Efeito da ploidia e da relação proteína-energia das dietas no crescimento e utilização do alimento em truta arco-íris (Salmo gairdneri R.)

Dissertação de Doutoramento em Biologia, área de especialização em Ecologia Animal, apresentada à Faculdade de Ciências da Universidade do Port

Solid-state fermentation of Ulva Rigida for production of cellulases, xylanases and ß-glucosidase

Seaweeds are important organisms in marine ecosystems, are included on blue biorefinery category and they are rich in different polysaccharides, that can induce enzyme production by solid-state fermentation (SSF). Ulva rigida was used as substrate in SSF to produce cellulases and xylanases and to increase its protein content. The SSF were performed with previously washed and with unwashed seaweed by Aspergillus ibericus. Xylanase and cellulase activities were higher in fermented unwashed seaweed, correspondingly 359.8±6.1 U/g dry substrate and 59.9±2.4 U/g, respectively. In fermented washed U. rigida, these values achieved 212.4±17.7 U/g and 43.6±3.7 U/g, respectively. -glucosidase activity was similar in washed seaweed (6.94±0.21 U/g) and in unwashed seaweed (6.87±0.18 U/g). After SSF, protein content of unwashed seaweed was increased by 14%. Unwashed U. rigida resulted in better enzymatic activities, being a promising option to produce these commercially-valued enzymes.Helena Fernandes was supported by PhD grant SFRH/BD/131219/2017, funded by the Portuguese Foundation for Science and Technology (FCT). José M. Salgado was supported by grant CEB/N2020 – INV/01/2016 from Project “BIOTECNORTE - Underpinning Biotechnology to foster the north of Portugal bioeconomy” (NORTE-01- 0145-FEDER-000004). This study was partially supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145- FEDER-006684) and BioTec-Norte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and by the project SPO3 (Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products reference POCI-01-0145-FEDER-030377 funded by FEDER-Operational Programme Competitiveness and Internationalization and FCT.info:eu-repo/semantics/publishedVersio

Improved lignocellulolytic enzyme production and antioxidant extraction using solid-state fermentation of olive pomace mixed with winery waste

Olive pomace is characterized by its low nutritional value and high phenolic content, which hinders its direct use as animal feed, fertilizer, or as a substrate in bioprocesses such as solidstate fermentation (SSF). A possible strategy for bioprocessing olive pomace by SSF is the mixture of olive mill wastes with other wastes produced in the same region, such as winery wastes. This may improve the production of bioactive compounds like enzymes and antioxidant phenolics. A simplexcentroid design was used to evaluate the use of olive mill and winery wastes alone or in combination as a substrate for SSF with Aspergillus niger and Aspergillus ibericus. Synergistic effects of combinations of crude olive pomace (COP), exhausted olive pomace (EOP), vine trimming shoots (VTS), and exhausted grape marc (EGM) were observed in the production of xylanases, cellulases, glucosidases, and in the variation in total phenolics and antioxidant activity of SFF extracts. A multiple response optimization was carried out, leading to the following optimal mixture of substrates: for A. niger, 23% (w/w) COP, 30% EGM, 33% VTS, 14% EOP; for A. ibericus, 30% EGM, 36% VTS, 34% EOP. The scaleup to tray bioreactor with optimal substrate made it possible to achieve the maximum xylanase, cellulase, and glucosidase production of 189.1 ± 26.7, 56.3 ± 2.1 and 10.9 ± 0.8 U/g, respectively. The antioxidant activity of fermented wastes was also improved 2.2fold as compared with unfermented wastes. Thus, a combination of olive mill and winery wastes in SSF is a potential strategy to increase their value and to develop a circular strategy in these industries. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd.José Manuel Salgado was supported by grant CEB/N2020 – INV/01/2016 from Project ‘BIOTECNORTE – Underpinning Biotechnology to foster the north of Portugal bioeconomy’ (NORTE-01-0145-FEDER-000004). This study was supported by SPO3 project, reference POCI-010145-FEDER-030377, funded by European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT), by the InovFeed project, reference MAR-02.01.01- FEAMP-0111. This study was supported by the FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the ERDF under the scope of North 2020 – Northern Regional Operational Program. Helena Fernandes and Carolina Castro were supported by PhD grant SFRH/BD/131219/2017 and post-doctoral grant SFRH/BDP/114942/2016, funded by the Portuguese Foundation for Science and Technology (FCT), respectivelyinfo:eu-repo/semantics/publishedVersio

In vitro evaluation of the interaction between exogenous carbohydrases produced by solid-state fermentation of brewers’ spent grain and digestive enzymes

[Excerpt] Introduction The presence of non-starch polysaccharides (NSP) in most plant feedstuffs (PF) has been associated with adverse effects on carnivorous fish nutrient digestibility and, ultimately, on growth and health. The lack of enzymatic machinery and well-developed microbiota in the digestive tract of carnivorous fish for processing NSP are possible causes for such effects. A promising nutritional strategy to improve nutrient digestibility of plant-based diets is the use of exogenous enzymes (e.g. carbohydrases, proteases) as feed additives. Solid-state fermentation (SSF) is an eco-friendly and cost-effective biotechnology process that allows converting inexpensive agro-industrial by-products into added-value products such as carbohydrases. As a low-cost and lignocellulosic by-product of the brewing industry, brewery spent grain (BSG) is an attractive substrate for microbial enzyme production by SSF. The present study was designed to assess the potential of a carbohydrase enzyme extract obtained by SSF to release total amino acids (AA) and monosaccharides (pentoses) from plant-based diets in an in vitro gastrointestinal model with enzyme extracts of European sea bass (Dicentrarchus labrax).[...]Supported by POCI-01-0145-FEDER-030377 and MAR-02.01.01-FEAMP-0111. HF and CC supported by grants SFRH/BD/131219/2017; SFRH/BPD/114942/2016, respectively.info:eu-repo/semantics/publishedVersio

Production of enzymatic extracts for aquafeeds by solid-state fermentation with Aspergillus ibericus of winery and olive mill wastes

[Excerpt] Introduction The replacement of fishmeal by plant ingredients in aquafeeds imposed new formulation strategies to overcome some nutritional restrictions associated with these alternative ingredients. Thus, supplementation of plant-based diets with feed additives, as exogenous enzymes and antioxidants compounds, has recently attracted increasing interest to improve feed utilization and to promote animal health. Solid state fermentation (SSF) of agro-industrial wastes has a high potential for the production of these additives, being a practical, economical, and environmentally-friendly process. Olive mill and winery wastes have valuable compounds that may be valorized thought SSF and that may be used as additives for aquafeeds. This study was conducted to optimize the production of non-starch carbohydrases through the SSF of the olive mill and winery wastes and to test its efficacy to improve the release of pentoses during digestion of a plant-based diet in European seabass.[...]Work supported by FCT PhD grant SFRH/BD/131219/2017, SFRH/BDP/114942/2016, IJFCT-POCI 01-0145-FEDER-030377 and MAR-02.01.01-FEAMP-0111info:eu-repo/semantics/publishedVersio

Black Soldier Fly (Hermetia illucens) pre-pupae meal as a fish meal replacement in diets for European seabass (Dicentrarchus labrax)

[EN] A feeding trial was carried out to assess the effect of partially replacing fish meal (FM) by Black soldier fly pre-pupae meal (HM) in diets for European seabass Dicentrarchus labrax juveniles. A FM-based diet was used as a control and three other diets were formulated to include 6.5%, 13%, and 19.5% of HM, replacing 15%, 30% and 45% of FM respectively. Each diet was fed to triplicate groups of fish (initial weight: 50 g) for 62 days. At the end of the trial, there were no differences among groups in growth performance or feed utilization. Plasma metabolic profiles also remained unaffected, except that plasma cholesterol was reduced with dietary HM inclusion. The apparent digestibility coefficients (ADC) of protein, lipids, dry matter, organic matter, and energy were generally high, and not affected by the dietary treatment. The ADC of arginine, histidine, and valine were higher in HM diets when compared to the control. Amylase and protease activities were not affected by dietary HM, while lipase activity was lower in HM 6.5 diets than in the control and HM 19.5 diets. In conclusion, up to 19.5% of HM, corresponding to 22.5% of total dietary protein, may successfully replace FM in diets for juvenile European seabass, without adverse effects on growth performance, feed utilization or digestibility.This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT - Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. We would like to express our thanks to P. Correia for assistance during the growth and digestibility trial. Magalhaes, R. was supported by a VEGICOR-AMINOAQUA project grant (ref. 31-03-05-FEP-43)Magalhães, R.; Sanchez-Lopez, A.; Leal, RS.; Martínez-Llorens, S.; Oliva-Teles, A.; Peres, H. (2017). Black Soldier Fly (Hermetia illucens) pre-pupae meal as a fish meal replacement in diets for European seabass (Dicentrarchus labrax). Aquaculture. 476:79-85. https://doi.org/10.1016/j.aquaculture.2017.04.021S798547

Effect of short chain fructooligosaccharides (scFOS) on immunological status and gut microbiota of gilthead sea bream (Sparus aurata) reared at two temperatures

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets.This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT Foundation for Science and Technology, under the projects "PEst-C/MAR/LA0015/2013 and UID/Multi/04423/2013". I. Guerreiro, P. Enes, A. Couto and B. Costas were supported by grants (SFRH/BD/76139/2011; BPD/39688/2007, BD/47495/2008 and BPD/77210/2011, respectively) from FCT, Portugal. C. Serra and A. Salvador were recipient of grants within the Project AQUAIMPROV (reference NORTE-07-0124-FEDER-000038) and Project BioEnv (REF. NORTE-07-0124-FEDER-000048), co-financed by the North Portugal Regional Operational Programme (ON.2 - O Novo Norte). We would like to express our thanks to Pedro Correia for the assistance during the growth trial. Authors would also thank to Jefo Species-specific additives France, for providing the prebiotic

Bio-enrichment of oilseed cakes by Mortierella alpina under solid-state fermentation

"Available online 3 August 2020"Oilseed cakes have potential for new applications as substrates for solid-state fermentation (SSF), to increase their nutritional value by increasing its polyunsaturated fatty acids (PUFAs) or protein content. In this sense, it was performed a screening of oilseed cakes to be used as substrates for the production of PUFAs by Mortierella alpina Peyronel MUM 9412. Of all by-products tested, linseed cake (LSC) was the oilseed cake that achieved the highest production of total PUFAs: 153.09 ± 2.25 mg/g. Overall, the PUFAs and protein contents of fermented LSC increased 33% and 11%, respectively. Further, supplementation of the rapeseed cake with linseed oil, prior to the SSF, proved to increase the PUFAs production in about 26%. This study demonstrated the potential of SSF for improving linseed and linseed cakes nutritional composition and the positive effect of linseed oil as inductor to improve the PUFAs production by M. alpina.José Manuel Salgado was supported by grant CEB/N2020 – INV/01/ 2016 from Project “BIOTECNORTE - Underpinning Biotechnology to foster the north of Portugal bioeconomy” (NORTE-01-0145-FEDER000004). This work was supported by the project “Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products”, reference POCI-01-0145- FEDER-030377, funded by European Regional Development Fund (ERDF). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Solid-state fermentation of distiller’s dried grains with solubles improves digestibility for european seabass (dicentrarchus labrax) juveniles

Aquaculture requires new, economical, and eco-friendly protein sources to replace traditional fisheries and plant ingredients. Using agriculture by-products as protein sources would reduce land-based feed production pressure and waste production, promoting a circular economy and sustainable aquaculture. Distiller’s dried grains with solubles (DDGS) is the main by-product of bioethanol production. Corn DDGS has a high protein level, but its high fiber content limits its use as a feed ingredient, particularly for carnivorous fish. Solid-state fermentation (SSF) uses lignocellulosic-rich substrates, such as DDGS, for microbial growth in the near absence of water, promoting enzyme production that degrades the lignocellulosic matrix, increasing free reducing sugars, protein, and antioxidant levels of the substrate. In the present work, the SSF of corn DDGS with Aspergillus carbonarius, A. ibericus, and A. uvarum was tested. Then, the digestibility of the most promising fermented DDGS (in terms of upgraded nutritional composition) was tested by including it in a reference diet (70% of a reference diet; 48% crude protein; 15% crude lipids) for European seabass (Dicentrarchus labrax) juveniles (171 g averaged weight; trial duration of 52 days). Among the fungi tested, Aspergillus ibericus led a generally higher upgrading of the DDGS nutritional composition, leading to a high amount of protein (from 42.7 to 49.7 g N/kg DM), phenolic compounds (1.49 to 4.86 mg/g caffeic acid equivalents), free sugars (9.5 to 31.9 mg/g), and enzyme production (45 U/g and 68 U/g of cellulase and xylanase, respectively), and a high reduction in acid detergent fiber and neutral detergent fiber content (up to 29 and 43%, respectively). Compared to the unfermented DDGS, fermented DDGS presented increased protein, lipids, starch, and energy digestibility, while phosphorous digestibility was similar. Compared to the reference diet, dietary inclusion of unfermented or fermented DDGS increased trypsin and chymotrypsin activities. The activity of digestive enzymes was not affected by the inclusion of fermented DDGS, except for amylase activity, which was lower with the fermented DDGS than with the unfermented DDGS diet. In conclusion, SSF of DDGS enhanced its nutritional value, increasing DDGS digestibility when included in diets for European seabass juveniles.This research was funded by the R&D&I project MB4Aqua, reference FCT.2022.06587.PTDC from Fundação para a Ciência e Tecnologia (FCT) and by the structural projects CEBpest (UIDB/04469/2020) and LABBELS (LA/P/0029/2020). Diogo Filipe was supported by a grant reference 2020.05525info:eu-repo/semantics/publishedVersio