Optimisation of stir bar sorptive extraction and liquid desorption combined with large volume injection-gas chromatography–quadrupole mass spectrometry for the determination of volatile compounds in wines

Stir bar sorptive extraction and liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE–LD/LVI-GC–qMS) had been applied for the determination of volatiles in wines. The methodology was optimised in terms of extraction time and influence of ethanol in the matrix; LD conditions, and instrumental settings. The optimisation was carried out by using 10 standards representative of the main chemical families of wine, i.e. guaiazulene, E,E-farnesol, β-ionone, geranylacetone, ethyl decanoate, β-citronellol, 2-phenylethanol, linalool, hexyl acetate and hexanol. The methodology shows good linearity over the concentration range tested, with correlation coefficients higher than 0.9821, a good reproducibility was attained (8.9–17.8%), and low detection limits were achieved for nine volatile compounds (0.05–9.09 μg L−1), with the exception of 2-phenylethanol due to low recovery by SBSE. The analytical ability of the SBSE–LD/LVI-GC–qMS methodology was tested in real matrices, such as sparkling and table wines using analytical curves prepared by using the 10 standards where each one was applied to quantify the structurally related compounds. This methodology allowed, in a single run, the quantification of 67 wine volatiles at levels lower than their respective olfactory thresholds. The proposed methodology demonstrated to be easy to work-up, reliable, sensitive and with low sample requirement to monitor the volatile fraction of wine.

Enrichment of bioactive compounds in microalgae for aquaculture

Microalgae are promising microorganisms for the production of food and fine chemicals. Several species of microalgae are used in aquaculture with the purpose of transfer bioactive compounds up to the aquatic food chain. The main objective of this project was to develop a stress–inducement strategy in order to enhance the biochemical productivity of Nannochloropsis gaditana, Rhodomonas marina and Isochrysis sp. for aquaculture purposes having in account their growth and organizational differences. In this regard, two experiments were design: the first one consisted on the alteration of overall nutrient availabilities in growth medium; and the second one comprised changes in nitrogen and sulfur concentrations maintaining the concentrations of the other nutrients present in a commercial growth medium (Nutribloom plus), which is frequently used in aquaculture. Microalgae dried biomass was characterized biochemically and elemental analysis was also performed for all samples. In first experimental design: linear trends between nutrient availability in growth media and microalgae protein content were obtained; optimum productivities of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) were attained for both R. marina and N. gaditana in growth media enriched with 1000 L L-1 of nutrient solution whereas for Isochrysis sp. the double of Nutribloom plus was needed; the decrease of glucans and total monosaccharides with nutrient availability for R. marina and Isochrysis sp. showed the occurrence of a possible depletion of carbohydrates towards lipids and proteins biosynthesis. Second experimental desing: N. gaditana exhibited the highest variation in their biochemical composition against the applied perturbation; variations observed for microalgae in their biochemical composition were reflected in their elemental stoichiometry; in N. gaditana the highest nitrogen concentrations lead to overall maximum productivities of the biochemical parameters. The results of the present work show two stress-inducement strategies for microalgae that may constitute a base for further investigations on their biochemical enhancement.

Anti-leishmanial effects of purified compounds from aerial parts of Baccharis uncinella C. DC. (Asteraceae)

Species of Baccharis exhibit antibiotic, antiseptic, wound-healing, and anti-protozoal properties, and have been used in the traditional medicine of South America for the treatment of several diseases. In the present work, the fractionation of EtOH extract from aerial parts of Baccharis uncinella indicated that the isolated compounds caffeic acid and pectolinaringenin showed inhibitory activity against Leishmania (L.) amazonensis and Leishmania (V.) braziliensis promastigotes, respectively. Moreover, amastigote forms of both species were highly sensible to the fraction composed by oleanolic + ursolic acids and pectolinaringenin. Caffeic acid also inhibited amastigote forms of L. (L.) amazonensis, but this effect was weak in L. (V.) braziliensis amastigotes. The treatment of infected macrophages with these compounds did not alter the levels of nitrates, indicating a direct effect of the compounds on amastigote stages. The results presented herein suggest that the active components from B. uncinella can be important to the design of new drugs against American tegumentar leishmaniases.

Comparative toxicity of antifouling compounds on the development of sea urchin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1) to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2) to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10 mM SrCl2 for 5 h) alone or in combination: ethanol + strontium (ES) and strontium + ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (M). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P > 0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P > 0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 It, respectively; P < 0.05) and strontium after 30 It (53.3%) was superior to 24 and 26h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P > 0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82 +/- 5.7 and 89.5 +/- 7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P > 0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl2 induces activation and parthenogenetic development in bovine oocytes. (C) 2003 Elsevier B.V. All rights reserved.

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (1) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and 1 (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22h; and in ID at 26h (45.0%) and 30h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Activation of bovine oocytes by strontium combined or not with an electric pulse

O objetivo deste trabalho foi avaliar as taxas de ativação e de clivagem de oócitos bovinos tratados com estrôncio (10mm de SrCl2), após maturação in vitro por 27-28 horas. No experimento 1, os tratamentos foram: S4 - ativação pelo estrôncio por 4 horas; S12 - ativação pelo estrôncio por 12 horas; S30 - ativação pelo estrôncio por 30 horas; e P - ativação por pulso elétrico (3 pulsos de 1,0kv/cm). No experimento 2 os tratamentos foram: PS4 - ativação combinada pelo pulso elétrico e pelo estrôncio por 4 horas; S4P - ativação pelo estrôncio por 4 horas e pelo pulso elétrico; e PS30 - ativação pelo pulso elétrico e pelo estrôncio por 30 horas. No experimento 1, todos os tratamentos apresentaram taxas similares de ativação (83-90%; P>0,05). Para clivagem, P foi melhor (53%; P<0,05) do que todos os tratamentos com estrôncio (6 a 28%). No experimento 2, o tratamento S4P apresentou melhor taxa de ativação (88%; P<0,05) do que PS4 e PS30 (60 e 68%, respectivamente). Para clivagem, observou-se o mesmo padrão, S4P (65%; P<0,05) e PS4 e PS30 (37% e 44%, respectivamente). Conclui-se que o estrôncio é capaz de ativar oócitos bovinos e sua combinação com pulso elétrico não melhora a ativação. Este é o primeiro relato demonstrando que o estrôncio ativa oócitos bovinos.

Selection of surfactant compounds to enhance the dispersion of Beauveria bassiana

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)