Genetic diversity and epidemiology of antimicrobial resistant Streptococcus pneumoniae isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality in Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Brazilian fruit pulps as functional foods and additives: Evaluation of bioactive compounds

Eight tropical fruit pulps from Brazil were simultaneously characterised in terms of their antioxidant and antimicrobial properties. Antioxidant activity was screened by DPPH radical scavenging activity (126–3987 mg TE/100 g DW) and ferric reduction activity power (368–20819 mg AAE/100 g DW), and complemented with total phenolic content (329–12466 mg GAE/100 g DW) and total flavonoid content measurements (46–672 mg EE /100 g DW), whereas antimicrobial activity was tested against the most frequently found food pathogens. Acerola and açaí presented the highest values for the antioxidant-related measurements. Direct correlations between these measurements could be observed for some of the fruits. Tamarind exhibited the broadest antimicrobial potential, having revealed growth inhibition of Pseudomonas aeruginosa. Escherichia coli, Listeria monocytogenes, Salmonella sp. and Staphylococcus aureus. Açaí and tamarind extracts presented an inverse relationship between antibacterial and antioxidant activities, and therefore, the antibacterial activity cannot be attributed (only) to phenolic compounds.

Plant derived and dietary phenolic antioxidants: Anticancer properties

n this paper, a review of the literature on the phenolic compounds with anticancer activity published between 2008 and 2012 is presented. In this overview only phenolic antioxidant compounds that display significant anticancer activity have been described. In the first part of this review, the oxidative and nitrosative stress relation with cancer are described. In the second part, the plant-derived food extracts, containing identified phenolic antioxidants, the phenolic antioxidants isolated from plants and plant-derived food or commercially available and the synthetic ones, along with the type of cancer and cells where they exert anticancer activity, are described and summarized in tables. The principal mechanisms for their anti-proliferative effects were also described. Finally, a critical analysis of the studies and directions for future research are included in the conclusion.

SEROVARS AND ANTIMICROBIAL RESISTANCE OF Salmonella spp. ISOLATED FROM TURKEY AND BROILER CARCASSES IN SOUTHERN BRAZIL BETWEEN 2004 AND 2006

Salmonella spp. causes diseases in fowls, when species-specific serovars (Salmonella Pullorum and S.Gallinarum) are present in flocks, and public health problems, when non-typhoid serovars are isolated, as well as possible bacterial resistance induced by the preventive and therapeutic use of antimicrobials in animal production. This study describes the serovars and bacterial resistance of 280Salmonella spp. strains isolated from turkey and broiler carcasses in Southern Brazil between 2004 and 2006. SalmonellaEnteritidis was the most prevalent serovar (55.7%), followed by Heidelberg (5.0%), Agona (4.3%), Bredeney (3.9%), Hadar (3.2%), and Typhimurium (2.9%). Tennessee and S. Enterica subspecies enterica(O: 4.5) were isolated only in turkeys, and Hadar (18.6%) was the most prevalent serovar in this species. Antimicrobial susceptibility tests were performed in 178 isolates (43 from turkeys and 135 from broilers). All isolates were sensitive to amoxicillin + clavulanic acid, polymyxin B, ciprofloxacin, and norfloxacin, and were resistant to bacitracin and penicillin. Broiler carcass isolates showed resistance to nalidixic acid (48.9%), nitrofurantoin (34.3%), neomycin (9.6%), tetracycline (5.2%), and kanamycin (8.9%); and turkey carcass isolates were resistant to nalidixic acid (62.8%), tetracycline (34.9%), and neomycin (30.2%), with a significant difference in turkeys when compared to broiler carcass isolates. These results indicate the need for judicious use of antimicrobials in livestock production, given that the serovars identified are potential causes of food poisoning.

Quantification and characterization of the phenolic compounds of kenaf leaves

Dissertation for the Master Degree in Technology and Food Security

Effect of an 8-weeks aerobic training program in elderly on oxidative stress and HSP72 expression in leukocytes during antioxidant supplementation.

OBJECTIVE: To investigate the effect of aerobic training in the context of antioxidant supplementation on systemic oxidative stress and leukocytes heat shock protein (Hsp)72 expression in the elderly. DESIGN: Sixteen septuagenarians (8 males and 8 females, mean age 74.6) were supplemented with Vitamin C and E (respectively 500 and 100mg per day) and randomly assigned either to sedentary (AS) or individualized aerobically trained (AT) group for 8 weeks. METHODS: Plasma Vitamin C and E concentrations and aerobic fitness, as well as resting and post graded exercise (GXT) Hsp72 expression in leukocytes, plasma levels of thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein product (AOPP) were measured pre and post training / supplementation. RESULTS: At the end of the intervention, the two groups showed a significant increase in resting plasma vitamin C and E (approximately 50 and 20% increase respectively) and a significant decrease in both resting and post GXT plasma TBARS and AOPP (approximately 25 and 20% decrease respectively). These changes were of similar magnitude in the two groups. The reduced oxidative stress was concomitant with a 15% decreased expression of Hsp72 in monocytes and granulocytes in both groups. CONCLUSION: This study provides evidence that in elderly, increased concentration of antioxidant vitamins C and E is associated with a reduction in oxidative stress and leukocytes Hsp72. In this context, 8 weeks of aerobic training has no impact on oxidative stress or leukocytes Hsp72 expression in elderly people.

Characterization and multivariate classification of grapes and wines of two Cabernet Sauvignon clones

The objective of this work was to assess and characterize two clones, 169 and 685, of Cabernet Sauvignon grapes and to evaluate the wine produced from these grapes. The experiment was carried out in São Joaquim, SC, Brazil, during the 2009 harvest season. During grape ripening, the evolution of physical-chemical properties, phenolic compounds, organic acids, and anthocyanins was evaluated. During grape harvest, yield components were determined for each clone. Individual and total phenolics, individual and total anthocyanins, and antioxidant activity were evaluated for wine. The clones were also assessed regarding the duration of their phenological cycle. During ripening, the evolution of phenolic compounds and of physical-chemical parameters was similar for both clones; however, during harvest, significant differences were observed regarding yield, number of bunches per plant and berries per bunch, leaf area, and organic acid, polyphenol, and anthocyanin content. The wines produced from these clones showed significant differences regarding chemical composition. The clones showed similar phenological cycle and responses to bioclimatic parameters. Principal component analysis shows that clone 685 is strongly correlated with color characteristics, mainly monomeric anthocyanins, while clone 169 is correlated with individual phenolic compounds.

Neuronal activity controls the antagonistic between PGC-1α and SMRT in regulating antioxidant defences

Transcriptional coactivators and corepressors often have multiple targets and can have opposing actions on transcription and downstream physiological events. The coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is under-expressed in Huntington's disease and is a regulator of antioxidant defenses and mitochondrial biogenesis. We show that in primary cortical neurons, expression of PGC-1α strongly promotes resistance to excitotoxic and oxidative stress in a cell autonomous manner, whereas knockdown increases sensitivity. In contrast, the transcriptional corepressor silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) specifically antagonizes PGC-1α-mediated antioxidant effects. The antagonistic balance between PGC-1α and SMRT is upset in favor of PGC-1α by synaptic activity. Synaptic activity triggers nuclear export of SMRT reliant on multiple regions of the protein. Concommitantly, synaptic activity post-translationally enhances the transactivating potential of PGC-1α in a p38-dependent manner, as well as upregulating cyclic-AMP response element binding protein-dependent PGC-1α transcription. Activity-dependent targeting of PGC-1α results in enhanced gene expression mediated by the thyroid hormone receptor, a prototypical transcription factor coactivated by PGC-1α and repressed by SMRT. As a consequence of these events, SMRT is unable to antagonize PGC-1α-mediated resistance to oxidative stress in synaptically active neurons. Thus, PGC-1α and SMRT are antagonistic regulators of neuronal vulnerability to oxidative stress. Further, this coactivatorcorepressor antagonism is regulated by the activity status of the cell, with implications for neuronal viability.

Andean indigenous food crops: nutritional value and bioactive compounds

The Andean area of South America is a very important center for the domestication of food crops. This area is the botanical origin of potato, peanut and tomato. Less well- known crops, such as quinoa (Chenopodium quinoa), kañiwa (Chenopodium pallidicaule) and kiwicha (Amaranthus caudatus), were also domesticated by ancient Andean farmers. These crops have a long history of safe use with the local populations and they have contributed to the nutrition and wellbeing of the people for centuries. Several studies have reported the nutritional value of Andean grains. They contain proteins with a balanced essential amino acid composition that are of high biological value, good quality oil and essential minerals, for example iron, calcium and zinc. They are potential sources of bioactive compounds such as polyphenols and dietary fiber. The main objective of the practical work was to assess the nutritional value of Andean native grains with a special emphasis on the bioactive components and the impact of processing. The compounds studied were phenolic acids, flavonoids, betalains and dietary fiber. The radical scavenging activity was measured as well. Iron, calcium and zinc content and their bioavailability were analyzed as well. The grains were processed by extrusion with the aim to study the effect of processing on the chemical composition. Quinoa, kañiwa and kiwicha are very good sources of dietary fiber, especially of insoluble dietary fiber. The phenolic acid content in Andean crops was low compared with common cereals like wheat and rye, but was similar to levels found in oat, barley, corn and rice. The flavonoid content of quinoa and kañiwa was exceptionally high. Kiwicha did not contain quantifiable amounts of these compounds. Only one variety of kiwicha contained low amounts of betalains. These compounds were not detected in kañiwa or quinoa. Quinoa, kañiwa and kiwicha are good sources of minerals. Their calcium, zinc and iron content are higher than the content of these minerals in common cereals. In general, roasting did not affect significantly mineral bioavailability. On the contrary, in cooked grains, there was an increase in bioavailability of zinc and, in the case of kañiwa, also in iron and calcium bioavailability. In all cases, the contents of total and insoluble dietary fiber decreased during the extrusion process. At the same time, the content of soluble dietary fiber increased. The content of total phenolics, phytic acid and the antioxidant activity decreased in kiwicha varieties during the extrusion process. In the case of quinoa, the content of total phenolic compounds and the radical scavenging activity increased during the extrusion process in all varieties. Taken together, the studies presented here demonstrate that the Andean indigenous crops have excellent potential as sources of minerals, flavonoids and dietary fiber. Further studies should be conducted to characterize the phenolic compound and antioxidant composition in processed grains and end products. Quinoa, kañiwa and kiwicha grains are consumed widely in Andean countries but they also have a significant, worldwide potential as a new cultivated crop species and as an imported commodity from South America. Their inclusion in the diet has the potential to improve the intake of minerals and health-promoting bioactive compounds. They may also be interesting raw materials for special dietary foods and functional foods offering natural sources of specific health-promoting components.

Composition of Hawthorn (Crataegus spp.) Fruits and Leaves and emblic Leafflower (Phyllanthus emblica) Fruits

Hawthorn (Crataegus sp.) is widely distributed in the northern hemisphere (Asia, Europe and North America). It has been used as a medicinal material and food for hundreds of years both in Europe and in China. Clinical investigations and other research suggest that extracts of hawthorn fruits and leaves have multiple health effects including hypolipidaemic, anti-atherosclerotic, hypotensive, cardioprotective and blood vessel relaxing activities. Hawthorn fruit extracts have also displayed antioxidant and radical scavenging activities. Emblic leafflower fruit (Phyllanthus emblica) is widely used in Chinese and Indian traditional medicine. It has been found to have anti-cancer, hypoglycaemic and hypolipidaemic activities as well as cardioprotective effects and antioxidant activity. The fruit is currently used as a functional food targeted at obese people in China. Phenolic compounds, procyanidins (PCs), flavonols and C-glycosyl flavones in hawthorn and hydrolysable tannins in emblic leafflower fruits are considered among the major bioactive compounds in these berries. Moreover, hawthorn and emblic leafflower fruits are rich in vitamin C, triterpenoids, fruit acids, sugar alcohols and some other components with beneficial effects on the health of human beings. The aim of the thesis work was to characterise the major phenolic compounds in hawthorn fruits and leaves and emblic leafflower fruits as well as other components contributing to the nutritional profile and sensory properties of hawthorn fruits. Differences in the content and compositional profile of the major phenolic compounds, sugars, acids and sugar alcohols within various origins and species of hawthorn were also investigated. Acids, sugars and sugar alcohols in the fruits of different origins/cultivars belonging to three species (C. pinnatifida, C. brettschneideri and C. scabrifolia) of hawthorn were analysed by gas chromatography (GC-FID) and mass spectrometry (Publication I). Citric acid, quinic acid, malic acid, fructose, glucose, sorbitol and myo-inositol were found in all the subspecies. Sucrose was present only in C. scabrifolia and three cultivars of C. pinnatifida var. major. Forty-two phenolic compounds were identified/tentatively identified in fruits of C. pinnatifida var. major by polyamide column chromatography combined with high-performance liquid chromatograph-electrospray ionisation mass spectrometry (HPLC-ESI-MS) (Publication II). Ideain, chlorogenic acid, procyanidin (PC) B2, (-)-epicatechin, hyperoside and isoquercitrin were the major phenolic components identified. In addition, 35 phenolic compounds were tentatively identified based on UV and mass spectra. Eleven major phenolic compounds (hyperoside, isoquercitrin, chlorogenic acid, ideain, (-)-epicatechin, two PC dimers, three PC trimers and a PC dimer-hexoside) were quantified in the fruits of 22 cultivars/origins of three species of Chinese hawthorn by HPLC-ESI-MS with single ion recording function (SIR) (Publication III). The fruits of the hawthorn cultivars/origins investigated fell into two groups, one rich in sugars and flavonols, the other rich in acids and procyanidins. Based on the compositional features, different biological activities and sensory properties may be expected between cultivars/origins of the two groups. The results suggest that the contents of phenolic compounds, acids, sugars and sugar alcohols may be used as chemotaxonomic information distinguishing the hawthorn species from each other. Phenolic compounds in fruits and leaves of C. grayana and their changes during fruit ripening/harvesting were investigated using HPLC-UV-ESI-MS (Publication IV). (-)-Epicatechin, PC B2 and C1, hyperoside and a quercetin-pentoside were the major phenolic compounds in both fruits and leaves. Three C-glycosyl flavones (a luteolin-C-hexoside, a methyl luteolin-C-hexoside and an apigenin-C-hexoside) were present in leaves in abundance, but only at trace levels in fruits. Ideain and 5-O-caffeoylquinic acid were found in fruits only. Additionally, eleven phenolic compounds were identified/tentatively identified in both leaves and fruits (three B-type PC trimers, two B-type PC tetramers, a quercetin-rhamnosylhexoside, a quercetin-pentoside, a methoxykaempferol-methylpentosylhexoside, a quercetin-hexoside acetate, a methoxykaempferol-pentoside, chlorogenic acid and an unknown hydroxycinnamic acid derivative). The total content of phenolic compounds reached the highest level by the end of August in fruits and by the end of September in leaves. The compositional profiles of phenolic compounds in fruits and leaves of C. grayana were different from those of C. pinnatifida, C. brettschneideri, C. scabrifolia, C. pinnatifida. var. major, C. monogyna, C. laevigata and C. pentagyna. Phenolic compounds in emblic leafflower fruits were characterised by Sephadex LH-20 column chromatography combined with HPLC-ESI-MS (Publication V). A mucic acid gallate, three isomers of mucic acid lactone gallate, a galloylglucose, gallic acid, a digalloylglucose, putranjivain A, a galloyl-HHDP-glucose, elaeocarpusin and chebulagic acid represented the major phenolic compounds in fruits of emblic leafflower. In conclusion, results of this study significantly increase the current knowledge on the key bioactive and nutritional components of hawthorn and emblic leafflower fruits. These results provide important information for research on the mechanism responsible for the health benefits of these fruits.

Comparison of total phenolic content and antiradical capacity of powders and "chocolates" from cocoa and cupuassu

Polyphenolic compounds seem to be related to the health benefits produced by the cocoa due to their antioxidant properties. Cupuassu powder, prepared from Theobroma grandiflorum seeds, is a very promising cocoa powder substitute. In order to assess the potential health benefits of the cupuassu powder, a comparison was performed between cocoa, chocolate, and cupuassu powders in relation to the content of total phenolic compounds, flavonoids and DPPH scavenging capacity of methanolic extracts. Cupuassu "chocolates" (milk, dark, and white) were also analyzed. Mineral, lipid, protein, and moisture determinations were made in cocoa and cupuassu powders and in cupuassu "chocolates". Results showed that the phenolic contents of cocoa and chocolate powders are more than three times higher than those of cupuassu powder; however, flavonoid contents were significantly lower. The DPPH scavenging capacity varied hugely among the different samples, from 0.5 (white cupuassu "chocolate") to 120 (cocoa powder) μg of Trolox equivalent per 100 g (FW), and presented a significant correlation (r = 0.977) with the total phenolic contents but not with the flavonoid contents (r = -0.035).

Comparison of phenol content and antioxidant capacity of nuts

Frequent nut intake is associated with protective effects against cardiovascular diseases. In addition to the generally high contents of unsaturated fatty acids, polyphenol compounds seem to be also implicated in health promoting effects of nuts due to their antioxidant properties. In this way, eleven different kinds of nuts, including pinhao seeds (Araucaria angustifolia) and Brazil nuts (Bertholletia excelsa), typical of Brazil, were analyzed for the content of phenol compounds, including the potent anti-mutagenic and anti-cancer ellagic acid, and antioxidant capacity of methanolic extracts. The antioxidant capacity varied a hundred times among the different nuts, from 1.2 to 120 mg of Trolox equivalents.100 g-1 (FW). Total ellagic acid was determined after acid hydrolysis of ellagitannins and ellagic acid glycosides, and it was detected in only 3 of the 11 samples. The content of free and total ellagic acid in nuts varied from 0.37 to 41 and from 149 (chestnuts) to 823 (walnuts) mg.100 g-1 (FW), respectively. Among nuts, samples with the highest contents of ellagic acid (walnuts and pecans) also presented the highest total phenol contents and DPPH radical scavenging capacities. Pinhao seeds and Brazil nuts did not present significant amounts of phenols nor antioxidant capacity.

Evaluation of nutritional compounds in new amaranth and quinoa cultivars

This study evaluated the fatty acid quantification, proximate and amino acid compositions, antioxidant activity, total phenolic compounds, vitamin E, and mineral contents of new amaranth (BRS Alegria) and quinoa (BRS Piabiru) cultivars which were produced in order to adapt these pseudocereals to the climatic conditions of central-western Brazil. They showed superior levels of protein and total lipids in comparison to their native counterparts. The lipid profile of the amaranth BRS Alegria was revealed to be better than those of other native cultivars. Quinoa BRS Piabiru presented a higher antioxidant capacity and phenolic content than the studied amaranth cultivar, but lower contents of tocopherols. All of the obtained results confirm that these new grains possess an overall amount of nutritional compounds that is better than those reported for many native counterparts of the studied samples. The employed analytical methods in this work contributed to a better understanding of the quinoa BRS Piabiru and amaranth BRS Alegria chemical compositions. Therefore, the diversity and quantity of all of the studied compounds in the samples denote the considerable importance of these grains for food science research area.

Antioxidant capacity of amaranth products: effects of thermal and enzymatic treatments

The effect of different process -defatting, protein concentration, thermal treatment, hydrolysis with Alcalase and in vitro digestion- on the antioxidant capacity of amaranth seeds was studied. The antioxidant capacity of the products was determined in methanolic and aqueous extracts and varied from 1.00 to 21.22 and 4.97 to 369.18 µ mol TE/g sample for DPPH and ORAC assays, respectively. The combination of protein concentration and hydrolysis with Alcalase led to products with higher antioxidant activity. However, after in vitro digestion, protein concentrate and its hydrolysate showed similar antioxidant capacity. A high correlation was observed between the antioxidant capacity and the total phenolic content for methanolic extracts, with r² values ranging from 0.6133 to 0.9352.

Phenols, flavonoids and antioxidant activity of aqueous and methanolic extracts of propolis (Apis mellifera L.) from Algarve, South Portugal

Propolis is a resinous substance collected by honeybees to seal honeycomb, which has been used in folk medicine due to its antimicrobial and antioxidant properties. In the present study, water and methanol were used to extract phenols and flavonoids from propolis collected in thirteen different areas in the Algarve region during the winter and spring. The ABTS•+, DPPH•, and O2•- scavenging capacity, and metal chelating activity were also evaluated in the propolis samples. Methanol was more effective than water in extracting total phenols (2.93-8.76 mg/mL) (0.93-2.81 mg/mL). Flavones and flavonols were also better extracted with methanol (1.28-2.76 mg/mL) than with water (0.031-0.019 mg/mL). The free radical scavenging activity, ABTS (IC50=0.006-0.036 mg/mL), DPPH (IC50=0.007-0.069 mg/mL) and superoxide (IC50=0.001-0.053 mg/mL), of the samples was also higher in methanolic extracts. The capacity for chelating metal ions was higher in aqueous extracts (41.11-82.35%) than in the methanolic ones (4.33-29.68%). Propolis from three locations of Algarve region were richer in phenols and had better capacity for scavenging free ABTS and DPPH radicals than the remaining samples. These places are part of a specific zone of Algarve known as Barrocal.