Partial purification, immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus

Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CNBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.

Características da carne de cordeiros alimentados com glicerina proveniente da produção de biodiesel

Pós-graduação em Zootecnia - FCAV

Chemical and Fatty acid composition of different cuts cooked or uncooked from yearling bulls fed oil sources

This study evaluated the chemical and lipid composition of uncooked or cooked loin (Longissimus thoracis) and rump (Biceps femoris) in samples of 2.54 cm thick from 35 carcasses of Nellore young bulls finished in feedlot for 96 days and slaughtered at an average weight of 532.17 ± 30.25 kg and 24 months of age. The rump had the lowest level of protein and ash (18.57 and 0.90%, respectively) and the highest level of ether extract compared to loin (3.37 and 1.90%, respectively). Higher levels of cholesterol were found in rump compared to loin (40.91 e 30.93 mg 100 g-1, respectively). The uncooked loin showed lower content of saturated fatty acids and higher content of polyunsaturated fatty acids. The best values for the omega-6: omega-3 ratio was observed in the uncooked beef. In the present study, the loin was healthier due to the higher amount of polyunsaturated fatty acids compared to rump. Cooking the meat decreases the levels of polyunsaturated fatty acids, omega-3, omega-6 and the omega-6: omega-3 ratio.

Efeitos da ractopamina e da imunocastração de suínos nas características da barriga e na qualidade do bacon

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Produção, imobilização e aplicação de lipase de Fusarium verticillioides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro propagation of Sacha inchi

The aim of this study was to perform an in vitro evaluation of the auxin: cytokinine ratio in different segments of the epicotyl and hypocotyl of Sacha inchi (Plukenetia Volubilis Linneo) seeds germinated in vitro. The segments apical (A), median (B) and basal (C) were introduced into semi-solid MS culture medium (2.0g L-1 Phytagel), supplemented with MS vitamins, sucrose (30.0g L-1) and submitted to three doses of auxin indolebutyric acid - IBA (0; 0.1; 0.5mg L-1), associated with four doses of the cytokinine benzylaminopurine - BAP (0; 0.1; 0.5; 1.0mg L-1), totaling 36 treatments. After nine weeks of in vitro cultivation, the apical segment ( A) presented shoot formation by direct organogenesis at the concentrations of 0.5 and 1.0 of BAP associated with 0.0 and 0.1 of IBA. It is feasible to use in vitro cultivation with the apical region of seeds germinated in vitro used as explants.

Effect of Echium oil compared with marine oils on lipid profile and inhibition of hepatic steatosis in LDLr knockout mice

Abstract Background In an effort to identify new alternatives for long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) supplementation, the effect of three sources of omega 3 fatty acids (algae, fish and Echium oils) on lipid profile and inflammation biomarkers was evaluated in LDL receptor knockout mice. Methods The animals received a high fat diet and were supplemented by gavage with an emulsion containing water (CON), docosahexaenoic acid (DHA, 42.89%) from algae oil (ALG), eicosapentaenoic acid (EPA, 19.97%) plus DHA (11.51%) from fish oil (FIS), and alpha-linolenic acid (ALA, 26.75%) plus stearidonic acid (SDA, 11.13%) from Echium oil (ECH) for 4 weeks. Results Animals supplemented with Echium oil presented lower cholesterol total and triacylglycerol concentrations than control group (CON) and lower VLDL than all of the other groups, constituting the best lipoprotein profile observed in our study. Moreover, the Echium oil attenuated the hepatic steatosis caused by the high fat diet. However, in contrast to the marine oils, Echium oil did not affect the levels of transcription factors involved in lipid metabolism, such as Peroxisome Proliferator Activated Receptor α (PPAR α) and Liver X Receptor α (LXR α), suggesting that it exerts its beneficial effects by a mechanism other than those observed to EPA and DHA. Echium oil also reduced N-6/N-3 FA ratio in hepatic tissue, which can have been responsible for the attenuation of steatosis hepatic observed in ECH group. None of the supplemented oils reduced the inflammation biomarkers. Conclusion Our results suggest that Echium oil represents an alternative as natural ingredient to be applied in functional foods to reduce cardiovascular disease risk factors.

Determinants of specific food consumption in the Canary Islands (Spain)

The consumption of specific functional foods (FF) and some determinants of FF item selection were assessed using a questionnaire administered to 1112 individuals in the Canary Islands (Spain). Food items considered were Milk products: easily digestible milk (or milk low in lactose), milk enriched with vitamins and/or minerals, skimmed milk with soluble fiber, milk with royal jelly, milk with modified fatty acids (omega 3), milk products low in fat, pro-biotic foods (yoghurt and fermented milk) and yoghurt with phytosterols; Cereals: fortified breakfast cereals, wholemeal cereals and energy bars; Drinks: juices and enriched drinks, stimulating drinks and isotonic drinks; DHA-enriched, low cholesterol eggs; Meat products: low salt sausages and cooked low fat ham; Fats: enriched margarine, margarine rich in phytosterols and

Bioactive and toxic compounds in different foodstuffs: analytical techniques and sensory analysis

Foods that provide medical and health benefits or have a role in disease risk prevention are termed functional foods. The functionality of functional foods is derived from bioactive compounds that are extranutritional constituents present in small quantities in food. Bioactive components include a range of chemical compounds with varying structures such as carotenoids, flavonoids, plant sterols, omega-3 fatty acids (n-3), allyl and diallyl sulfides, indoles (benzopyrroles), and phenolic acids. The increasing consumer interest in natural bioactive compounds has brought about a rise in demand for these kinds of compounds and, in parallel, an increasing number of scientific studies have this type of substance as main topic. The principal aim of this PhD research project was the study of different bioactive and toxic compounds in several natural matrices. To achieve this goal, chromatographic, spectroscopic and sensorial analysis were performed. This manuscript reports the main results obtained in the six activities briefly summarized as follows: • SECTION I: the influence of conventional packaging on lipid oxidation of pasta was evaluated in egg spaghetti. • SECTION II: the effect of the storage at different temperatures of virgin olive oil was monitored by peroxide value, fatty acid activity, OSI test and sensory analysis. • SECTION III: the glucosinolate and phenolic content of 37 rocket salad accessions were evaluated, comparing Eruca sativa and Diplotaxis tenuifolia species. Sensory analysis and the influence of the phenolic and glucosinolate composition on sensory attributes of rocket salads has been also studied. • SECTION IV: ten buckwheat honeys were characterised on the basis of their pollen, physicochemical, phenolic and volatile composition. • SECTION V: the polyphenolic fraction, anthocyanins and other polar compounds, the antioxidant capacity and the anty-hyperlipemic action of the aqueous extract of Hibiscus sabdariffa were achieved. • SECTION VI: the optimization of a normal phase high pressure liquid chromatography–fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa powder and chocolate samples was performed.

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Modulation der Genexpression und des neuronalen Zelltods durch das Amyloid-Vorläufer-Protein (APP)

Das Amyloid-Vorläufer-Protein (APP) spielt eine zentrale Rolle in der Entstehung und Entwicklung von Morbus Alzheimer. Hierbei ist die proteolytische Prozessierung von APP von entscheidender Bedeutung. Das Verhältnis von neurotoxischen und neuroprotektiven Spaltprodukten, die über den amyloidogenen und nicht-amyloidogenen Weg der APP-Prozessierung gebildeten werden, ist für das Überleben von Neuronen und deren Resistenz gegen zytotoxische Stress-Stimuli von hoher Relevanz. Störungen der Calcium-Homöostase sind ein bekanntes Phänomen bei Morbus Alzheimer. Im ersten Teil der vorliegenden Arbeit wurde die Rolle von überexprimiertem APP in der Regulation des neuronalen Zelltods nach Calcium Freisetzung untersucht. Die Calcium Freisetzung aus dem endoplasmatischen Retikulum wurde durch die Inhibition der sarko- und endoplasmatischen Calcium-ATPasen (SERCA) ausgelöst. Dies führt zur Induktion der sogenannten „unfolded protein response“ (UPR) und zu einer Aktivierung von Effektor-Caspasen. Für APP-überexprimierende PC12 Zellen konnte bereits zuvor eine im Vergleich zur Kontrolle nach der durch Calcium Freisetzung-induzierten Apoptose eine erhöhte intrazelluläre Calcium Konzentration nachgewiesen werden. Über die Messung der Aktivierung von Effektor-Caspasen konnte zudem ein gesteigerter Zelltod in den APP-überexprimierenden Zellen gemessen werden. Zudem konnte gezeigt werden, dass der pro-apoptotische Transkriptionsfaktor CHOP, nicht aber die klassischen UPR-Zielgene spezifisch hochreguliert wurden. Die APP-modulierte gesteigerte Induktion von Apoptose nach Calcium Freisezung konnte durch Komplexierung der intrazellulären Calcium Ionen und durch Knockdown von CHOP im Vergleich zur Kontrolle gänzlich unterdrückt werden. Ferner bewirkte die Inhibition der Speicher-aktivierten Calcium-Kanälen (SOCC) eine signifikante Unterdrückung der beobachteten erhöhten intrazellulären Calcium Konzentration und der gesteigerten Apoptose in den APP-überexprimierenden PC12 Zellen. In diesem Teil der Arbeit konnte eindeutig gezeigt werden, dass APP in der Lage ist den durch Calcium-Freisetzung-induzierten Zelltod zu potenzieren. Diese Modulation durch APP verläuft in einer UPR-unabhängigen Reaktion über die Aktivierung von SOCC’s, einer erhöhten Aufnahme von extrazellulärem Calcium und durch erhöhte Induktion des pro-apoptotischen Transkriptionsfaktors CHOP. Im zweiten Teil dieser Arbeit wurde die sAPPα-vermittelte Neuroprotektion untersucht. Dabei handelt es sich um die N-terminale Ektodomäne von APP, die über die Aktivität der α-Sekretase prozessiert wird und anschließend extrazellulär abgegeben wird. Ziel dieser Versuchsreihe war die neuroprotektive physiologische Funktion von APP im Hinblick auf den Schutz von neuronalen Zellen vor diversen für Morbus Alzheimer relevanten Stress-Stimuli bzw. Apoptose-Stimuli zu untersuchen. Durch die Analyse der Effektor-Caspasen konnte gezeigt werden, dass sAPPα in der Lage ist PC12 Zellen potent vor oxidativem Stress, DNA-Schäden, Hypoxie, proteasomalem Stress und Calcium-Freisetzung zu schützen. Außerdem konnte gezeigt werden, dass sAPPα in der Lage ist den pro-apoptotischen Stress-induzierten JNK/Akt-Signalweg zu inhibieren. Eine Beteiligung des anti-apoptotischen PI3K/Akt-Signalwegs bei der sAPPα-vermittelten Protektion konnte über die Inhibition der PI3-Kinase ebenfalls demonstriert werden, die eine Aufhebung der sAPPα-vermittelten Neuroprotektion bewirkte. Diese Daten zeigen neue molekulare Mechanismen auf, die dem sAPPα-vermittelten Schutz vor pathophysiologisch relevanten Stress-Stimuli in neuronalen Zellen zugrunde liegen. Im letzten Teil der Arbeit wurden verschieden Gruppen von pharmakologischen Substanzen im Hinblick auf ihre neuroprotektive Wirkung untersucht und mit ihren Effekten auf den APP-Metabolismus korreliert. Die Untersuchungen ergaben, dass Galantamin, ein schwacher Acetycholinesterase Inhibitor und allosterisch potenzierender Ligand von nikotinischen Acetylcholin-Rezeptoren in der Lage war, naive, und mit noch höherer Effizienz APP-überexprimierende Zelllinien vor dem Stress-induzierten Zelltod zu schützen. Zudem bewirkte Galantamin in APP-überexprimierenden HEK293 Zellen eine rasche Erhöhung der sAPPα Sekretion, so dass hier von einer Rezeptor-vermittelten Modulation des APP Metabolismus ausgegangen werden kann. Omega-3 Fettsäuren wirken sich positiv auf die Membranfluidität von Zellen aus und es konnte bereits gezeigt werden, dass die Bildung des toxischen Aβ Peptids hierdurch vermindert wird. In Analogie zu Galantamin schützte die Omega-3 Fettsäure Docosahexaensäure (DHA) neuronale Zellen vor dem Stress-induzierten Zelltod, wobei der Schutz in APP-überexprimierenden Zellen besonders effizient war. Diese Daten legen nahe, dass die Aktivierung des antiamyloidogenen Wegs der APP-Prozessierung ein viel versprechender Ansatz für die Entwicklung neuer Therapien gegen Morbus Alzheimer sein könnte.

Autenticazione d’origine, stima della Freshness quality e studio del profilo nutrizionale come elementi fondanti per la valorizzazione dell’alimento ittico

Questa tesi è dedicata alla qualità dell'alimento ittico in tre delle sue possibili accezioni. Dopo aver spiegato il complicato rapporto del consumatore con gli alimenti ittici e come l'Unione Europea abbia cercato di fare chiarezza al riguardo, gli argomenti di discussione saranno: Autenticazione d'origine La polpa di 160 esemplari di spigola (Dicentrachus labrax), suddivisi tra selvatici, allevati intensivamente e allevati estensivamente, provenienti dall'Italia e dall'estero per un totale di 18 fonti indagate, è stati analizzata individualmente per caratterizzarne la componente lipidica, isotopica e minerale e verificare le potenzialità di queste informazioni ai fini della autenticazione di origine in senso lato. Stima della Freshness Quality Numerosi lotti di seppia (Sepia officinalis), nasello (Merluccius merluccius) e triglia di fango (Mullus barbatus) sono stati sottoposti a due possibili modalità di stoccaggio sotto ghiaccio fondente, per indagare come, nell’arco della loro vita commerciale, ne evolvessero importanti connotati chimici (cataboliti dell’ATP e loro rapporti), fisici (proprietà dielettriche dei tessuti) e sensoriali (Quality Index Methods specie-specifici. Studio del profilo nutrizionale La componente lipidica di numerosi lotti di mazzancolla (Penaeus kerathurus), canocchia (Squilla mantis) e seppia (Sepia officinalis) è stata caratterizzata allo stato crudo e dopo cottura secondo tecniche “dedicate” per stabilire il contributo di queste matrici come fonte di acidi grassi polinsaturi della serie omega 3 e per pervenire alla determinazione dei loro coefficienti di ritenzione vera.

Strategie alimentari atte a incrementare il contenuto di Omega3 e CLA del latte

Tra i componenti naturali biologicamente attivi, e con cui è possibile arricchire il latte, notevole importanza rivestono gli acidi grassi Omega3 e i CLA. I ruoli benefici svolti da questi particolari lipidi si manifestano soprattutto nella regolazione dei processi infiammatori, nella prevenzione del diabete e delle malattie cardiovascolari; è inoltre dimostrato come i CLA abbiano attività anticancerogena e ipocolesterolemica, stimolino il sistema immunitario e prevengano l’insorgenza del diabete e delle malattie croniche non trasmissibili. Lo scopo di questo lavoro è stato quello di individuare, in condizioni sperimentali controllate e nelle comuni realtà di allevamento, le possibili strategie alimentari atte ad aumentare le concentrazioni di Omega-3 e CLA del latte vaccino, senza penalizzare i titoli di grasso del latte. I lavori sperimentali effettuati possono essere distinti in 3 fasi: una prima fase in cui l’oggetto principale delle ricerche svolte è stato l’incremento in acidi grassi Omega3 del latte, una seconda fase in cui l’obiettivo si è allargato anche all’incremento delle concentrazioni in CLA, e una terza fase che ha avuto come scopo quello di incrementare i livelli di Omega3 e CLA del latte senza penalizzare i titoli di grasso. Le strategie alimentari più efficaci si sono basate sulla modificazione degli apporti lipidici della razione attraverso la supplementazione con seme di soia o relativo olio, semi di lino o relativo olio, olii di pesce e acido stearico. Le ricerche svolte hanno evidenziato come, attraverso opportune ed accurate modulazioni degli apporti lipidici delle razioni, sia di fatto possibile innalzare i contenuti di acidi grassi della serie Omega3 e CLA nel latte vaccino, senza penalizzare i titoli del grasso del latte.

Effetto della carenza di Azoto su crescita e composizione di Phaeodactylum tricornutum

La diatomea presa in esame in questo studio è Phaeodactylum tricornutum, diatomea marina con simmetria pennata. L’interesse commerciale verso Phaeodactylum tricornutum nasce dal suo alto contenuto di acidi grassi polinsaturi (PUFA), tra cui troviamo alcuni omega 3, come l’acido eicosapentaenoico (EPA) e l’acido docosaesaenoico (DHA) e dalla quantità del polisaccaride di riserva prodotto, il β-1,3 glucano crisolaminarina. E’stata studiata la capacità dei β-1,3 glucani, in particolare della laminarina, di inibire l’attacco cellulare da parte di alcuni batteri dannosi per la salute umana. Anche i pigmenti accessori di questa diatomea in particolare il β-carotene (appartenente ai caroteni) e la fucoxantina (appartenente alle xantofille) possono essere impiegati nella nutraceutica. Negli studi svolti precedentemente sull’effetto di fattori ambientali sulla composizione di questa specie, è stata presa in considerazione solo la produzione di lipidi e non è mai stato seguito contemporaneamente l’andamento di lipidi e polisaccaridi: questo progetto di tesi prevede la valutazione della possibilità di ottenere colture di P. tricornutum ad alto contenuto sia polisaccaridico sia lipidico per applicazioni industriali. Tutto il lavoro di tesi si è svolto presso l’azienda Micoperi Blue Growth (MBG), nello stabilimento di Ortona (CH) in due fasi: nella prima fase l’esperimento è stato condotto in batch su piccola scala e la crescita e la composizione di P. tricornutum sono state seguite in due diversi terreni di coltura: uno ricco in azoto, denominato N, per mezzo del quale si è voluta incrementare la crescita e la biomassa ed uno a ridotto contenuto di azoto, denominato N/3, per indurre la produzione di lipidi e polisaccaridi. Ne è stata seguita la crescita per mezzo di misure di assorbanza, peso secco, pH, conta cellulare, determinazione dei macronutrienti ed è stata analizzata la composizione biochimica con determinazione dei composti polisaccaridici totali, determinazione qualitativa dei polisaccaridi, determinazione della clorofilla a, valutazione quantitativa e qualitativa dei lipidi. E' stato notato che la condizione con un contenuto polisaccaridico e lipidico maggiore è quella con un ridotto contenuto di azoto. Con la seconda fase si è voluto verificare la riproducibilità su larga scala di quanto notato nel primo esperimento in sistemi chiusi industriali. E’stata avviata una monocoltura di Phaeodactylum tricornutum in fotobioreattore (PBR) da 70L in semicontinuo e ne è stata monitorata la crescita misurando assorbanza, peso secco, pH, quantità di macronutrienti nel terreno; la composizione biochimica è stata valutata determinando i polisaccaridi totali e la loro composizione qualitativa, le proteine totali, i lipidi totali e la composizione qualitativa. In conclusione con questo lavoro si è visto il terreno migliore per la produzione di polisaccaridi e lipidi e le tempistiche di produzione in Phaeodactylum tricornutum, e in aggiunta, abbiamo dimostrato che sia contenuti che tempistiche sono riproducubili in un sistema industriale chiuso per produrre biomassa ad alto valore commerciale.

Pharmacological prevention and treatment in clinical at-risk states for psychosis

Over the last couple of decades, the treatment of psychoses has much advanced; yet, despite all progress, the individual and societal burden associated with psychosis and particularly schizophrenia has largely remained unchanged. Therefore, much hope is currently placed on indicated prevention as a mean to fight these burdens before they set in. Though the number of studies investigating pharmacological interventions is still limited, encouraging results have been reported from the pioneering trials, despite several methodological limitations. Furthermore, it has become clear that persons characterized by the at-risk criteria are already ill and do not only need preventive intervention, but also treatment. In consequence, outcome criteria have to be broadened to cover the current needs of the patients. As is indicated by a recent study successfully using Omega-3 fatty acids for both purposes, it may be promising to develop and investigate interventions especially for the at-risk state, independent of their effectiveness in manifest disease states. Treatment studies may become promoted by the proposed introduction of a new disorder category into DSM-V. Future prevention studies, however, need to solve the challenge of changing immediate transition rates, demanding for new risk enrichment strategies as a prerequisite for feasible trial designs.