ESB - Teses de Doutoramento / Doctoral Theses
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- Effect of polyphenols on saccharomyces cerevisiae fed-batch fermentationPublication . Carvalho, Luís Carlos Rocha; Oliveira, Carla Cristina Marques de; Oliveira, Ana Lúcia da Silva; Çarsanba, ErdemThe industrial production of β-farnesene is traditionally achieved through Saccharomyces cerevisiae fed-batch fermentation, often employing sugarcane syrup as the feedstock. However, the presence of phenolic compounds in sugarcane syrup potentially impacts yeast performance. This thesis aimed to study the evolution of phenolic compounds over β-farnesene production by S. cerevisiae in bioreactor fermentations with sugarcane syrup, assess the phenolic compounds’ impact on this process, and explore the potential of beet syrup, a lower phenolic content alternative feedstock, for β-farnesene production via a similar fermentation process. The phenolic compounds present within both the sugarcane syrup and the fermentation broth were systematically identified and quantified. Sugarcane syrup presented 50.7 mg/L of total phenolic content (TPC), comprising hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. Among two kinetic models assessed, the Weibull model exhibited superior fitting capacity (R2 ≥ 0.85) and accurately depicted phenolic compounds’ accumulation during the 13-day fed-batch fermentation in 2 L bioreactors. The concentration of most phenolic compounds ascended until day three, stabilizing until the end of the fermentation process. Nevertheless, hydroxybenzaldehyde and protocatechuic, caffeic, ferulic and p-coumaric acids declined after the second day, indicating yeast metabolism. This work provides a model that can be applied to describe the phenolic accumulation during similar fed-batch processes. To investigate the phenolic influence on the fermentation, sugarcane syrup phenolic compounds were effectively removed with activated charcoal. The syrup purification process was optimized with a central composite design, varying the type of charcoal, concentration of charcoal and contact time. Optimization yielded purification conditions with charcoal pellets at 115 g/L and 12.5 h of contact time, while removing 96.7 % of phenolic compounds and recovering 43.7% of syrup mass. Purified syrup introduction into fermentations displayed divergent scale-dependent outcomes. Although in shake-flasks there was an 11 % enhancement in β-farnesene productivity, no significant increase was detected in bioreactors. In contrast, an increment in biomass productivity was exclusive to bioreactors, registering an increase of 12 %. Therefore, for the conditions tested, the phenolic compounds did not influence the β-farnesene production at large-scale. Moreover, β-farnesene production using an alternative feedstock, sugarbeet syrup, was successful. Despite presenting lower TPC (21.3 mg/L) compared to sugarcane syrup (50.7 mg/L), beet syrup contained high glucose (193 g/L) and hydroxymethylfurfural (HMF; 1.4 g/L) concentrations. Inoculum production in the second bioreactor step required optimization due to slow growth and the desired cell density was achieved with a fed-batch mode with initial 40 g/L of sugars from beet syrup and 10 g/L of sugar pulses. Over 8 days, fed-batch fermentation yielded 127 g/L β-farnesene concentration, with 19.44 % cumulative yield and 1.70 g/L/h cumulative productivity, affirming the viability of using beet syrup as the fermentation feedstock. In conclusion, this thesis showcased the dynamic relationship between phenolic content and β-farnesene fed-batch process highlighting an increase in the fermentation broth's phenolic content. Notably, specific phenolic compounds were metabolized by the yeast. The effects of sugarcane syrup phenolic compounds on S. cerevisiae were dependent on the fermentation scale. While valuable for mitigating yeast oxidative stress, phenolic compounds’ removal did not enhance bioreactor fermentation productivity. On the other hand, the utilization of beet syrup as feedstock may enable β-farnesene production in mild climates without extensive sugarcane cultivation, fostering sustainable production across diverse regions.
- The bioactive effects of cannabinoids on skin : from daily care to disease management applicationsPublication . Veiga, Mariana da Luz Cabral; Fernandes, João Carlos Azevedo Cruz Gonçalves; João Pedro Azevedo, Silva; Sara Nunes da Costa e, SilvaCannabinoids were first isolated in the 1960s from the Cannabis sativa L. plant. The study into the pharmacological and therapeutic potential of cannabinoids has gained traction in the past decades. This comes as a result of their recognized bioactivity, including antimicrobial, anti-inflammatory, and immunomodulatory activity, and due to the decriminalization of Cannabis-derived products continuing to expand worldwide. Cannabidiol (CBD) is one of the most researched cannabinoids. In particular, its anti-inflammatory activity has been vastly investigated, with a consequent unravelling of direct and indirect interactions with a myriad of receptors, broadening the potential therapeutic applications of CBD. More recently, cannabigerol (CBG), the main precursor of all phytocannabinoids, has also been in focus, although reports on this cannabinoid are still scarce in comparison with its counterpart CBD. Studies concerning the use of CBD and CBG for different therapies have increased, although the current role of cannabinoids in treating dermatological conditions has yet to be defined. Therefore, this thesis aims to explore the use of CBD and CBG for topical applications and to enlighten their potential as promotors of skin health and skin care. To this end, we first assessed their safety through a range of chemical and biological assays, including sensitization and mutagenicity tests. Although CBD extracted from the plant has solidified its use for topical applications, the CBG used in this work is produced through fermentation technology, and its safety profile has yet to be elucidated. After concluding both molecules were safe for topical application, we evaluated their potential as cosmetic ingredients. Several assays were performed to determine if CBD and CBG could be used for skin care and hair care, with both compounds demonstrating their ability to prevent inflammation in keratinocytes after exposure to pollution, and CBG presenting the capacity to induce hair growth in an ex vivo hair follicle model. Next, and considering the antimicrobial activity reported for cannabinoids, we focused on evaluating the antimicrobial and antibiofilm activity of CBD and CBG against bacteria and fungi belonging to the skin microbiota or associated with skin disorders and assessed their anti-adhesion effect against Staphylococci in keratinocytes. For the first time, we reported the MIC and MBC for Cutibacterium acnes, Pseudomonas aeruginosa and Escherichia coli. Thus, this study cemented the use of cannabinoids as antimicrobial agents, besides reporting on their potential as preservative ingredients and their positive lack of impact on the normal skin microbiota. We then evaluated the capacity of CBD and CBG to ameliorate the profile of inflammatory skin conditions, including psoriasis, irritant contact dermatitis (ICD) and atopic dermatitis (AD). Firstly, we developed, optimized, and validated ex vivo skin models with these conditions, and then assessed the impact of cannabinoids on them. Regarding the psoriatic model, CBD and CBG significantly reduced the inflammation and the hyperproliferation of keratinocytes, two of the main characteristics of this disease. Finally, the treatment of CBD and CBG for ICD and AD was also successful, with a reestablishment of the epidermal barrier and a reduction in inflammatory markers. These results are important as inflammatory skin conditions are associated with a low quality of life, and although corticosteroids are the go-to medications to alleviate symptoms, it has been demonstrated these medicines lose efficiency over time and have several side effects. We also attested that neither CBD nor CBG have a side effect common to betamethasone, hypertrichosis. Overall, the works herein reported provide valuable contributions in the field of dermatology, providing a step towards developing new applications for cannabinoids and potentially uncovering new topical treatments for skin disorders.
- Bioactive compounds from nannochloropsis oculata grown under modulated stress : from production to validationPublication . Sousa, Sérgio Daniel da Cruz e; Gomes, Ana Maria Pereira; Carvalho, Ana Paula Taboada da Costa SantosNannochloropsis oculata is a microalga widely recognized as a potential source of polyunsaturated fatty acids, namely the omega-3 eicosapentaenoic acid (EPA), which beneficial health effects have been demonstrated. In order to maximize the EPA amount that may be obtained from a culture, different strategies may be pursued, such as increasing the microalgas’ biomass EPA content and the efficiency of its extraction process. Based on the above rationale, the main goal of the current thesis was to apply different stress conditions to N. oculata culture, which would increase EPA content, but without hindering growth. The strategy employed entailed the application of stress in a modulated sinusoidal fashion, to stimulate the response, although providing the microalga the possibility to adapt and not cease its growth. Two abiotic factors were independently utilized (temperature and light intensity), and conditions were alternated between optimum and stress levels, in multiday cycles. The factors were applied at lower-than-optimum values (5 and 10 ºC; 30 and 50 μmol photons/m2/s for temperature and light intensity, respectively), and it was found that the approach was successful at achieving the desired goals. While temperature stress (10 ºC) presented the best results, with an 158% EPA content increase, light intensity (30 μmol photons/m2/s) also increased EPA content significantly by 126%. Moreover, the increases in EPA content originated from individual cells contents and not just from overall biomass increase, and these were obtained in a relatively short period of time, which is of utmost importance regarding its industrial viability. Eicosapentaenoic acid is conventionally extracted with hazardous organic solvents. Therefore, an additional line of research was undertaken, aiming at extracting EPA by using less hazardous solvent mixtures (SM), namely hexane:isopropanol (Hxn:2-PrOH; 3:2) and diethyl ether:ethanol (Et2O:EtOH; 2:1). The use of alternative technologies, namely high hydrostatic pressure (HHP) and moderate electric fields (MEF), was also assessed as adjuvants to increase the extraction efficiency of the SM. Concerning SM, Et2O:EtOH was able to increase 1.3-fold the EPA yield of the conventional Folch SM. Regarding the technologies, neither increased extraction yields when used independently; however their sequential combination was able to increase EPA yield by 162%. When associated, Et2O:EtOH and the combined technologies (HHP - 200 MPa, 21 °C, 15 min – followed by MEF processing at 40 °C, 15 min) enabled to extract higher amounts of EPA from N. oculata wet biomass, which is extremely relevant for the industries as no expensive drying step is required. Moreover, it is of dire importance to provide such alternatives to the “classical” extraction processes and solvents, as the industries require higher yields and lower environmental impact in their downstream processing. Since the biological potential of a microalga (or an extract thereof) is intrinsically dependent on its composition, the impact of the stress modulation and the SM utilized for extraction of lipid fraction were studied by characterization and assessment of biochemical and biological activities. Concerning the lipid extracts, it was revealed that the one obtained from the stressed culture presented the best results, namely in terms of anti-steatosis (hepatic lipid accumulation < 8% than that of steatosis-induced cells) and anti-inflammatory activities (decreased expression of interleukine (IL)-6, IL-10, and interferon- in LPS-stimulated RAW 264.7 macrophages). Moreover, both extracts presented oxygen radical absorbance capacity (within the range of 49- 50 μmolTrolox equivalent/mgextract), no metabolic inhibition of several cell lines (Caco-2, HT29-MTXE12, Hep G2, 3T3-L1, and RAW 264.7), and inhibition of triacylglycerols hepatic accumulation (glycerol release ca. 2-fold higher than in non-treated cells). Overall, the results showed that the modulated stress did indeed enhance the potential beneficial effects of the N. oculata lipid extracts on human health, as well as maintain their safety. Once the lipid fraction was extracted from the stressed culture, remaining biomass may still contain other compounds with interesting biological activities. Hence, by applying a biorefinery concept, the defatted biomass was enzymatically hydrolyzed with cellulase Celusoft Supreme for 3 h, followed by the protease NewPro, for 6 h, both at 53 ºC, after which the soluble fraction of hydrolysate (SFH) was characterized in terms of peptide and polysaccharide profiles, and assessed regarding its biochemical and biological activities. The results showed that the defatted biomass may still be utilized as a resource, since the SFH presented antioxidant capacity (3.2 μmolTrolox equivalent/mgFDB), antidiabetic (19.4% inhibition of α-glucosidase) and immunosuppression potential (NO production by LPS-stimulated Raw 264.7 cells decreased to < 10% that of the control), anti-inflammatory activity (decreased expression of IL-6, IL-8 and tumor necrosis factor-α by Caco-2 cells in LPS-induced inflammation) and antimicrobial activity, particularly against Gram-positive bacteria. In conclusion, it was demonstrated how modulated stress may enhance the production of bioactive compounds from N. oculata (specifically EPA) and new strategies to efficiently extract them were identified, as well as their biological potential was unveiled. The strategies presented herein for production of N. oculata with increased EPA content, and extraction thereof, may constitute solutions for the food and nutraceutical industries, allowing them to obtain EPA faster and with higher yields, concomitantly decreasing the process environmental impact. Ultimately, that will increase the economic viability of obtaining EPA from N. oculata.
- 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.
- Strategies to overcome industrial stress factors in engineered Saccharomyces Cerevisiae and impact on bioreactor fermentationPublication . Lopes, Ana Catarina Barros; Oliveira, Carla; Silva, João Pedro Azevedo; Carsanba, ErdemSaccharomyces cerevisiae fermentation is a valuable process to produce industrially relevant compounds sustainably and cost-effectively. However, biomolecule production by fermentation is usually impaired by harsh environmental conditions that may lead to yeast stress resulting in stuck or sluggish fermentations. Thus, enhance yeast tolerance to environmental stresses through genetic engineering or culture media supplementation may improve biomolecule productivity. Accordingly, this thesis focuses on the study of solutions to overcome the impact of oxidative and ethanol stresses on β-farnesene producer yeast in bioreactor fermentation. Culture medium supplementation with antioxidant compounds is a strategy commonly applied to overcome yeast oxidative stress. Therefore, the impact of an antioxidant peptide extract (APE), obtained from industrial spent yeast, was evaluated in the fermentation of S. cerevisiae in the presence and absence of the oxidative stress inducer hydrogen peroxide (H2O2). Under 2 mM of H2O2 exposure, the addition of 0.7 g/L APE led to the reduction of intracellular reactive oxygen species (ROS) levels up to 3-fold in shake-flasks and 1.7-fold in batch bioreactor fermentations. As a result, cell density and biomolecule concentration were enhanced up to 2-fold and 2.8-fold in shake-flasks, while, in bioreactors were increased up to 1.5-fold and up to 1.6-fold, respectively. Culture medium supplementation with APE showed to be a promising strategy to overcome yeast oxidative stress and improve βfarnesene production, while allowing for the valorisation of biomass waste as a sustainable and eco-friendly solution for the biotechnology industry. Yeast stress tolerance is also often improved using genetic engineering. Since yeast membrane is the first target of ethanol toxicity, improving the synthesis of mono-unsaturated fatty acids can be a good strategy to overcome ethanol stress. The effect of overexpressing OLE1 and TniNPVE genes, coding homologous and insect delta-9 fatty acids desaturases, respectively, on oleic acid production was evaluated in strains modified with these genes under a range of different pGAL promoter strengths (pGAL3 << pGAL2 < pGAL10 < pGAL1). OLE1 overexpression with pGAL10 (pGAL10-OLE1) increased oleic acid content between 30.9% and 77.3% compared to control, while TniNPVE overexpression with pGAL2 (pGAL2-TniNPVE) enhanced oleic acid between 18.4 and 34.7% in shake-flasks fermentations. Additionally, pGAL10-OLE1 and pGAL2-TniNPVE grew faster than control strain with a max 6 to 7% higher and produced more 55% of -farnesene. Besides, pGAL10-OLE1 and pGAL2-TniNPVE were exposed to 12.5% and 15% ethanol at different stages of fermentation and demonstrated to be more sensitive to ethanol than the control strain. Therefore, the higher oleic acid levels obtained by genetic modification improved yeast growth and productivity but not its resistance to ethanol. To clarify if high levels of mono-unsaturated fatty acids content can positively affect yeast growth and productivity at a larger scale, fed-batch fermentations with pGAL10-OLE1 were conducted for 13 days in 2-L bioreactors. pGAL10-OLE1 presented increments up to 59.5% in oleic acid and 49.1% in palmitoleic acid relative to control. These changes in monounsaturated fatty acids content resulted in β-farnesene productivity improvements between 5.5% (day 13) and 21.2% (day 2). In conclusion, improve yeast tolerance to oxidative stress, by reducing ROS accumulation with APE medium supplementation, benefited yeast growth and productivity. In contrast, OLE1 overexpression did not improve yeast tolerance to ethanol. However, the increase in mono-unsaturated fatty acids content led to higher farnesene productivity, which can benefit the industrial production of the biomolecule.
- Fractionation of sugarcane fermentation spent broths aqueous phase for production of value-added components towards an integrated biorefinery concept in synthetic biotechnologyPublication . Horta, Bruno Miguel Guedes; Oliveira, Catarina Silva Simão de; Burgal, João Porfírio da Silva; Cálix, Carla Maria Dias Marques de OliveiraFarnese aqueous waste (FAW) consists of the fermentation spent broth generated from the production of trans-β-farnesene, by Amyris, Inc.. It is generated in high scale and is usually disposed of by land disposal and fertirrigation, causing environmental problems. The main objective of this PhD project was to study and fractionate FAW, developing value-added products in line with the circular economy concept. Consequently, an integrated downstream process was developed, and the compositional and bioactive characteristics of the resulting products were assessed to determine their potential applications. FAW has an average dry matter content of 63.56±18.67 g/L, 10.08±3.75 g/L of ash content, 18.71±5.97 g/L of proteins, 1.15±0.47 g/L of ammonia, 2.08±1.35 g/L of total lipids, 12.83±7.35 g/L of organic acids, 10.63±4.95 g/L of sugars, 1.00±0.56 g/L of volatile compounds and 23.31±15.8 mg/L phenolic compounds. FAW proteinaceous compounds were comprised of small peptides with molecular weight below 1 kDa, and a great variety of amino acids (AA). The most interesting compounds to target in subsequent fractionation were the proteinaceous fractions, which could be used as ingredients for animal feed; minerals, with potential benefits for plant fertilisation; and phenolic compounds, known for their bioactive properties and their potential applications in cosmetic formulations. Chemical methods, such as precipitation, salting-out, aqueous two-phase systems, were initially used to recover protein and mineral-rich fractions. However, the use and disposal of chemicals could give rise to environmental concerns and compromise the sustainability of the valorisation strategy. Therefore, a chemical-free and environmentally friendly process was developed to remove nutrients from FAW creating a potential product for agriculture. In this regard, natural zeolites were used to treat FAW and removing and recovering nutrients, mainly potassium (K), peptides and AA (nitrogenous compounds – N). The optimal conditions for the simultaneous recovery of K and total N were obtained with a 0.2 Z/F (zeolite/FAW) ratio, 50 rpm mixing, 10 hours of contact and the use of 2 contact units, in a counter-current scheme. This process resulted in the production of zeolites loaded with 3597.23±368.33 mg/kg of ammoniacal N, 255.84±28.99 mg/kg of P, 36626.45±4215.54 mg/kg of K, 113.06±4.75 mg/kg of free AA and 4700.31±346.90 mg/kg of total AA. Peptides were also detected in the loaded zeolites. The composition displayed promising attributes for agricultural practices and exhibited encouraging results in preliminary phytotoxicity assessments, indicating its safety application on plants. The secondary output obtained from this process, consisted of treated FAW, which was an aqueous fraction that still contained a considerable nutrient load. Therefore, a cascade membrane filtration, using membranes with different molecular-weight cut-off (MWCO), was developed to recover compounds for application in cosmetic and animal feed formulations. It consisted of a sequential filtration employing micro-, ultra- and nano- filtration membranes. Two stages of ultrafiltration generated two retentates with compositional similarities in terms of the protein content (15.79±0.06 g/L and 6.96±0.03 g/L, respectively), minerals (7.83±0.14 g/L and 5.30±0.05 g/L, respectively), total analysed phenolic compounds (22.65±0.44 mg/L and 27.15±0.06 mg/L, respectively), and exhibited an interesting antioxidant activity. These fractions were hypothesised as aquafeed ingredients. As for the nanofiltration stage, a retentate with a protein content of 8.34±0.01 g/L, minerals content of 10.67±0.14 g/L and the total analysed phenolic compounds of 56.35 ± 0.29 mg/L, was obtained. The peptide profile of this sample consisted of 2.59% of peptides with MW between 1000 and 5000 Da, 50.58% in the 500- 1000 Da range and 46.83% with MW below 500 Da. This fraction also demonstrated interesting antioxidant activity. Due to its significant content in small peptides and the need for more extensive sample processing and capital investment, this retentate was targeted towards more lucrative markets, such as cosmetics. The permeate had a low nutrient load and could be considered as treated effluent. Thus, an integrated process for the valorisation of FAW was developed, which allows obtaining five products with potential commercial interest – an agricultural additive, ingredients for aquafeed and ingredients for cosmetics – additionally to a treated effluent.
- Establishment of in vitro gastrointestinal models platform for human food and animal feed ingredients developmentPublication . Carvalho, Nelson Fernando Mota de; Madureira, Ana Raquel Mendes Ferreira Monteiro; Oliveira, Diana Luazi Matos deGlobally, the human diet significantly relies on animal-derived products. The agri-food industry needs to optimize animal growth and performance to meet consumers demands, while reducing costs, ensuring product quality, and addressing sustainability and health concerns. Enhancing diets, through feed/food fortification with bioactive ingredients targeting gut microbiota is a promising strategy to promote the well-being and animal’s performance. Agro-industrial byproducts are potential sources of these bioactive ingredients, however, their effects and potential application on animal/human diets must be carefully evaluated. The main goal of this doctoral research was to create a platform of in vitro models that simulate the human and animal gastrointestinal tract (GIT), the GIOTA platform. Thus, models were implemented to assess and validate the impact of feed/food supplemented with bioactive ingredients on the gut microbiota, before resorting to in vivo trials. This supports the development of new products and promotes the inclusion of more sustainable ingredients, such as some of the byproducts tested in these models. The experimental work focused on 3 types of GIT: Human, Poultry and Bovine. In the first part, the preservation of human fecal inoculum was optimized, and the metabolic profile of colonic fermentations was compared between fresh and frozen inoculum. Storage at -20 oC with a 30% (v/v) glycerol solution allowed maintaining the viability of the inoculum for 90 days without affecting colonic fermentations. Furthermore, with the in vitro model already implemented, the impact of food matrices on microbiota modulation was evaluated. Skim milk (SKM) supplemented at 1% (w/v) by different well-known functional ingredients that modulate the gut microbiota were tested. The combination of SKM with fructo-oligosaccharides proved to be most beneficial for consumer’s health, promoting Lactobacillus, Bifidobacterium and Clostridium cluster IV, increasing the production of short-chain fatty acids, and reducing ammonia production. In second part, a poultry GIT in vitro model, specifically chickens, was designed to predict the effects of feed formulations on their microbiota. Two preservation methods of cecal inoculum were studied: (1) 5 % (v/v) dimethyl sulfoxide (DMSO) at -80 oC and (2) 30% (v/v) glycerol at -20 oC. Fresh and frozen inoculums were used in this model, and their fermentation profile were compared. The DMSO preservation method proved to be the most appropriate for cecal inoculum preservation for 90 days. Additionally, to validate the results of the in vitro model, a broiler chicken in vivo assay and the chicken in vitro model were performed, comparing the effect of incorporating in feed 1% (w/w) sugarcane bagasse lignin, a byproduct of the sugarcane industry. The tested supplementation, in the in vivo assay, had a positive impact on broiler chicken cecum, increasing the acetate and butyrate concentrations and reducing the presence of Enterobacteriaceae. The GIT model showed a similar trend in microbiota modulation of the in vivo assay, but still requires adjustments in inoculum preparation. In third part, to assess the impact of diet on the bovine intestinal microbiota’s, 2 in vitro models were developed, one for adult cow (ruminant) and another for calf (monogastric). Preservation studies of rumen and fecal inoculum from adult cows were also performed. In the adult cow model, ruminal and hindgut fermentations were performed with fresh and frozen inoculum, while in the calf model only hindgut fermentations were performed. Preservation at -80 oC was the condition that least affected inoculum’s bacterial viability and did not affect their fermentative capacity. Simultaneously, a calf in vivo assay and a calf model were conducted and compared on the evaluation of the effect of supplementing 1% (w/w) autolyzed spent yeast (AY) from farnesene production on milk replacer (MR). The AY supplementation, in the in vivo assay, had a positive impact on calf’s hindgut microbiota, increasing the presence of Bifidobacterium. However, this result was not observed in the model, which requires adjustments in the methodology associated with the digestion of liquid feed matrices. Scientific research and innovation play a crucial role in addressing modern challenges in the agri-food industry. The developed models serve as valuable and sustainable tools in evaluating the impact and validating the potential of new products of new products in the animal and human feeding industries.
- Development of sustainable processes for obtaining lignin from sugarcane by-products and its valorisationPublication . Antunes, Filipa Alexandre Soares Gomes; Costa, Patrícia Filipa Santos; Mota, Maria Inês Ferreira da; Burgal, João Porfírio da SilvaThe sugarcane industry generates several by-products, including straw (SCS) and bagasse (SCB). Their conversion into value-added products is of the utmost importance from a circular economy perspective. Lignin - one of the three main components of biomasses - has attracted considerable interest as a versatile ingredient for different applications. Therefore, this thesis aims to valorise, in a sustainable way, sugarcane residues (SCS and SCB), through the development of lignin-based products. In Chapter 1, it is presented a literature review focused on the valorisation of lignin obtained from sugarcane by-products. In Chapter 2, the detailed objectives of the project are presented. Chapter 3 presents different pre-treatments for lignin extraction, namely, alkaline, organosolv and deep eutectic solvents (DES). Furthermore, the combination of pretreatments was carried out following an integrated process approach: hydrothermal pretreatment followed by an alkaline pretreatment. The alkaline pretreatment of SCB proved to be the most effective among all pretreatments, producing lignin with distinct properties, such as purity (89%), colour (light brown; L*/a*/b*: 60.3/6.1/19.1), odour (woody sweet) and production cost (29 €/kg). Regarding sustainability, all processes achieved excellent scores ranging from 83 to 93%, with the alkaline extraction achieving the highest value. Chapter 4 describes the purification of the lignin obtained with lower purity. In this context, the alkaline liquor from the SCS was subjected to a two-stage membrane filtration step: first the liquor was filtered with a polyvinylidene fluoride microfiltration membrane (500-1000 kDa), followed by filtration with a 10 kDa polyethersulfone ultrafiltration membrane to concentrate the permeate flux. After acid precipitation at pH 2, the lignin purity increased from 61% to 84%. Chapter 5 details the characterisation of the lignins obtained by the pre-treatments in terms of physicochemical and structural properties. The results confirmed the presence of the main functional groups in the seven lignins, with some differences attributed to the biomass. The highest content of total hydroxyl phenolic units was obtained for the organosolv SCB and integrated process lignins. It was also concluded that guaiacyl (G) units were predominant in the SCB pretreatments, followed by syringyl (S) units. The organosolv lignin showed lower molecular weight, while the highest molecular weight distribution was observed for the SCS lignins with DES and alkaline pretreatments. In Chapter 6, the antioxidant and antibacterial activities of the SCB and SCS lignins from the different optimised pretreatments were evaluated, and the relationship between chemical structure and lignin functionality was elucidated. The total phenolic content and antioxidant activity were significantly higher for the alkaline, organosolv and integrated process SCS and SCB lignins. Regarding the antibacterial activity, the alkaline lignins showed activity against both representative bacteria evaluated (Escherichia coli and Staphylococcus aureus), with the SCB alkaline lignin showing the best performance. Chapter 7 explores the development of two products in the areas of cosmetics and materials. For the cosmetics field, lignin was tested as a natural UV blocker, antioxidant, and pigment in an blemish balm (BB) cream formulation. Remarkable results were achieved in terms of in vitro and in vivo SPF of 9.5 ± 2.9 and 9.6 ± 0.8, respectively. Furthermore, the lignin-based BB cream showed a broad-spectrum UV protection (critical wavelength 378 ± 0.5 nm). Lignin also showed antioxidant capacity after incorporation, being safe for topical applications. For materials, lignin was explored as a waterproofing agent in coatings for cardboard packaging. The results showed that the lignin-based coating was able to increase the water resistance of paper by up to three times, suggesting that this coating is a promising solution to improve the performance of paper-based packaging. The coating also demonstrated resistance to cracking by 90° folding. The results of this study corroborate the potential of sugarcane residues as a natural and sustainable source of new ingredients for the development of innovative lignin-based products.
- Early feeding patterns, nutritional status and growth in Portuguese children 12-36 months : a national representative studyPublication . Sampaio, Maria Margarida Lobo Machado Sousa Nazareth Almeida; Rêgo, Carla Maria Barreto da Silva de Sousa; Pinto, Elisabete Cristina BastosMetabolic programming results from the influence of early environmental experiences, particularly nutritional, on the future expression of the health-disease binomial, notably of non-communicable diseases. The period before conception, pregnancy and the first years of life constitutes a window of high sensitivity in respect of the environmental effect on genetic plasticity, with repercussions on general health and especially future nutritional status. Knowledge about each modifiable factor is essential for the establishment of recommendations for paediatric age, as well as for adults of reproductive age. EPACI Portugal 2012, Estudo do Padrão Alimentar e de Crescimento na Infância (Study of the Childhood Feeding Patterns and Growth)'s purpose was to study Portuguese children's dietary patterns and nutritional status. It was a nationally representative study that included 2230 children between 12-36 months and took place in 2012-2013, in 128 randomly selected Health Units of mainland Portugal. Through a team of trained interviewers, a questionnaire was applied, which included the collection of data on the parents, health history, dietary practices and anthropometric data of the children at different time milestones based on the consultation of the Child and Youth Health Bulletin, as well as the measurement of the weight, length/height and head circumference at the assessment moment. The body mass index (BMI) was afterwards calculated, and the nutritional status was characterised based on the World Health Organization (WHO) criteria. Information regarding the current diet was obtained through a 3-day food diary. A sub-sample of 2009 full-term children was analysed to describe early feeding practices. Most started breastfeeding (BF), but the prevalence decreased over time. Only about 20% met the WHO recommendation regarding exclusive BF for six months. Most infants complied with the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) recommendations regarding the timing of introduction of complementary feeding (CF). The foods introduced and the age of introduction of each respected the recommendations, with the exception of foods rich in sugar, which were early and excessively consumed. No association was found between the BF or timing of CF and the expression of overweight (OW)/obesity (OB). To determine the prevalence of nutritional inadequacy, and in the absence of national recommendations or the official adoption of a reference, the existing nutritional recommendations were analysed. After the review of the most commonly used recommendations and its systematisation, the comparison of the three main international recommendations - Food and Nutrition Board/Institute of Medicine (FNB/IOM), Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) and European Food Safety Authority/European Commission (EFSA/EC) - was made. EFSA's recommendations, established for the European reality, are the most recent, and present a great methodological robustness, were chosen to assess the nutritional adequacy of Portuguese children. For such purpose, nutritional intake and inadequacy and main suppliers of energy, macronutrients and some micronutrients were assessed in a sub-sample of 853 children, corresponding to those who filled in the food diaries. It was observed that most of the children exceeded the recommendations for energy but consumed a low proportion of energy provided by fat. The average daily protein intake was about five times higher than recommended, milk being the main supplier of energy and protein. Most children exceeded their sodium intake and none reached the recommended intake for vitamin D. At the time of the interview, 32.0% of children were at risk of OW, BMI z-score (zBMI) >1, and more than half of the parents were OW/OB. Higher maternal age and gestational weight gain (GWG) below recommendations were associated with lower zBMI in the offspring. In contrast, maternal OB at the time of assessment and high BMI before pregnancy were associated with higher zBMI in the offspring. About half of the mothers with OW or OB had a GWG above the recommendations. It becomes crucial to develop tools to identify and make early interventions in the life cycle aiming at preventing non-communicable diseases in adulthood, particularly the transgenerational transmission of OB.