Microbial fructo-oligosaccharides as emergent prebiotics

Introduction. Fructo-oligosaccharides (FOS) have been associated to prevention and treatment of many XXI centurys diseases. There is therefore a huge interest in successfully developing bioprocesses for their production. FOS have been industrially produced via fermentation, by several microorganism enzymes, with low yields and purities in a two-step bioprocess. To obtain high-content FOS we explored an integrated strategy using a co-culture of an Aspergillus ibericus as FOS producer strain, with Saccharomyces cerevisiae, with the gene responsible for sucrose hydrolysis disrupted, as small saccharides removal. The functionality of the microbial FOS produced as prebiotics was further assessed. Methodology. The wholecell microorganisms were used in a one-step bioprocess. Parameters such as initial yeast concentration, inoculation time, fermentative broth composition, temperature and pH were optimized in shake-flask previously scale-up to a lab bioreactor size. FOS prebiotic potential was evaluated in anaerobic batch cultures for 24 h using human faeces from 5 healthy volunteer individuals. The dynamic bacterial populations changes were assessed by PCR-real time, as well as the production of short chain fatty acids (SCFA) by HPLC. Sugars were analysed by HPLC. Results. Fermentations run under 30 ºC, initial pH of 6.0 and 17 g.L-1 yeast extract led to FOS mixtures with 97.4 ± 0.2% (w/w) purity in shake-flask. The scaledup fermentations in bioreactor yielded 64 ± 2% of FOS, after 141 h, with a FOS content of 118 ± 5 g.L-1 and a purity of 93.0 ± 0.5% (w/w). The microbial FOS triggered a beneficial effect on gut microbiota composition. SCFA including succinate, acetate, propionate and valerate were produced by the five faecal inoculum tested, at high concentrations. Lower amount of formate and butyrate were also produced. Conclusions. The one-step fermentation using the specific co-culture studied showed to be more efficient, economical and fast than the conventional two-step bioprocesses, thus avoiding the need of the conventional downstream treatments. The microbial FOS produced exhibited promising potential as nutraceutical ingredients for gut microbiota modulation with likely prebiotic features.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE–01–0145–FEDER-000004) funded by the European Regional Development Fund under the scope of Norte 2020–Programa Operacional Regional do Norte and the Project ColOsH PTDC/BTM–SAL/30071/2017 (POCI–01–0145–FEDER–030071).info:eu-repo/semantics/publishedVersio

Strategies for Fructo-oligosaccharides production with high-content and purity

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] The consumers’ interest in healthy and high nutritional food has significantly increased in the recent years. This trend towards the adoption of healthier lifestyles has been the main driver for the great demand of functional ingredients, such as the prebiotics fructo-oligosaccharides (FOS). Industrially, FOS are produced from sucrose through purified enzymes, in two-step bioprocesses, with low theoretical yields (0.50-0.55 gFOS.gSucrose-1) and purities (50-55%). Downstream steps are therefore needed to remove the non-prebiotic sugars and enable the incorporation of these FOS mixtures in diabetic, dietetic and healthy foods. In the last ten years, we have been investigating new strategies to produce FOS with higher contents, purities and differentiated functionalities. We have been exploring Aureobasidium pullulans and Aspergillus ibericus as FOS producers, in one-step fermentation processes, using the whole cells of the microorganisms instead of the isolated enzymes. This strategy proved to be efficient, fast and economic, yielding 0.64 gFOS.gSucrose-1. The FOS mixtures produced were able to stimulate the growth of probiotic strains and were simultaneously resistant to hydrolysis along the gastrointestinal system confirming their health claims as prebiotics. The probiotic strains preferentially metabolized the FOS mixture synthesized by A. ibericus, followed by the one from A. pullulans and lastly the commercial FOS. [...]info:eu-repo/semantics/publishedVersio

Fructo-oligosaccharides: production, characterization and purification

GLUPOR 12 - 12nd International Meeting of the Portuguese Carbohydrate Chemistry GroupThe consumers interest in healthy and high nutritional food has significantly increased in the recent years. This trend towards the adoption of healthier lifestyles has been the main driver for the great demand of functional ingredients, such as the prebiotics fructo-oligosaccharides (FOS). Industrially, FOS are produced from sucrose through purified enzymes, in two-step bioprocesses, with low theoretical yields (0.50-0.55 gFOS.gSucrose-1) and purities (50-55%). Downstream steps are therefore needed to remove the non-prebiotic sugars and enable the incorporation of these FOS mixtures in diabetic, dietetic and healthy foods. In the last ten years, we have been investigating new strategies to produce FOS with higher contents, purities and differentiated functionalities. We have been exploring Aureobasidium pullulans and Aspergillus ibericus as FOS producers, in one-step fermentation processes, using the whole cells of the microorganisms instead of the isolated enzymes. This strategy proved to be efficient, fast and economic, yielding 0.64 gFOS.gSucrose-1. The FOS mixtures produced were able to stimulate the growth of probiotic strains and were simultaneously resistant to hydrolysis along the gastrointestinal system confirming their health claims as prebiotics. The probiotic strains preferentially metabolized the FOS mixture synthesized by A. ibericus, followed by the one from A. pullulans and lastly the commercial FOS. The purification of FOS is not straightforward due to the physicochemical similarities between the different oligosaccharides and the smaller saccharides. To increase the FOS purity, we have been exploring different strategies including microbial treatments and downstream treatments as activated charcoal and ion-exchange chromatography. As microbial treatments, we studied the use of a Saccharomyces cerevisiae strain, able to metabolize the small saccharides without FOS hydrolyse, in co-culture with the FOS microorganism producer or in a two-step fermentation, in which FOS are firstly synthesized and then purified by the S. cerevisiae. Fermentations in two-steps were found to be more efficient than the co-culture ones and purities of 82% (w/w) in FOS were obtained [1]. To avoid competition by the subtract in the co-culture, we are now evaluating the use of a S. cerevisiae strain with the SUC2 gene for invertase expression repressed. Using this strategy, FOS are being produced with yields of 0.64 gFOS.gSucrose-1 and purities up to 93% (w/w). As downstream treatment we optimized an adsorption/desorption process of sugars using activated charcoal and ethanol as eluent. Mixtures containing 50.6% (w/w) of FOS were purified to 92.9% (w/w) with a FOS recovery of 74.5% (w/w) and some fractions were obtained with purities up to 97% (w/w) [2]. Acknowledgements: Clarisse Nobre acknowledges the Portuguese Foundation for Science and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. References [1] Nobre, C, Castro, CC, Hantson, A-L, Teixeira, JA, Weireld, G, Rodrigues, LR, Strategies for the production of high-content fructo-oligosaccharides through the removal of small saccharides by co-culture or successive fermentation with yeast, Carbohydrate Polymers, 136, 274281, 2016. [2] Nobre, C, Teixeira, JA, Rodrigues, LR, Fructo-oligosaccharides purification from a fermentative broth using an activated charcoal column. New Biotechnology, 29(3), 395401, 2012.Clarisse Nobre acknowledges the Portuguese Foundation for Science and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER027462), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER016403) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Valorization of rejected plantain fruit as an isomaltooligosaccharides syrup

Introduction. The generation of agricultural waste, in particular from plantain fruit, have been increasing. Its accumulation and poor valorization limits the environmental, economic and social development of communities. The main goal of this work was to characterize physicochemically and functionally the flour obtained from rejected DominicoHartón pulp (Musa AAB Simmonds). As well as to evaluate the production of a prebiotic isomaltooligosaccharides syrup from this waste feedstock. Methodology. Moisture, total protein, ethereal extract and ash contents of the raw flour were determined applying AOAC methods. Thermal properties of the raw flour were determined through rheometry, differential scanning calorimetry and thermogravimetric analysis. The functionality and kinetics hydrolysis of the raw and cooked flour was evaluated by in vitro gastrointestinal digestion using the INFOGEST protocol. The production of isomaltooligosaccharides was made from plantain flour solution 18% (p/p) that was treated with thermostable -amylase at 95 ºC for 3 h. Starch dextrins were then treated with -amylase (barley) and pullulanase at 50 ºC for 24 h. Finally, the syrup was treated with -glucosidase (transglucosidase) at 50 ºC for 24 h. Saccharides were analyzed by HPLC. Results. Moisture, total protein, ethereal extract and ash contents determined for the flour were 9.8±0.5%, 2.83±0.01%, 0.46±0.03% and 2.15±0.02%, respectively. Starch and amylose content was 87±2% and 31.2 ± 0.8%, respectively. The pasting temperature, gelatinization temperature, gelatinization enthalpy, mass loss at the first decomposition stage and mass loss at the second decomposition stage were 79.26±0.02 ºC, 69.9±0.24 ºC, 2.48±0.04 J×g-1, 10.79±0.04% and 59.3 ± 0.1%, respectively. When cooked, the starch content changed for rapid (0.5±0.1 to 2.4±0.3), slow (15.6±1.2 to 32±1) and resistant digestion (59±1 to 48±2). The glycemic index predicted for raw and cooked flour was 47.7±2.2 and 84.2±1.8, respectively. The greatest amount of production of isomaltooligosaccharides was of degree of polymerization (DP) 3 (isomaltotriose) at 6 h of transglycosylation step, in a content of 25.75 g/L. Conclusions. The rejected plantain fruit showed potential as a prebiotic source, which may be a good strategy for the valorization of this waste.This work was supported by the Ministry of Science Technology and Innovation of Colombia (Minciencias) through the research project “Design of a Biorefinery for the Use of the Lignocellulosic and Starchy Waste of the Plantain Agribusiness” (Call 757 for Funding of National Doctorates) [grant 1640318]; and the Universidad de Caldas through the research project “Basic Process Development for the Use of Agro– industrial and Urban Waste Under the Concept of Biorefineries” [grant 0240518]. The study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE–01–0145– FEDER-000004) funded by the European Regional Development Fund under the scope of Norte 2020– Programa Operacional Regional do Norte and the Project ColOsH PTDC/BTM–SAL/30071/2017 (POCI– 01–0145–FEDER–030071).info:eu-repo/semantics/publishedVersio

Symbiosis Specificity of the Preceding Host Plant Can Dominate but Not Obliterate the Association Between Wheat and Its Arbuscular Mycorrhizal Fungal Partners

The symbiosis established between arbuscular mycorrhizal fungi (AMF) and roots of most land plants plays a key role in plant nutrient acquisition and alleviation of environmental stresses. Despite the ubiquity of the symbiosis, AMF and host species display significant specificity in their interactions. To clarify preferential associations between wheat (Triticum aestivum) and AMF, we characterized root AMF communities in the transition from two first host species, ryegrass (Lolium rigidum) and yellow-serradella (Ornithopus compressus), grown separately or together, to a second host (wheat), by sequencing the large subunit ribosomal DNA (LSU rDNA) gene. The response of AMF communities in wheat to prior soil disturbance – and consequently of the mycelial network [intact extraradical mycelium (ERM) vs. disrupted mycelium] established with either of the first hosts – was also investigated. Since the outcome of a specific host– symbiont interaction depends on the molecular responses of the host plant upon microbial colonization, we studied the expression of six key symbiosis-related genes in wheat roots. AMF communities on L. rigidum and O. compressus roots were clearly distinct. Within an undisturbed ERM, wheat AMF communities were similar to that of previous host, and O. compressus-wheat-AMF interactions supported a greater growth of wheat than L. rigidum-wheat-AMF interactions. This effect declined when ERM was disrupted, but generated a greater activation of symbiotic genes in wheat, indicating that plant symbiotic program depends on some extent on the colonizing symbiont propagule type. When a mixture of L. rigidum and O. compressus was planted, the wheat colonization pattern resembled that of O. compressus, although this was not reflected in a greater growth. These results show a lasting effect of previous hosts in shaping wheat AMF communities through an efficient use of the established ERM, although not completely obliterating host–symbiont specificity

Development of a functional prebiotic strawberry preparation by in situ enzymatic conversion of sucrose into fructo-oligosaccharides

Food industry has been pressed to develop products with reduced sugar and low caloric value, while maintaining unchanged their rheological and physicochemical properties. The development of a strawberry preparation for the dairy industry, with prebiotic functionality, was herein investigated by in situ conversion of its intrinsic sucrose content into prebiotic fructo-oligosaccharides (FOS). Two commercial enzymatic complexes, Viscozyme® L and Pectinex® Ultra SP-L, were evaluated for the synthesis of FOS. Operational parameters such as temperature, pH, and enzyme:substrate ratio (E:S) were optimized to maximize FOS yield. The rheological and physicochemical properties of the obtained strawberry preparation were evaluated. For functional analysis, the resistance of FOS to the harsh conditions of the gastro-intestinal digestion was evaluated by applying the standardized INFOGEST static protocol. At optimal conditions (60 , pH 5.0), Pectinex® produced 265±3 g·L1 FOS, yielding 0.57±0.01 gFOS·gin.GF1 after 7hours reaction (E:S:1:40); and Viscozyme® produced 295±1 g·L1 FOS, yielding 0.66±0.00 gFOS·gin.GF1 after 5 hours (E:S:1:30). The obtained strawberry preparations contained more than 50%(w/w) prebiotic FOS incorporated (DP 35), with 80 % reduction of its sucrose content. The caloric value was therefore reduced by 2631%. FOS showed resistance to gastrointestinal digestion being only slightly hydrolysed (< 10%). Fructo-furanosylnystose was not digested at any phase of the digestion. Although the physicochemical properties of the prebiotic preparations were different from the original one, parameters such as the lower °Brix, water activity, consistency and viscosity, and its different color, may be easily adjusted. Results indicate that in situ synthesis strategies are efficient alternatives in the manufacture of reduced sugar and low-caloric food products with prebiotic potential.This work was supported by the FCT under the scope of the strategic funding of UIDB/04469/2020 unit, by National Funds through the FCT under the project cLabel+ (POCI01-0247-FEDER-046080) co-financed by Compete 2020, Lisbon 2020, Portugal 2020 and the European Union, through the European Regional Development Fund (ERDF) and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Daniela A. Gonçalves acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD Grant (2022.11590.BD). The authors acknowledge Frulact SA for all support concerning the materials and information related to the industry sector.info:eu-repo/semantics/publishedVersio

Development of a prebiotic strawberry preparation for the dairy industry

Background. Food industry has been pressed to develop products with reduced sugar and caloric value, with the challenge of keeping rheological and sensory characteristics. Herein we developed a functional strawberry preparation for the dairy industry, by in-situ enzymatic conversion of sucrose into prebiotic fructo-oligosaccharides (FOS). Methodology. Two enzymatic complexes (Pectinex®Ultra SP-L and Viscozyme®L) were applied in the preparation. Operational parameters were optimized to maximize FOS yield: temperature, pH, enzyme:substrate ratio (E/S). Rheological, physicochemical and functional properties (INFOGEST gastrointestinal digestion protocol) were evaluated. Results. At optimal conditions (60 , pH 5.0), Pectinex produced 265±3 g·L1 FOS, yielding 0.581±0.006 gFOS·gin.GF1 after 7 h reaction (E/S:1/40); and Viscozyme produced 295±1 g·L1 FOS, yielding 0.664±0.004 gFOS·gin.GF1 after 5 h (E/S:1/30), both resulting in preparations with 50% (w/w) FOS. The caloric value was reduced 24%, including 80% sucrose reduction. Differences in colour, water activity and ºBrix were not relevant, while consistency and viscosity decreased 70% and pH increased from 4.4 to 4.7. FOS showed resistance to gastrointestinal digestion; only kestose was slightly hydrolysed at intestinal phase. Conclusions. A prebiotic strawberry preparation was successfully produced at lab scale, by in-situ enzymatic conversion of caloric into functional sugars. Next, the process will be scaled-up at industrial level. Background. Food industry has been pressed to develop products with reduced sugar and caloric value, with the challenge of keeping rheological and sensory characteristics. Herein we developed a functional strawberry preparation for the dairy industry, by in-situ enzymatic conversion of sucrose into prebiotic fructo-oligosaccharides (FOS). Methodology. Two enzymatic complexes (Pectinex®Ultra SP-L and Viscozyme®L) were applied in the preparation. Operational parameters were optimized to maximize FOS yield: temperature, pH, enzyme:substrate ratio (E/S). Rheological, physicochemical and functional properties (INFOGEST gastrointestinal digestion protocol) were evaluated. Results. At optimal conditions (60 , pH 5.0), Pectinex produced 265±3 g/L FOS, yielding 0.581±0.006 g(FOS)/g(initial.GF) after 7 h reaction (E/S:1/40); and Viscozyme produced 295±1 g/L FOS, yielding 0.664±0.004 g(FOS)/g(initial.GF) after 5 h (E/S:1/30), both resulting in preparations with 50% (w/w) FOS. The caloric value was reduced 24%, including 80% sucrose reduction. Differences in colour, water activity and ºBrix were not relevant, while consistency and viscosity decreased 70% and pH increased from 4.4 to 4.7. FOS showed resistance to gastrointestinal digestion; only kestose was slightly hydrolysed at intestinal phase. Conclusions. A prebiotic strawberry preparation was successfully produced at lab scale, by in-situ enzymatic conversion of caloric into functional sugars. Next, the process will be scaled-up at industrial level.info:eu-repo/semantics/publishedVersio

Probiotic potential of fructo-oligosaccharides produced by Aspergillus ibericus

The gastrointestinal tract harbours a diverse and dynamic microbial community that directly impacts human health. Prebiotics, such as fructo-oligosaccharides (FOS), play a crucial role in the modulation of colonic microbiota, reducing pathophysiological disorders and associated chronic diseases. The prebiotic potential of FOS produced by a newly isolated strain Aspergillus ibericus was studied. FOS fermentability by the probiotic Lactobacillus rhamnosus was evaluated. L. rhamnosus was grown in de Man-Rogosa-Sharpe (MRS) broth, with different carbon sources: glucose (positive control), no sugar (negative control), microbial-derived FOS from A. ibericus and Raftilose®P95, a non-microbial commercial FOS sample (from Beneo-Orafti, Belgium). A final concentration of 2 % (w/v) in sugar was used. Fermentation was carried out in a 96-well microplate and a shake flask, for 24 h, at 37 ºC, with an agitation of 120 rpm. Biomass growth was analysed by optical density at 620 nm. The consumption of sugars and the production of short chain fatty acids (SCFA) and lactate was quantified by HPLC. Maximum cell growth was reached at approximately 12 h, for all carbon sources. The highest growth was achieved for glucose samples, followed by the microbial-derived FOS, then Raflitose and finally the negative control. Although the microbial-derived FOS promoted great cellular growth, only kestose (GF2), together with residual amounts of glucose and sucrose presented in the sample, were consumed. This may explain the two different slopes exhibited during the exponential phase growth. Most likely hypothesis is that probiotic bacteria was cleaving GF2 in the first hours of fermentation, using only the smallest sugars present for growing. And Nystose (GF3) and fructofuranosylnystose (GF4) were not consumed, even when prolonging the fermentation up to 48 h. SCFA identified were valerate and propionate, as well as succinate, formate, acetate, iso-butyrate and nbutirate, although in lower amount. Higher amount of SCFA and lactate were determined while growing in the microbial-derived FOS, as compared to the commercial sample. Overall, lactate was the main metabolite produced during the fermentations. In conclusion, the prebiotic potential of microbial-derived FOS synthesized by A. ibericus was demonstrated, providing promising indication of its usability as food ingredient with strong prebiotic features.info:eu-repo/semantics/publishedVersio

In-situ enzymatic conversion of sucrose into prebiotic fructooligosaccharides for the development of a functional strawberry preparation

The increased search for reduced-sugar and healthier food products has driven the growth of the functional food market [1]. This opened space for the development of novel functional products. Frulact SA, a partner in this project, is specialized in the development and production of fruit-based preparations, which are mainly utilized in the dairy industry for incorporation in flavored yogurts. Its market is expected to increase at a compound annual growth rate of 6.1% until 2030 [2]. However, despite being rich in nutrients, these preparations have a high amount of caloric added sugar. To reduce this sugar in a strawberry preparation, we herein propose an in-situ enzymatic conversion of its sucrose content into prebiotic fructooligosaccharides (FOS) [3,4]. Two commercial enzymatic complexes were evaluated for the in-situ synthesis of FOS. At optimal conditions (60 °C and pH 5.0), Pectinex® Ultra SP-L yielded 0.57 ± 0.01 gFOS/gini.sucrose after 7 h reaction and Viscozyme® L, 0.66 ± 0.00 gFOS/gini.sucrose after 5 h. The resultant strawberry preparations contained more than 50 % (w/w) of FOS in total carbohydrates. Also, more than 80 % of the original sucrose content was reduced, diminishing its caloric value by 31 %. The data show that consumption of dairy products containing 10 % of the developed prebiotic preparation would result in the ingestion of >2.5 grams of FOS per 100 mL of product. The prebiotic preparation showed also to resist the harsh conditions of the gastrointestinal tract since more than 90 % of FOS were not hydrolyzed during digestion. The conversion of sucrose into FOS changed some physicochemical and textural attributes of the original product (i.e., sweetness, color, viscosity, consistency), yet those can be easily adjusted. The in-situ technological approach here developed shown great potential as an innovative strategy for the development of low-sugar and low-calorie prebiotic food.This work was supported by the FCT under the scope of the strategic funding of UIDB/04469/2020 unit, by National Funds through the FCT under the project cLabel+ (POCI-679 01-0247- FEDER-046080) co-financed by Compete 2020, Lisbon 2020, Portugal 2020 and the European Union, through the European Regional Development Fund (ERDF) and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Daniela A. Gonçalves acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD Grant (2022.11590.BD).info:eu-repo/semantics/publishedVersio

How prebiotics have been produced from agro-industrial waste: an overview of the enzymatic technologies applied and the models used to validate their health claims

Background The disposal of waste from the food industry represents a major environmental concern. Nonetheless, agro-industrial by-products can be enzymatically converted into low-cost high-value-added products, such as prebiotics, while contributing to a circular economy. As a first approach for health claims validation of these novel products, several gastrointestinal models have been used. Scope and approach The main objective of this review is to provide a comprehensive overview of the advances in the enzymatic technologies applied to the production of prebiotics from agro-industrial wastes. The strategies used in the conversion of the wastes, including pre-treatment processes, type of enzymes applied, and the reaction conditions used are revised. Finally, from data obtained by in vivo trials and in vitro gastrointestinal simulation, the functionality of the produced prebiotics and their biological mechanisms of action are discussed. Key findings and conclusions Enzymatic processes have proven their efficiency for the conversion of low-cost agro-industrial wastes into commercial valuable compounds, such as prebiotics. Still, the potential of enzymes for the bioconversion of the vast diversity of existing wastes has yet to be explored. By researching different setups of the enzymatic reaction and optimization of the reaction conditions, greater yields of the prebiotic extraction or synthesis may be achieved. Also, despite the many available gastrointestinal models, few studies have been done on the biological function of the prebiotics obtained from agro-industrial wastes, which has been a drawback in the validation of health claims associated with these novel products.Daniela Gonçalves, Dalila Roupar, and Abigail González acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD grants (2022.11590.BD, SFRH/BD/139884/2018, and 2021.06268.BD, respectively). Clarisse Nobre acknowledges FCT for the Assistant Research contract 2021.01234.CEECIN. This study was supported by the FCT under the scope of the strategic funding of UIDB/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Also by the project cLabel+ (POCI-01-0247-FEDER-046080) co-financed by Compete 2020, Lisbon 2020, Portugal 2020, and the European Union, through the European Regional Development Fund (ERDF), as well as the project AgriFood XXI (NORTE-01-0145-FEDER-000041), under the ERDF through the Competitiveness factors Operational program – Norte 2020, COMPETE and by National Funds through the FCT.info:eu-repo/semantics/publishedVersio