Wladimir Ronald Lobo Farias

Extraction of lipids from microalgae by ultrasound application: Prospection of the optimal extraction method

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry will depend on the selection of appropriate microalgal strains and the selection of the most suitable lipid extraction method. In this paper, five extraction methods were evaluated regarding the extraction of lipids from Chlorella vulgaris: Bligh and Dyer, Chen, Folch, Hara and Radin, and Soxhlet. Furthermore, the addition of silica powder was studied to evaluate the introduction of more shear stress to the system as to increase the disruption of cell walls. Among the studied methods, the Bligh and Dyer method assisted by ultrasound resulted in the highest extraction of oil from C. vulgaris (52.5% w/w). Addition of powder silica did not improve the extraction of oil.

Bioprospecting for oil producing microalgal strains: Evaluation of oil and biomass production for ten microalgal strains

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3 L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.

Antitumor properties of a sulfated polysaccharide from the red seaweed Champia feldmannii (Diaz‐Pifferer)

In recent years, much attention has been focused on polysaccharides isolated from natural sources. The aim of this study was to investigate the in vitro and in vivo antitumor properties of a sulfated polysaccharide isolated from the seaweed C. feldmannii (Cf‐PLS). Hematological, biochemical and histopathological analyses were performed in order to evaluate the toxicological aspects related to Cf‐PLS treatment. Its effects on the immunological system were also investigated. The Cf‐PLS did not show any significant in vitro cytotoxicity at the experimental exposure levels that were used, but showed in vivo antitumor effect. The inhibition rates of sarcoma 180 tumor development were 48.62 and 48.16% at the doses of 10 and 25 mg kg−1, respectively. In addition, Cf‐PLS was also able to increase the response elicited by 5‐fluorouracil (5‐FU) from 48.66 to 68.32%. The histopathological analysis of liver and kidney showed that both organs were moderately affected by Cf‐PLS‐treatment. Neither enzymatic activity of alanine aminotransferase nor urea or creatinine levels were significantly altered. In hematological analysis, leucopeny was observed after 5‐FU treatment, but this effect was prevented when the treatment was associated with the Cf‐PLS. It was also demonstrated that Cf‐PLS acts as an immunomodulatory agent, raising the production of specific antibodies, and increasing the production of OVA‐specific antibodies. It also induced a discreet hyperplasia of lymphoid folicules of the white pulp in the spleen of treated mice. In conclusion, Cf‐PLS has some interesting anticancer activity that could be associated with its immunostimulating properties. Copyright

HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family

HCA and HML represent lectins isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis, respectively. Hemagglutination inhibition assays suggest that HML binds GalNAc/Gal substituted with a neutral sugar through 1–3, 1–4, or 1–2 linkages in O‐linked mucin‐type glycans, and Fuc(α1–6)GlcNAc of N‐linked glycoproteins. The specificity of HCA includes the epitopes recognized by HML, although the glycoproteins inhibited distinctly HML and HCA. The agglutinating activity of HCA was inhibited by GalNAc, highlighting the different fine sugar epitope‐recognizing specificity of each algal lectin. The primary structures of HCA (9193±3 Da) and HML (9357±1 Da) were determined by Edman degradation and tandem mass spectrometry of the N‐terminally blocked fragments. Both lectins consist of a mixture of a 90‐residue polypeptide containing seven intrachain disulfide bonds and two disulfide‐bonded subunits generated by cleavage at the bond T50–E51 (HCA) and R50–E51 (HML). The amino acid sequences of HCA and HML display 55% sequence identity (80% similarity) between themselves, but do not show discernible sequence and cysteine spacing pattern similarities with any other known protein structure, indicating that HCA and HML belong to a novel lectin family. Alignment of the amino acid sequence of the two lectins revealed the existence of internal domain duplication, with residues 1–47 and 48–90 corresponding to the N‐ and C‐terminal domains, respectively. The six conserved cysteines in each domain may form three intrachain cysteine linkages, and the unique cysteine residues of the N‐terminal (Cys46) and the C‐terminal (Cys71) domains may form an intersubunit disulfide bond.

In vivo anti-inflammatory effect of a sulfated polysaccharide isolated from the marine brown algae Lobophora variegata.

Context: Lobophora variegata J.V. Lamouroux (Dictyotaceae) is a brown marine alga widely encountered in the Brazilian sea coast that presents high content of fucans. Anti-inflammatory effects of fucans are reported mostly in models in vitro, but little is known about its effects in vivo. Objective: To investigate vascular and cellular effects of a sulfated polysaccharide from the brown marine algae L. variegata (SP-Lv) in acute inflammatory models. Materials and methods: SP-Lv was isolated by DEAE-cellulose and analyzed by agarose gel electrophoresis and evaluated for its inhibitory effect on paw edema, vascular permeability, leukocyte migration and peritoneal nitrite content induced by zymosan in Wistar rats. Anticoagulant activities and possible systemic toxicity were also evaluated. Results: SP-Lv inhibited the paw edema (120 min: 1.42 ± 0.11 vs. 0.95 ± 0.05 mL), plasma exudation (21.53 ± 0.62 vs. 11.96 ± 0.68 μg/g), nitrite content (4.42 ± 0.33 vs. 2.86 ± 0.003 μM) and leukocyte migration (5.15 ± 1.21 vs. 1.99 ± 0.16 cells/103 mL) induced by zymosan. SP-Lv and l-NAME reduced the paw edema (60–120 min) elicited by l-arginine. However, at 180 min SP-Lv effect was more accentuated and sustained until 240 min, while that of l-NAME was abolished. Similarly to indomethacin, SP-Lv inhibited the entire edema time-course induced by phospholipase A2, except for the time of 60 min. Discussion and conclusion: The anti-edematogenic effect of SP-Lv seems to occur via inhibition of nitric oxide synthase and cyclooxygenase activities. These results suggest a potential applicability of polysaccharides from alga origin in acute inflammatory conditions.

New affinity procedure for the isolation and further characterization of the blood group B specific lectin from the red marine alga Ptilota plumosa

The red marine alga Ptilota plumosa has been shownto contain an anti-human blood group B lectin. We report here a new isolationprocedure by affinity chromatography on Sephadex G-200 and characterisation ofthe isolated lectin. The Mr, determined by gelfiltration, was 52,500. SDS-PAGE revealed a single protein band withMr 17,440, indicating the native lectin was atrimer of subunits with the same Mr, as reported for the lectinsfromtwo other Ptilota species, P.filicinaand P. serrata. Analysis of amino acid composition showedslightly more basic than acidic amino acids. This was in contrast to theP. filicina and P. serrata lectinspreviously found to contain a higher proportion of acidic than basic aminoacids. Haemagglutination inhibition tests showed the P.plumosa lectin was inhibited by galactose, glucose and theirderivatives with p-nitrophenyl-α-D-galactoside moststrongly inhibitory. All glycoproteins tested failed to inhibit the lectin. Theamino acid composition, human blood group-B specificity and lack of inhibitionby glycoproteins indicate the lectin from P. plumosapossesses unique characteristics among marine algal lectins.

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised.

Efeito do eugenol como agente mitigador do estresse no transporte de juvenis de tilápia do Nilo

Brazil stands out in the American scene as one of the countries with the greatest potential for fish farming mainly in fresh water. Nile tilapia (Oreochromis niloticus) stands out being the most cultivated fish species in our country. Methodologies to reduce the interference of stressors agents in vital and physiological functions of fishes are important during handling. This study aimed to evaluate the effect of eugenol in the metabolic and ionic responses of juvenile Nile tilapia, submitted to transport in plastic bags, at different densities in order to verify the efficiency of the product as an mitigate agent of stress. Eugenol was used at a concentration of 15mg L-1 in water. The evaluated densities were 4, 7 and 10 fish L-1, which were equivalent to 140, 245 and 350g L-1. After four hours of transport the metabolic (glucose and lactate) and ions parameters (chloride, magnesium and calcium) were evaluated, as well as the water quality in the plastic bags. For the two metabolic parameters, the use of the eugenol in order to reduce the stress response was not satisfactory. There was an increase in blood glucose level at the densities of 140 and 350g L-1 immediately after termination of the transport, and the level of fish lactate content at the density of 245g L-1 had increased after 24 hours indicating that the animals could not maintain the initial homeostasis. Among the concentrations of the evaluated ions magnesium suffered the greater variation. We can conclude that the addition of 15 mg L-1 of eugenol in the water during the transport of juvenile Nile tilapia at densities of 140, 245 and 350g L-1 was not able to minimize stress responses.

Desempenho zootécnico de alevinos de tilápia do Nilo cultivados em um sistema de recirculação de água com a microalga Spirulina platensis

The present work aimed to evaluate the zootechnical parameters of Nile tilapia, water quality and rate of nitrogen removal compounds by Spirulina platensis in a closed system of water circulation. The experiment took place at the Biotechnology Aquaculture Applied Center (CEBIAQUA) of The Fishing Engineering Department of the Federal University of Ceara. Tilapias culture was conducted in three treatments with four replicates at densities of 010, 0,15 and 0,20 juveniles L-1. The tilapias were fed ad libitum with a commercial ration (0,8mm) containing 55% crude protein (CP), administered daily at 9, 13 and 17 h. Throughout the experiment, the water of fishes boxes and of the respective microalgae basins was reused in the closed recirculation system between the two cultures, with only replacement of water due to evaporation. The results showed that the most efficient stocking density was 0,10 L-1, because it presented the lowest feed conversion ratio. With respect to water quality, levels of N-ammonia (0,26 ± 0,03mg L-1), N-nitrates (2,55 ± 0,71mg L-1) and N- nitrites (0,19 ± 0,01mg L-1) remained within acceptable values for the cultivation of tilapia Nile. Moreover, were reduced 95.57 ± 1.1% of ammonia, 87.9 ± 4.4% of nitrate and 42.63 ± 8.5% of nitrite of water fish culture.