Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values ≤ 125.0 μg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 μg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 μg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Enhancing bioactive fatty acids of the meat from lambs reared in intensive systems through nutritional modulation

Tese de Doutoramento em Ciências Veterinárias, especialidade de Produção Animal

Bioactive compounds of citrus as health promoters

Citrus are a group of fruit species, quite heterogeneous in many aspects, including chemical composition of the fruit. Since ancient times, some citrus fruits were used to prevent and cure human diseases. In the recent decades, it has been demonstrated that fruits can actually help prevent and cure some diseases and above all, they are essential in a balanced diet. Citrus fruits, as one of the groups of fruit species, with greater importance in the world, have been studied for their effects on human health. Some species of citrus were referred as potential antioxidant based therapy for heart disease, cancer and inflammation. Fruit peels and seeds have also high antioxidant activity. The health benefits of citrus fruit have mainly been attributed to the high level of bioactive compounds, such as phenols (e.g., flavanone glycosides, hydroxycinnamic acids), carotenoids and vitamin C. These compounds are present in the fruit pulp and hence in the juice. But some bioactive compounds can be found in parts of the fruit which usually are not used for human food. The content of bioactive compounds depends on the species and cultivar, but also depends on the production system followed in the orchard. Citrus fruits, their derivatives and their by-products (peel, pulp and oil) are reach in different bioactive compounds and its maturity, postharvest and agroindustry processes influence their composition and concentration. The aim of this chapter was to review the main bioactive compounds of the different components of citrus and their relationship to health.

Coating bioattivi su zirconia per applicazioni in campo biomedicale: stato dell'arte e prospettive

I materiali bioceramici, in base alla loro capacità d’interazione con l’osso e i tessuti del corpo umano, possono essere classificati in bioinerti e bioattivi. I materiali bioinerti, una volta impiantati, formano uno strato fibroso, non aderente, all’interfaccia con l’osso. Tale strato è una forma naturale di protezione che l’organismo adotta per isolare il materiale che viene, inizialmente, percepito come estraneo. Al contrario, i materiali bioattivi, una volta impiantati, mostrano una risposta biologica immediata, creando un legame attivo con l’osso e i tessuti nel quale vengono impiantati, favorendo e velocizzando la guarigione. La zirconia è un materiale ceramico altamente biocompatibile, definito come bioinerte per la sua scarsa capacità d’integrazione con l’osso ed i tessuti dell’organismo umano. Questa sua particolarità può, nel lungo termine, comprometterne la funzione fino ad arrivare, in alcuni casi, al totale malfunzionamento dell’impianto. Negli ultimi anni, diversi studi sono stati condotti con lo scopo di aumentare la capacità di biointegrazione della zirconia ed alcuni brevetti sono stati depositati. L’obiettivo del presente lavoro è quello di condurre un’analisi bibliografica ed una ricerca brevettuale sul tema dei coating bioattivi su zirconia per impianti dentali ed ortopedici. La necessità di condurre questo studio deriva dalla crescente richiesta di utilizzo della zirconia, in particolare, nel settore dentale. La zirconia rappresenta, infatti, ad oggi, il migliore candidato per la sostituzione dei metalli negli impianti dentali. Le buone proprietà meccaniche, l’eccellente biocompatibilità e l’aspetto estetico molto simile a quello dei denti naturali, rendono questo materiale particolarmente adatto a questo genere di applicazioni. La possibilità di rendere la sua superficie bioattiva rappresenta un importante miglioramento delle prestazioni in termini di biointegrazione, durabilità, sicurezza ed affidabilità.

Bioactive Phenols in Small Fruits and Berries

Bioactive compounds are extra nutritional constituents occurring naturally in plant foods in small amounts, however in quantities enough to produce bioactive effects. Among bioactive compounds the phenolic compounds are a very large set of molecules, which include several groups such as for example flavonoids, phenolic acids or tannins. Small fruits and berries include a wide diversity of fruits, like grapes, strawberries, blackberries, blueberries, raspberries, cherries, hardi kiwi, gooseberries, cranberries, currants (black, white, red), physalis, crowberries, açaí, elderberries, dates or goji berries, and these frequently have been reported as having particularly high concentrations of phenolic compounds with antioxidant activity. Hence, the objective of this chapter is to review the literature about the type and contents of different phenolic compounds present in small fruits and berries, as well as their bioactive properties, including antioxidant capacity. All the fruits and berries investigated in this chapter were particularly rich in bioactive compounds, including phenolic compounds that provide the fruits with high antioxidant properties. The most relevant health promoting effects include anti-cancer, anti-inflamatory, neuro protective, cardio protective or anti-diabetes, thus indicating that these foods are a valuable resource to prevent and treat diseases.

Chemical Composition and Bioactive Compounds in Bananas and Postharvest Alterations

Bananas arise as one of the most popular fruits consumed all around the world. Banana belongs to the genus Musa from the family Musaceae. It is original from tropical regions and presents a strong ability to protect itself from the oxidative stress caused by extreme climatic conditions such as intense sunshine and high temperature. For this protection, bananas increase the production of bioactive compounds with antioxidant activity, which protect the fruit from the oxidative damage. Scientific studies have demonstrated that bananas (both in the pulp and peel) contain different antioxidant compounds, like vitamins (A, B, C and E), β-carotene and phenolic compounds (catechin, epicatechin, lignin, tannins, anthocyanins). Furthermore, banana is also notably rich in minerals, like potassium and phosphorus. The knowledge about the chemical composition and the contents in compounds with biological activity is of high interest given the importance of bananas as a valuable food all over the world. However, because bananas are perishable due to some factors like chemical reactions, including those that result in the production of ethylene, their postharvest conservation in pivotal for the commercialization. The effects of postharvest treatments and storage conditions on the composition of bananas are, therefore, essential. In this way, the present chapter focus on the composition of bananas, including macronutrients, micronutrients and bioactive compounds, as well as the effect of postharvest treatments and storage conditions in the quality of bananas.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Streptococcus thermophilus APC151 strain is suitable for the manufacture of naturally GABA-enriched bioactive yogurt

Consumer interest in health-promoting food products is a major driving force for the increasing global demand of functional (probiotic) dairy foods. Yogurt is considered the ideal medium for delivery of beneficial functional ingredients. Gamma-amino-butyric acid has potential as a bioactive ingredient in functional foods due to its health-promoting properties as an anti-stress, anti-hypertensive, and anti-diabetic agent. Here, we report the use of a novel Streptococcus thermophilus strain, isolated from the digestive tract of fish, for production of yogurt naturally enriched with 2 mg/ml of gamma-amino-butyric acid (200 mg in a standard yogurt volume of 100 ml), a dose in the same range as that provided by some commercially available gamma-amino-butyric acid supplements. The biotechnological suitability of this strain for industrial production of yogurt was demonstrated by comparison with the reference yogurt inoculated with the commercial CH1 starter (Chr. Hansen) widely used in the dairy industry. Both yogurts showed comparable pH curves [ΔpH/Δt = 0.31-0.33 h-1], viscosity [0.49 Pa-s], water holding capacity [72–73%], and chemical composition [moisture (87–88%), protein (5.05–5.65%), fat (0.12–0.15%), sugar (4.8–5.8%), and ash (0.74–1.2%)]. Gamma-amino-butyric acid was not detected in the control yogurt. In conclusion, the S. thermophilus APC151 strain reported here provides a natural means for fortification of yogurt with gamma-amino-butyric acid.

Combined Use of NMR, LC-ESIMS and Antifungal Tests for Rapid Detection of Bioactive Lipopeptides Produced by Bacillus

The science and technology interact with the art in several ways. Biotechnological coupled with analytical approaches can play an important role in protecting and preserving cultural heritage for future generations. Many microorganisms influenced by environmental conditions are the main responsible for biological contamination in built heritage. Biocides based on chemical compounds have been used to mitigate this problem. Thus, it is vitally important to develop proper remediation actions based on environmentally innocuous alternative. Bacillus specie is emerging as an optimistic alternative for built heritage treatment due to their capacity to produce secondary metabolites with antagonistic activities against many fungal pathogens. Therefore, the intent of this work was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using spectroscopic (NMR) and chromatographic techniques (LCESI- MS). The high antifungal activity obtained for Bacillus sp. active compounds produced in this study confirms the great potential to suppress biodeteriogenic fungi growth on historical artworks. Additionally, the proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and could be used as a viable alternative to be employed for screening and production of new green biocides.

Determination of bioactive amines in tropical wines produced at Brazilian's Northeast.

The wine production in the region of the Sub-middle São Francisco river Valley has started there are few years ago.The region has a climate variability that allows to obtain two or three harvests per year, which influences significantly the composition of the grapes. This work aimed to evaluate the amount of amines present in white and red tropical wines produced in the Northeast of Brazil. The winemaking was done by the traditional method. After stabilizing the wines were bottled and then analyzed for the determination of the amines putrescine, spermidine and spermine, by thin layer chromatography (TLC). The harvests were in June (harvest I) and November (harvest II) 2009. The results showed that the edaphoclimatic conditions of the two harvests had significant influence (p> 0.05) on bioactive amines concentration, with high values for red wines. In addition, tropical wines from Sub-middle São Francisco river Valley showed adequate correlation between bioactive amines as compared to other wines in the world.