C than commercial starch, which encourages the use of acorn starch as an additive in fermented yogurt and dairy products. As extraction by HP and PEF led to obtaining acorn starches with different properties, a dairy product was developed to try to identify the most suitable starch for this purpose. The effect of replacing commercial corn starch with acorn starch extracted by HP and PEF under optimal conditions on the nutritional composition, functional and sensory properties, and shelf-life of chocolate puddings was studied. Acorn starch improved the rheological properties of puddings without negatively affecting their nutritional composition, internal structure, or in-vitro digestibility. The sensory analysis revealed that 61% of tasters preferred the puddings made with acorn starch over commercial corn starch (control). As for the shelf life, acorn starch led to color stabilization and improved texture of the puddings after 28 days of storage at 4°C compared to the control without compromising the microbiological safety. Of the acorn starches used, PEF-extracted acorn starch led to the greatest improvement in pudding texture. In addition, the chocolate pudding made with PEF-extracted acorn starch also had a higher acceptability index than the HP-extracted starch. Thus, PEF-extracted acorn starch seems to be the most suitable for pudding production. In its entirety, the work described in this Doctoral thesis marks important steps in the valorization of acorns in Portugal, that is, in the valorization of its polyphenols and starch. This work will also contribute to the sustainability and transformation of the acorn in a circular economy context, as it demonstrates the potential of using PEF technology, as a sustainable alternative to alkaline extractions, in obtaining products with high added value and of great economic interest.
- Characterisation of staphylococcus aureus circulating in PortugalPublication . Castro, Ana Isabel Rodrigues Pereira de; Teixeira, Paula Cristina Maia; Silva, Joana Gabriela Laranjeira daStaphylococcus aureus is a pathogen responsible for skin infections and invasive diseases such as meningitis or pulmonary infection and staphylococcal food poisoning (SFP). The main goal of this study was to increase knowledge on S. aureus circulating in Portugal mainly concerning their virulence characteristics and antimicrobial resistance. According to EFSA-ECDC, Portugal is considered to have one of the highest levels of methicillin-resistant S. aureus (MRSA) from bacteraemia in Europe. The frequency of occurrence of S. aureus from different origins was determined. Swab samples were collected from hands and nose of health care professionals and food handlers - noses and hands are niches where S. aureus are likely to be present in humans - and particularly noses of children (3 to 6 years). Presence of S. aureus in food samples was also determined. Collected isolates were further characterized phenotypically and genomically. Nasal carriage of S. aureus was higher in children (48.6%) compared to health care professionals (39.6%) and food handlers (19.8%). The occurrence of S. aureus on the hands of health care professionals and food handlers was 8.9% and 11.1%, respectively. Globally, higher S. aureus occurrence rates were obtained for nasal carriage. The first case of Methicillin-Resistant Staphylococcus aureus (MRSA) was reported in 1961. Since then, it has been considered the leading cause of nosocomial infections, responsible for causing serious morbidity and mortality rates, worldwide. Globally, the occurrence of MRSA strains was higher in hospital health care professionals; no MRSA strains were detected in food handlers, children presented a carriage of 9.7% of MRSA and low occurrence rates were detected in the analysed food samples (between 0.68 and 5.5 %). Other virulence factors have been reported for S. aureus strains including antibiotic resistance and presence of enterotoxins, Panton-Valentine Leukocidin (PVL), haemolysins, exfoliative toxins and numerous factors involved in invasion of host cells among others. In the present study, resistance to oxacillin, penicillin and ampicillin and to antibiotics of classes other than betalactams, namely ciprofloxacin, gentamicin, rifampicinn, vancomycin, tetracycline, erythromycin, nitrofurantoin and chloramphenicol was investigated. As expected, high levels of resistance to ß-lactams were observed. Tested strains showed low resistance rates to gentamicin, chloramphenicol and rifampicin. Regardless of the source of isolates, resistance to erythromycin was evident. Enterotoxin genes sea - sej and tst were evaluated by multiplex PCR. Although sea is considered the most prevalent enterotoxin gene reported all over the world, in the present study this was not verified. tst gene was detected in children, hospital health care professionals and food handlers. PVL genes were analysed in all the MRSA strains collected from various origins, but only one food strain showed the presence of these genes. Typing by SCCmec has been performed and revealed the presence of type IV and V. SCCmec type IV is associated worldwide with PVL positive strains, but which was not verified in this study. PFGE typing was performed on S. aureus strains isolated from hands and from nose of the same individual of the health care professionals and food handlers; 60 and 30% of the individuals, respectively, presented the same S. aureus strain on hands and nose.
- Chemiomics : a systematic chemistry approach to unravel the interface pathways between oxidation and Maillard mechanism responsible for flavour modulation during wine storagePublication . Monforte, Ana Rita Araújo da Silva Monteiro; Ferreira, António César da Silva; Maaten, Sara Isabel da Fonseca Selgas Martins Van Der; Bro, RasmusWine is a widely consumed beverage with a high commercial value, which price can increase with aging for a certain type. However, the knowledge of the chemical processes occurring during aging that result in a specific chemical/sensory profile remains limited. This lack of knowledge and understanding significantly limits the ability to improve product quality and consistency. For that reason, unravelling the chemical changes occurring during aging that are responsible for wine flavour, constitutes a critical task when one attempts to address issues related with authenticity and sensory quality. These considerable chemical modifications are related in particular with the formation of aroma active substances such as Strecker aldehydes. Depending on the storage conditions, namely related with dissolved oxygen, and availability, quality and quantity of antioxidants as well as precursors concentrations of key odorants the chemical profile of wines changes. In that regards these work presents several studies in order to evaluate the impact of such factors in the formation of Strecker aldehydes, in particular phenylacetaldehyde (Chapters 3, 4, 5 and 6) and other molecules (Chapter 7 and 8). In chapters 3, 4, 5 and 6, studies were performed in wine model solutions and the results demonstrated that the presence of metals, are relevant in promoting the Strecker degradation through phenolics oxidation pathways at wine pH. Also, this route is the major pathway for phenylacetaldehyde formation. For the first time it was demonstrated that the presence of glucose inhibits the formation of phenylacetaldehyde in both wine model systems and in white wine. By quinone quantitation it was shown that glucose affects directly their concentration, which suggest that in white wine glucose has an antioxidant effect by inhibiting o-quinones formation. In order to study the phenomenon in high complex systems, in chapters 7 and 8, aged white wines were studied.
- Design of cellulose based micro- and nanostructures for encapsulation and controlled release of lipophilic biomoleculesPublication . Casanova, Francisca Casanova Cerqueira; Ramos, Óscar Leandro da Silva; Pereira, Carla Patrícia Fernandes; Fernandes, João Carlos Azevedo Cruz GonçalvesPoor aqueous solubility, stability and bioavailability of interesting active biomolecules is a challenge in the development of bioactive formulations. Cellulose micro- and nanostructures are promising and sustainable carriers with unique features that may be used in enabling delivery strategies. In the context of sustainable development, lignocellulosic biomass from industrial and agricultural wastes have attracted much attention as cellulose sources. Sugarcane plantations for the sugar and alcohol industries are known for their high volumes and large amounts of residues, such as sugarcane bagasse (SCB), a promising renewable and low-cost source of cellulose. This PhD project aims to valorize a major by-product of the agro-industrial sugarcane industry – SCB – by extracting the cellulose polymer and developing micro- and nanostructured systems for the delivery of lipophilic biomolecules as means to offer an efficient and controlled release of these molecules and promote their biological functions. Initially, a chemical and structural characterization of SCB was performed, followed by the evaluation of different approaches for the extraction and purification of cellulose from SCB. Considering the results obtained through the different and complementary techniques employed, the most promising approach revealed to be autohydrolysis (170 ºC for 1 h), followed by bleaching with hydrogen peroxide (12%, v/v, at 85 ºC for 1.5 h) and sodium chlorite (12%, w/v, at 85 ºC for 1.5 h). Through this method we were able to obtain a white cellulose rich fraction (87.5% ± 0.6 cellulose) with low contaminants, high crystallinity index (73.1% ± 1.1) and typical cellulose functional groups from SCB. Microcrystalline cellulose (MCC), cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) were then produced from the SCB cellulose rich fraction, contributing to the valorization of the agro-industrial byproduct. Optimized MCC, CNF and CNC were produced by mild acid hydrolysis, ultrasonication and acid hydrolysis followed by sonication, respectively. MCC had a particle size of ca. 30 μm, CNF of ca. 600 nm long and 8 nm wide, and CNC of ca. 120 nm long and 6 nm wide, which is in agreement with the definition of these types of structures in the literature. ATR-FT-IR and PXRD results confirm the cellulose structure for the three cellulosic materials. CNC had a higher crystalline fraction than CNF, which is attributed to the removal of the amorphous part of the microfibril during acid hydrolysis. The overall properties of our micro- and nanocellulose materials were found to be similar to those of commercial products. Subsequently micro- and nanocellulose were tested as carrier materials for the delivery of lipophilic biomolecules: curcumin, a model lipophilic biomolecule, and cannabigerol (CBG), a promising cannabinoid. Nanocellulose hydrophobic modifications with the surfactant cetyltrimethylammonium bromide (CTAB) and tannic acid/decylamine (TADA) and modification by TEMPO-mediated oxidation, were also tested and compared with the unmodified structures. The modified cellulose structures were characterized and found to successfully bind curcumin, with encapsulation efficiencies (EE) ranging from 63% to 99%. The CTAB and TADA modifications resulted in highly effective EE of 90-99% for curcumin. These modified systems provide a strategy for stabilizing and delivering curcumin by fixing it in hydrophobic domains, potentially improving its solubility and stability. Among the modifications, CNC-CTAB showed the most promising results, allowing for a sustained release of curcumin (ca. 50% released in 8 hours) and exhibiting high EE (ca. 80%) for both curcumin and CBG after scale-up by spray drying. The safety and biological potential of the CNC-CTAB delivery systems encapsulating curcumin and CBG were evaluated. In vitro cytotoxicity and genotoxicity tests were performed on the systems, which revealed to be safe for intestinal application at certain concentrations. Encapsulation reduced the cytotoxicity of both curcumin and CBG, highlighting the potential benefits of using CNC-CTAB as a delivery system for these compounds. The encapsulated biomolecules demonstrated antioxidant and anti-inflammatory properties, effectively reducing reactive oxygen species and cytokine production by intestinal cells. The delivery systems also exhibited antimicrobial properties against Campylobacter jejuni, suggesting its potential in mitigating inflammation in the gastrointestinal tract. Furthermore, the system showed ability to protect curcumin from degradation and facilitate its interaction with the intestinal epithelium, highlighting the potential of CNC-CTAB as a carrier to enhance the biological functions of curcumin and CBG, particularly in the context of intestinal inflammatory disorders. The valorization approach proposed in this work could be beneficial for the sugarcane industry to improve its environmental and economic sustainability in line with circular bioeconomy. This study emphasizes the potential of hydrophobic modified CNC as delivery systems for lipophilic biomolecules. This system effectively reduced the cytotoxicity of curcumin and CBG, protected from degradation, and enhanced biological activities, allowing for their beneficial effects to last longer and be more effective. This breakthrough has the potential to be applied to other compounds and ingredients, serving as an innovative step in utilizing agro-industrial by-products for value creation.
- Design of natural and sustainable ingredients from sugarcane byproducts for cosmetic and skin care industryPublication . Carvalho, Maria João Coimbra Branco de; Madureira, Ana Raquel Mendes Ferreira Monteiro; Oliveira, Ana Lúcia da Silva; Pedrosa, Sílvia Marlene Almeida SantosSugarcane straw is a byproduct of sugar and ethanol production. This material is rich in phenolic compounds, known to have bioactive properties, including antimicrobial and antioxidant, which make them attractive for developing natural and sustainable ingredients for the cosmetic industry, namely preservative and anti-aging ingredients. However, the processing and use of sugarcane straw extracts for the cosmetic industry is yet unknown. Also, the bioactive properties of a sugarcane extract enriched with phenolic compounds need to be investigated in the event of being interesting for cosmetic industry. The safety of these ingredients on the skin and their impact on skin microbiota, which are fundamental to skin health, remains to be assessed. To assess the impact on the skin microbiota, less expensive and time-consuming techniques need to be developed. Thus, the main objectives of this thesis are 1) develop and optimize a process of extraction and purification of phenolic compounds from sugarcane straw; 2) develop a cosmetic ingredient based on the extract; 3) assess the preservative and anti-aging properties both in vitro and in vivo, as well as their capacity for dermal permeation.; 4) develop an in vitro preclinical model to test cosmetic ingredients on the skin microbiota; and 5) test the impact of the sugarcane straw extract-based ingredient on the skin microbiota. Initially an extraction and purification process to obtain the phenolic compounds from sugarcane straw was developed. After ethanolic extraction of the phenolic compounds, amberlite XAD-2 resin was used for purification of the extract. The purified extract was analyzed, resulting in the identification of 54 secondary metabolites, mainly hydroxybenzoic acids, hydroxycinnamic acids and flavones. The main compounds detected were chlorogenic acid, neochlorogenic acid and 3-O-feruloylquinic acid. Purification aimed to reduce the sugar content, and after, only a small number of monosaccharides, such as glucose, cellobiose and xylose + arabinose, were detected in the extracts. Additionally, the extracts displayed antioxidant activity, measured by ABTS and DPPH methods. Subsequently, the development of a cosmetic ingredient based on the phenolic compounds extract aimed to improve the solubility of the dry extract powder, and for that purpose, different cosmetic solvents (1,2-pentanediol, 1,2-hexanediol, dipropylene glycol and 1,5-pentanediol) were tested. The antimicrobial activity of the solubilized ingredients was evaluated against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. 1,2-Hexanediol afforded the ingredient with the best antimicrobial performance, with a minimum inhibitory concentration between 5 and 3 % (w/v), being the first compliant with USP 51 standards for cosmetic preservatives. The resulting ingredient formulation was chosen for further testing. Regarding the anti-aging properties of the engineered cosmetic ingredient, it was able to inhibit elastase (55 %), collagenase (25 %) and tyrosinase (47 %) and promote the hyaluronic acid production in fibroblasts (HDF) at non-cytotoxic and low-sensitization concentrations. Additionally, the ingredient reduced the levels of the pro-inflammatory cytokines IL-1α and IL-6 in immortalized keratinocytes (HaCaT) exposed to urban pollution particles. The ingredient's ability to permeate the skin was tested on a synthetic membrane that simulates the different layers of the skin, and around 67 % of the phenolic compounds present in the ingredient formulation remained on the surface of the skin, indicating that the ingredient has low dermal permeation. Sugarcane straw extract-based ingredient was incorporated in O/W and W/O emulsions and its stability was assessed in accelerated conditions following ISO/TR18811 directive. The anti-aging performance was evaluated in a cosmetic clinical trial, and the findings were verified by an external entity. This external evaluation not only covered the same parameters but also included assessments for anti-wrinkle effects, skin roughness, and elasticity. In summary, the ingredient did not enhance skin hydration, firmness, or exhibit significant anti-wrinkle or firming effects. Nevertheless, it was well-received and well-tolerated by the study participants. Finally, a preclinical in vitro skin microbiota model was developed, with test conditions optimized and validated using a reference ingredient with known in vivo data. The sugarcane straw extract-derived ingredient was then assessed using skin microbiota samples from 12 healthy volunteers, with microbial group impact analyzed through qPCR and next-generation sequencing (NGS). These results aligned with the manufacturer's in vivo findings, thus affirming the validity of the in vitro model. Sugarcane straw extract-based ingredient was found to have no effect on community α-diversity, although affected the relative abundance of Staphylococcus epidermidis. Thus, in this thesis, a purified extract from sugarcane straw rich in phenolic compounds was obtained and formulated to develop a cosmetic ingredient that has been shown to have preservative and anti-aging properties in vitro. Furthermore, a preclinical in vitro model was developed and validated as a tool for evaluating the impact of new cosmetic ingredients on the skin microbiota.
- Development and characterization of functional ingredients from olive pomace : bioactivity and potential applicationPublication . Ribeiro, Tânia Isabel Bragança; Pintado, Maria Manuela Estevez; Vicente, António Augusto; Nunes, João Miguel dos Santos AlmeidaAtualmente, grande volume dos resíduos gerados ao longo da cadeia alimentar Europeia é produzido durante o processamento alimentar (19-39%). A indústria de extração de azeite não é exceção, gerando anualmente grandes quantidades de subprodutos e resíduos com elevado impacto fitotóxico, mas que são também ricos em diversos compostos bioativos e nutrientes com potencial aplicação como ingredientes alimentares. Assim sendo, é imprescindível a adoção de estratégias de redução do desperdício e de valorização dos subprodutos, no sentido de melhorar a sustentabilidade económica e ambiental da indústria alimentar. Atualmente, o bagaço de azeitona é o subproduto de maior relevo da indústria de azeite e a sua valorização adequada é também o maior desafio do sector para alcançar uma cadeia produtiva sustentável alinhada com os princípios da Bioeconomia Circular. Esta tese visou alcançar uma valorização “zero desperdício” para o bagaço de azeitona, priorizando os produtos de alto valor acrescentado sobre os de baixo valor através da produção e caracterização de ingredientes alimentares, nomeadamente ingredientes em pó ricos em vários compostos bioativos, seguida da validação in vitro das suas atividades biológicas ligadas a benefícios para a saúde, como a prevenção e redução de doenças cardiovasculares, diabetes, problemas intestinais e posterior aplicação no desenvolvimento de alimentos funcionais. Uma estratégia de “desperdício zero” foi alcançada para o bagaço de azeitona. Esta estratégia consiste num procedimento simples de fracionamento, em que se obteve uma fração líquida que revelou um alto teor de hidroxitirosol (513,61-625,76 mg/100 g de peso seco) e uma fração de polpa caracterizada por ser uma boa fonte de fibra alimentar antioxidante. Adicionalmente, obteve-se ainda uma fração de caroços que exibiu valores caloríficos significativamente elevados (18,65-18,94 MJ/kg). Estes resultados validaram o potencial valor acrescentado das frações líquidas e de polpa como ingredientes alimentares e como biocombustível no caso da fração de caroços. Após validação do potencial da fração líquida e de polpa do bagaço de azeitona como ingredientes alimentares, dois novos ingredientes em pó, estáveis e seguros, foram desenvolvidos sem qualquer etapa de extração, nomeadamente um pó enriquecido na fração líquida (LOPP) e um pó enriquecido na fração de polpa (POPP). O LOPP exibiu uma quantidade significativa de manitol (141 g/kg), potássio (54 g/kg) e hidroxitirosol/derivados (5 mg/g). O POPP exibiu uma grande quantidade de fibra alimentar (620 g/kg) associada a um teor significativo de fenólicos (7,41 mg equivalentes de ácido gálico/g fibra) com atividade antioxidante substancial. O POPP também revelou uma composição de ácidos gordos insaturados semelhante à do azeite (76% do total de ácidos gordos), sendo ainda uma fonte razoável de proteína (12%). As suas propriedades funcionais (solubilidade, capacidade de retenção de água e de óleo), capacidade antioxidante e atividade antimicrobiana foram também avaliadas, assim como a sua segurança biológica. Em síntese, a multifuncionalidade e aplicabilidade dos pós de bagaço de azeitona como fortificantes nutricionais, promotores de benefícios para a saúde e conservantes foram atestadas. A digestão simulada in vitro permitiu concluir que os fenólicos e minerais do LOPP foram afetados negativamente pela digestão, no entanto, uma bioacessibilidade significativa de potássio e hidroxitirosol foi verificada ( 57%) e as suas bioatividades foram apenas moderadamente afetadas. Em relação às suas bioatividades, pelo menos metade da atividade antioxidante (capacidade de Resumo x absorção do radical de oxigénio), de inibição da -glucosidase e de inibição da enzima conversora de angiotensina (91,98%) foram retidas e bioacessíveis após a digestão. Por sua vez, a digestão do POPP revelou uma perda significativa de fenólicos na digestão oral (62,48%), mas o papel da fibra como veículo de fenólicos permitiu recuperar uma quantidade significativa de fenólicos no estômago (77,11%) e um índice de bioacessibilidade de pelo menos 50% (tirosol e o seu glucosídeo). A fibra do POPP também interagiu positivamente com os ácidos gordos, diminuindo e facilitando bioacessibilidade dos saturados (5-6%) e dos insaturados (4-11%), respetivamente. Após realização da digestão in vitro dos pós de bagaço de azeitona, as frações disponíveis no cólon de ambos os pós exibiram uma composição abundante em compostos associados a potenciais benefícios para a saúde intestinal, nomeadamente fibra e compostos fenólicos e fibra. Assim sendo, procedeu-se a fermentação fecal in vitro das frações não digeridas retidas no cólon após a digestão gastrointestinal simulada in vitro do LOPP e POPP. Os resultados da análise do gene 16S rRNA e dos ácidos gordos de cadeia curta mostraram que estes pós não tiveram um impacto negativo na diversidade da microbiota intestinal e que promoveram uma maior produção de ácidos de cadeia curta (acetato>butirato>propionato) do que os frutooligossacarídeos. A identificação dos metabólitos fenólicos dos pós de bagaço de azeitona por LC-ESI-UHR-QqTOF-MS seguida das análises de clustering supervisionada e clustering hierárquico permitiram avaliar os padrões destes metabólitos ao longo da fermentação fecal in vitro, bem como explicar as potenciais atividades biológicas destes pós no intestino. Ambos os pós apresentaram um conteúdo fenólico e uma capacidade de absorção de radical de oxigénio significativas e capacidade de inibição da adesão de patógenos como o Bacillus cereus (22,03%) e Listeria monocytogenes (20,01%), sobretudo pelo POPP. Por fim, para validar o desempenho dos pós de bagaço de azeitona como ingredientes funcionais, LOPP e POPP foram incorporados em iogurte para aumentar o seu conteúdo em fibra alimentar, hidroxitirosol e ácidos gordos insaturados. A adição de POPP (2%) ao iogurte permitiu cumprir a condição de “fonte de fibra”, por sua vez LOPP (1%) forneceu 5 mg de hidroxitirosol/ derivados num iogurte (120 g). A adição de POPP e azeite juntamente com LOPP ou POPP permitiu uma melhoria do perfil de ácidos gordos insaturados dos iogurtes. Além disso, todos os iogurtes fortificados apresentaram um maior teor de fenólicos e atividade antioxidante. A digestão in vitro reduziu a atividade antioxidante de todas as formulações e mostrou que a incorporação de azeite juntamente com o LOPP e POPP aumentou a bioacessibilidade de hidroxitirosol e do hidroxitirosol glucosídeo, respetivamente. Contudo, todas as formulações com os pós de bagaço de azeitona podem ser consideradas boas fontes de hidroxitirosol, lípidos insaturados e fibras para os consumidores. Em suma, os resultados deste trabalho irão contribuir para a sustentabilidade da indústria do azeite no contexto da bioeconomia circular, através do desenvolvimento de ingredientes de valor acrescentado destinados à indústria alimentar com potencial impacto positivo na saúde, proporcionando ainda um biocombustível sólido.
- Development and characterization of functional ingredients through valorization of pineapple by-products : production, bioactivity and potential applicationPublication . Campos, Débora Andreia Campelo; Pintado, Maria Manuela Estevez; Pastrana, LorenzoAs indústrias de processamento de frutas produzem toneladas diárias de resíduos, sendo os subprodutos de abacaxi um dos mais importantes, devido ao tamanho do fruto e, devido ao fato de 60% do peso do seu peso ser descartado como resíduo. A valorização total de subprodutos de abacaxi, por meio da química verde e de tecnologias sustentáveis, visando a obtenção de zero resíduos, é uma das possíveis abordagens para realizar uma valorização integrada destes subprodutos através de uma abordagem de biorrefinaria. O objetivo principal deste trabalho de doutoramento consistiu em utilizar subprodutos do abacaxi (cascas e centros) no desenvolvimento de ingredientes funcionais, desde concentrados de enzimas, extratos prebióticos/ antioxidantes e farinhas funcionais, e caracterizar e validar as atividades biológicas e as capacidades tecnológicas desses novos ingredientes. Primeiro, os centros e cascas do abacaxi foram fracionados gerando dois sumos (frações líquidas) e duas frações sólidas. As frações líquidas do centro representam 5% e da casca 17% do peso total do abacaxi. Ambas as frações líquidas apresentaram alto teor de açúcares simples, representando ca. 65% no caule e ca. de 67% na casca; ambas frações contêm menos de 1% de fibra dietética solúvel. Além disso, foi encontrado alto conteúdo de compostos fenólicos totais ca. 1.3% para a fração líquida do caule e ca. 0.7% para a fração líquida da casca, com elevador teor de vitamina C, associada também a uma elevada atividade antioxidante medida pelos métodos de ABTS e ORAC. Por outro lado, a fração sólida do caule representa ca. 44%, enquanto que a fração sólida da casca representa ca. 38% de abacaxi total, contendo proporções muito diferentes de hidratos de carbono. A fração sólida do caule contém 23% de açúcares simples, 5% de fibra dietética solúvel e 45% de fibra dietética insolúvel, enquanto que fração sólida da casca contém 28% de açúcares simples, 8% de fibra dietética solúvel e 38% de fibra dietética insolúvel, demonstrando um grande potencial como ingrediente rico em fibra. A composição dos hidratos de carbono estruturais mostrou diferenças, principalmente no teor total de celulose e lignina insolúvel. Uma precipitação com polissacáridos foi aplicada às frações líquidas do abacaxi para extrair a principal enzima presente - a bromelaína, uma importante enzima natural que é amplamente aplicada na indústria. A metodologia de separação desenvolvida baseada na precipitação com carragenana, representa uma alternativa natural às metodologias tradicionais de separação. Uma otimização experimental foi aplicada aos subprodutos de abacaxi para definir os valores ótimos responsáveis pela separação da bromelaína. Alto rendimento de recuperação (cerca dos 80%) de bromelaína ativa foi obtida para ambos os sumos, sendo possível extrair 0.26 g de bromelaína a partir de 100 g de subprodutos de abacaxi usando baixas concentrações de polissacarídeo (0.2-0.3%, p/v). Ambas as frações líquidas foram submetidas a um sistema gastrointestinal (TGI) simulado para compreender o impacto na composição dos bioativos presentes. Observou-se um elevado grau de hidratos de carbono digeridos, uma vez que os hidratos carbono de alto peso molecular, após a digestão bucal e duodenal, transformaram-se em moléculas de menor tamanho (tri-, di- e monossacarídeos). O conteúdo total de polifenóis aumentou ao longo do TGI, assim como a atividade antioxidante, medida por ABTS e ORAC, devido à libertação de polifenóis complexados com outras moléculas. A análise de composição de polifenóis mostrou que o ácido clorogénico está em maior concentração, combinado com outros como os ácidos cafeíco, cumárico e ferúlico, principalmente libertados após a digestão com TGI. A fração líquida do caule foi selecionada para estudar o potencial prebiótico, revelando elevada atividade em cinco estirpes probióticas (duas de lactobacilos e três de bifidobactérias). A atividade prebiótica foi medida após cada compartimento do TGI, a qual diminuiu ligeiramente ao longo do sistema. No entanto, a fração residual evidenciou capacidade para modular o crescimento e o metabolismo das estirpes probióticas. As frações sólidas também foram submetidas à simulação do TGI e a fração remanescente foi fermentada in vitro num modelo de fezes humanas. Ambas as frações evidenciaram elevadas concentrações de ácidos ferúlico e cumárico (cascas: 70 mg 100 g-1 e 113 mg 100 g-1 e caules: 95 mg 100 g-1 e 68 mg 100 g-1, respetivamente) que foram detetados após a simulação do TGI, bem como após 48 h de fermentação com microbiota intestinal, demonstrando ter um alto potencial antioxidante durante todas as fases do TGI. A fração sólida do caule promoveu o crescimento de Lactobacillus spp. e Bifidobacterium spp., enquanto a fração sólida da casca promoveu principalmente o crescimento de Lactobacillus spp. O crescimento desses grupos levou à produção de ácidos gordos de cadeia curta (AGCC), principalmente ácido propiónico e acético, que modularam positivamente o metabolismo intestinal. A prova de conceito de um dos ingredientes desenvolvidos foi realizada através da aplicação de bromelaína natural em um processo de amaciamento de um fiambre tradicional. A concentração mais baixa de bromelina (1 mg mL-1) permitiu o amaciamento da carne de porco. Através de um desenho experimental fatorial, os intervalos de valores e as condições ótimas de aplicação de enzima foram selecionadas: 1 mg de bromelaína por mL de salmoura. Os resultados deste trabalho experimental contribuirão para a sustentabilidade das indústrias de processamento de frutas num contexto de economia circular, através do desenvolvimento de ingredientes de valor agregado com impacto bioativo e funcional. Além disso, o desenvolvimento de seis novos ingredientes funcionais, através da aplicação de um processo a jusante integrado com obtenção de resíduos ZERO, ajudará as indústrias de processamento de frutas a implementar soluções de baixo custo, com produtos finais de alto valor agregado, o que permite a reintegração do valor económico, com a geração de novos fluxos de receita e o incentivo a uma sociedade mais sustentada.
- Development of a bio-based process that converts lipid’s distillation and evaporation residue into polymers with potential application for further materials industryPublication . Sousa, Ana Paula Capêto Matos; Oliveira, Ana Lúcia da Silva; Teixeira, Ana Sofia Moreira dos Santos GuimarãesThe objective of this study was to valorize a microbial oil (FDR) with a composition akin to vegetable oils and a degree of unsaturation (»130 g I2/100 g) typical of semi-drying vegetable oils (e.g., soybean, corn, and sunflower oils). Considering this, it was deemed appropriate to apply the methodologies used for epoxides and polyols synthesis from vegetable oils commonly employed in condensation polymers (e.g., polyurethanes, polyhydroxyurethanes, and polyesters) production. A set of experiments using a two-step mechanism was performed starting with epoxide production (oxirane ring formation from C=C bonds) with in situ performic acid followed by insertion of -OH groups (polyol synthesis) by oxirane ring-opening with phosphoric acid or glycerol. Two epoxides were obtained (4.6 % of oxirane content) with reagent ratio FDR: HCOOH: H2O2 = 100:6:160, one from crude FDR at 60 ºC for 4 h, and another one after treatment by winterization (FDRw) at 85 ºC for 3 h with improved color, and lower viscosity. From those epoxides were produced two polyether polyols, UPT (f = 4.0, -OH = 82.3 mg KOH/g ) and JG (f = 8.9, -OH = 265.3 mg KOH/g), and one polyester polyol KCO obtained melting epoxide with castor oil (f = 5.5, -OH = 104.2 mg KOH/g). In a proof-of-concept, polyurethane rigid (PUR) foams with MDI (diisocyanate) were prepared, integrating the FDR-based polyol (UPT) in the backbone, and sugarcane bagasse ash (SCBA, 4.5 %) used by its high content in silica for added fire-retardant properties. Comparing with reference foam without ash (PF), the analysis of structure and thermo mechanical properties indicated that ash particles acted as nucleating points with no chemical interaction, reducing foam’s density (PF, 134.7 kg/m3 ; PFA, 105.7 kg/m3 ), cell's size (PF, 496 nm; PFA, 480 nm), and thermal conductivity (PF, 0.0482 W/m·K; PFA, 0.0461 W/m·K). The added plasticizing effect increased the polymer chain mobility, thus decreasing the glass transition (Tg) temperature (PF, 74.0 ºC; PFA, 71.8 ºC) and delaying the decomposition onset temperature (Td) (PF, 215 ºC; PFA, 230 ºC). The flammability test results suggest that SCBA has improved the foam performance in compliance with the fire protection standard UL 94, class HB. To remove the toxic isocyanates from that equation the safer route was to prepare non isocyanate polyurethanes (NIPUs) and their hybrids (HNIPUs). Two methods were explored (cycloaddition of CO2 to epoxide and transcarbonation with polyol JG) for cyclic carbonates synthesis (the monomeric units). The result of the latter was a hard HNIPU, with lower gel content (6.8 %) and Tg (60.6 °C) comparing with the glycerol-based reference (gel = 80.0 % e Tg = 106.8 ºC), though harder and with higher melting temperature, Tm (171.6 ºC vs 147.9 ºC) mainly due to wax crystals content. Using polyols KCO and UPT (from FDRw), were prepared two polyesters (KCO and UPT) with elastomeric properties, low Young’s modulus (1.92 × 10-3 and 3.9 MPa) and elongation up to 150 ad 185.4 %, respectively. Polyester KCO, was tailored for controlled release of active principles for wound dressing applications, with proved skin biocompatibility and degradability, hydrophilic character, low Tg (−51.2 ◦C), and high Tm (169.8 ◦C). The second (UPT) showed an amorphous structure and shape memory behavior between - 47 and 12 °C, apt for low-temperature applications where it is required to maintain the mechanical integrity of components, eventually subjected to large deformations during service conditions. Applying the same principles of sustainability and circularity to the building and construction (B&C) industry, an experimental campaign was envisioned integrating FDRw and SCBA into cementitious mortars to ultimately enhance the hygrothermal properties in 3 D impressed specimens. Thermal conductivity (λ), water absorption by capillary action (Aw), and in saturated state the moisture content (Wsat), and water absorption (Ab) were determined in cured specimens. The variation found when comparing the results for specimen containing only FDRw 1% (λ = 1.493 W/m·K, Aw = 0.048 kg/m2 . s0.5, , Wsat = 53.47 kg/m3 , and Ab = 2.58 %), and reference (λ = 1.411 W/m·K, Aw = 0.045 kg/m2 . s0.5 , Wsat = 36.15 kg/m3 , and Ab = 1.74 %) suggests that pore distribution, pore size and interconnectivity are driving factors inducing inner defects and major voids partially filled by the presence of SCBA (Mix SCBA/ FDRw = 5/1) improving the mortar’s hardened properties (λ = 1.382 W/m·K, Aw = 0.041 kg/m2 . s0.5 , Wsat = 41.43 kg/m3 , and Ab = 1.99 %).
- Development of a new functional dairy product enriched in microbial bioactive conjugated fatty acids through an industrial-based approachPublication . Fontes, Ana Luiza Rodrigues; Gomes, Ana Maria Pereira; Alcalá, Luís Miguel Rodríguez; Domingues, Maria do Rosário Gonçalves dos Reis MarquesThe search for innovative food products with added-value properties has been an increasing tendency in the last years. Accordingly, different food compounds with potential bioactive properties have been identified, such as, conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which have been described with anti-carcinogenic, anti-obesity and anti-inflammatory activities, among others. These compounds are naturally found in meat and milk of ruminants or vegetables oils, but, due to availability and concentration concerns, it is not feasible to attain beneficial effects from CLA/CLNA through these sources. Alternatively, in situ microbial enrichment of food matrices has been studied, since several probiotic strains have been described with the capacity to isomerize linoleic acid (LA) and α-linolenic acid (α-LNA) into their corresponding conjugated forms. Thus, the objective of this work was to study the development of a new added-value dairy product through in situ microbial production of CLA and CLNA using previously hydrolyzed commercial vegetable oils as substrate sources, so as to evaluate possible side fatty acid (FA) metabolites released during the process, study the biochemical and organoleptic properties, and assess the shelf-life stability of the developed dairy product. First the selection of CLA/CLNA-producing strains was performed from among 85 probiotic strains through molecular detection of genes encoding enzymes involved in CLA/CLNA formation, namely, linoleate isomerase (LAI), myosin-cross-reactive antigen (MCRA) and fatty acid-hydratase (FA-HY), using reported primers and primers designed based on conserved motifs. Meanwhile, it was determined the strains’ maximum tolerance to LA after exposure to increasing concentrations thereof, namely 1, 2 and 5 mg/mL. About 34 strains revealed the presence of at least one of the screened genes, where the designed primers were more effective, but no association was found between their LA-tolerance and the CLA production potential. Moreover, only 4 genotypically-positive strains revealed the capacity to convert 0.5 mg/mL LA into CLA isomers. These were further tested for CLNA production from 0.5 mg/mL α-LNA. The strain Bifidobacterium breve DSM 20091 demonstrated the best yields of CLA and CLNA isomers (>50% of substrate conversion), being selected for the following assays. In a second phase, the utilization of commercial vegetables oils as substrate sources for the development of a CLA/CLNA-enriched fermented milk was studied. To increase the bioavailability of LA and α-LNA present in the selected oils, i.e., soybean (43.7 g LA/100 g oil) and flaxseed (41.3 g α-LNA/100 g oil) oils, different lipases were tested. The Candida rugosa lipase showed the best yields for all of the tested oils (>90% of hydrolysis). Bifidobacterium breve DSM 20091 was thereafter assayed in milk containing 0.5 mg/mL of LA and/or α-LNA obtained from the hydrolyzed oils. Results showed that this strain was not able to produce CLA simultaneously with CLNA to the same extent. Therefore, higher substrate concentrations (i.e., α-LNA) were further tested with only hydrolyzed flaxseed oil. The best yields were attained at 2 mg/mL α-LNA, registering ca. 1 mg/g of mainly CLNA isomers. To evaluate possible FA metabolites, a gas chromatography-mass spectrometry (GC-MS) analysis of milk fermented with pure LA and/or α-LNA or hydrolyzed flaxseed oil was performed. No further FA compounds were found that could result from LA or α-LNA metabolization; however, two additional CLNA isomers never reported before for bifidobacteria strains were discovered. The biochemical and organoleptic properties of the developed CLNA-enriched fermented milk were thereafter evaluated through analysis of sugars and organic acids content, titratable acidity, pH, nutritional composition, volatile compounds profile and sensory properties. The developed product showed comparable compositional properties; however, it lacked important flavor contributors, and bitterness and astringency prevailed. Finally, the stability of the CLNA-enriched fermented milk was assessed throughout 28 days of refrigerated (4 ºC) storage. To obtain an alternative delivery system of CLNA isomers, the enriched fermented milk was further lyophilized, and its stability during storage (12 weeks at room temperature) was also evaluated. For both products a free saturated FAs loss and an increment of conjugated isomers content was observed. In conclusion, the selection of potential CLA/CLNA-producing strains cannot rely solely on genotypic and/or substrate tolerance screening techniques. Even though, it was possible to obtain a fermented milk enriched in conjugated FAs with B. breve DSM 20091 using hydrolyzed flaxseed oil, and this strain showed a preference to produce CLNA isomers. Moreover, two additional CLNA isomers never reported before were discovered. The biochemical and nutritional characteristics of the CLNA-enriched fermented milk were acceptable, but there are organoleptic features that remain to be tackled. Although conjugated isomers in the developed dairy products increased throughout storage, the alterations in free saturated FA suggested the occurrence of oxidation processes.
- Development of peptide-rich extracts from yeast by-products for nutraceutical applicationPublication . Oliveira, Ana Sofia da Silva; Carvalho, Ana Paula Taboada da Costa Santos; Ferreira, Carlos Miguel Henriques; Pereira, Joana Odila Mendes de SáAo longo dos anos, a biologia sintética cresceu com o uso de leveduras geneticamente modificadas para produção de ingredientes sustentáveis. À semelhança da indústria cervejeira, é gerada uma quantidade considerável de levedura excendentária, que possui um valor nutricional notável como fonte proteica alternativa. Considerando o aumento da população global e o consequente aumento da procura de proteína, a presente tese visa valorizar os péptidos de leveduras geneticamente modificadas, produzindo extratos ricos em péptidos quer da sua extração direta, quer a partir dos resíduos de extração de β-glucanas e mananas (componentes da parede celular das leveduras) numa abordagem de economia circular para a indústria nutracêutica. Relativamente à extração de péptidos diretamente da levedura, foram usadas metodologias escaláveis e sustentáveis como a homogeneização a alta pressão, sonicação, hidrólise enzimática e autólise. Os extratos de leveduras geneticamente modificadas mostraram excelentes perfis nutricionais, semelhantes aos das estirpes não-modificadas. A autólise destacou-se como uma abordagem promissora, embora outras métricas de sustentabilidade ainda precisem ser estudadas para a implementação industrial desse processo. Para aprofundar as propriedades biológicas dos péptidos do autolisado, testaram-se dois métodos de purificação: ultrafiltração e cromatografia por exclusão de tamanho. As frações resultantes exibiram diversos teores de proteína, distribuições de pesos moleculares e sequências de péptidos, apresentando uma elevada capacidade de inibição da enzima conversora da angiotensina, atividade antioxidante, e uma maior inibição da 3-hidroxi-3- metilglutaril-coenzima A (HMG-CoA) redutase do que o autolisado original, sugerindo a importância da purificação para produzir suplementos alimentares com efeito anticolesterolémico. Até onde sabemos, a inibição da HMG-CoA redutase associada a péptidos de levedura é aqui reportada pela primeira vez. Considerando o custo-benefício, a ultrafiltração parece ser o método mais adequado para a produção de extratos ricos em péptidos destinados ao mercado nutracêutico. A ultrafiltração foi também aplicada aos sobrenadantes dos resíduos de extração de β-glucanas e mananas da levedura, maximizando a abordagem de economia circular para a produção de extratos ricos em péptidos. Estes extratos apresentaram um desempenho nutricional e bioativo comparável aos obtidos diretamente da levedura, tornando-os excelentes ingredientes para o setor nutracêutico. Considerando a composição dos aminoácidos nos extratos peptídicos, identificou-se potencial para formar complexos Fe-péptidos com a fração Gpep > 1 kDa (fração peptídica com peso molecular maior que 1 kDa proveniente dos resíduos de extração de β-glucanas) em condições anóxicas. Do desenho fatorial aplicado mostrou-se que as condições ótimas de reação ocorrem a pH 6,0 durante 30 min, sendo a complexação confirmada por análise química e estrutural. Finalmente, os complexos foram submetidos à simulação in vitro da digestão gastrointestinal humana, e sua absorção foi analisada numa linha celular de adenocarcinoma do cólon humano, avaliando a absorção de Fe. Os resultados revelaram que os complexos se comportaram similarmente aos sais de Fe e bisglicinato de Fe comercial, indicando o seu potencial promissor para a suplementação dietética de Fe. Além disso, os péptidos dos complexos demonstraram atividade antioxidante, sugerindo a proteção do Fe contra a oxidação em diferentes ambientes gastrointestinais. Em suma, esta tese promove a valorização de levedura excedentária geneticamente modificada, explorando tanto a biomassa diretamente, como os resíduos de extração de outros dos seus componentes. Isso resulta na produção de diversos extratos peptídicos que podem servir como fonte alternativa de alimento e/ou suplementos para controlo da anemia, bem como para outras áreas, tais como a cosmética, ou ingredientes na produção de microrganismos por fermentação.