Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon)

BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.

Cytotoxic activity of extract and active fraction of Turbinaria decurrens Bory on colon cancer cell line HCT-116 / Actividad citotóxica del extracto y la fracción activa de Turbinaria decurrens Bory en la línea celular de cáncer de colon HCT-116

Turbinaria deccurrens Bory contains bioactive compound that is beneficial for health. Turbinaria deccurrens Bory is one of many species of brown seaweed that grows in Indonesian marine life and has been known to have cytotoxic activity. The aim of this study is to determine fucoxantin content and the cytotoxic activity of extract and fraction T. decurrens on colon cancer cell lines. Cytotoxic assay of ethanolic extract, n-hexane, ethyl acetate and ethanolic fractions against HCT-116 by MTS assay using Cell Counting Kit-8 (CCK-8). Fucoxantin content in extract and fraction were analyzed using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) analysis. Extract and fraction of T. decurrens contain fucoxanthin with the highest content of fucoxanthin was in ethyl acetate fraction. CCK-8 assay showed that extract, n-hexane and ethyl acetate fraction inhibited the growth of HCT-116. Brown seaweed Turbinaria decurrens was potential as an anticolon cancer agent.

Turbinaria deccurrens Bory contiene compuestos bioactivos que son beneficiosos para la salud. Turbinaria deccurrens Bory es una de muchas especies de algas pardas que crecen en aguas marinas de Indonesia y se ha estudiado su actividad citotóxica. El objetivo de este estudio fue determinar el contenido de fucoxantina y la actividad citotóxica del extracto y la fracción de T. decurrens en líneas celulares de cáncer de colon. Se llevó a cabo un ensayo citotóxico de extracto etanólico, nhexano, acetato de etilo y fracciones etanólicas contra HCT-116 mediante ensayo MTS utilizando Cell Counting Kit-8 (CCK-8). El contenido de fucoxantina en el extracto y la fracción se analizaron usando cromatografía líquida de alta resolución de fase reversa (RP-HPLC). El extracto y la fracción de T. decurrens contienen fucoxantina conmayor contenido de fucoxantina en la fracción de acetato de etilo. El ensayo CCK-8 mostró que la fracción de extracto, n-hexano y acetato de etilo inhibía el crecimiento de HCT-116. El alga marrón Turbinaria decurrens es un agente potencial contra el cáncer de colon.

Hydrolytic efficiency and isomerization during de-esterification of natural astaxanthin esters by saponification and enzymolysis

Background: Astaxanthin from natural sources is typically esterified with fatty acids; hence, it must be hydrolyzed to remove esters before identification and quantification by conventional HPLC. Alkaline-catalyzed saponification and enzyme-catalyzed enzymolysis are the most commonly used de-esterification methods. However, information on the efficiency and isomerization during de-esterification of natural astaxanthin esters by these two methods remains scarce. Therefore, we conducted two HPLC-based experiments to determine which method is better for hydrolyzing astaxanthin esters. Results: To assess the effect of enzymolysis (0.67 U/mL cholesterol esterase, at 37°C) and saponification (0.021 M NaOH, at 5°C) conditions on free astaxanthin recovery and destruction or structural transformation of astaxanthin, we varied the total treatment time across a range of 195 min. The results showed that enzymolysis and saponification were complete in 60 min and 90 min, respectively. After complete hydrolysis, the maximum free astaxanthin recovery obtained by enzymolysis was 42.6% more than that obtained by saponification. The identification of by-products, semi-astacene and astacene, during the process of saponification also indicated that a more severe degradation of astaxanthin occurred during saponification. Moreover, the composition of astaxanthin isomers during saponification was similar to that of the isomers during enzymolysis between 30 min and 75 min (all-trans:9-cis:13-cis = 21:3:1, approximately) but dramatically changed after 90 min, whereas the composition in the enzymolysis treatment remained relatively stable throughout. Conclusion: Compared with saponification, enzymolysis with cholesterol esterase was recommended as a more accurate method for de-esterification of natural astaxanthin esters for further qualitative and quantitative HPLC analysis.

Un método basado en la PCR para la identificación rápida de levaduras acumuladoras de astaxantina (Phaffia spp. ) / PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.)

It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts.

Recientemente, se ha encontrado que la distribución natural, el hábitat y la diversidad genética de levaduras productoras de astaxantina (p. ej., Phaffia rhodozyma, sinónimo Xanthophyllomyces dendrorhous) son mucho mayores de lo que se pensaba. P. rhodozyma se explota biotecnológicamente debido a su capacidad para producir el pigmento carotenoide astaxantina y, por lo tanto, se utiliza como una fuente natural de este pigmento para la acuicultura. También se encontró que esta levadura es capaz de sintetizar el potente protector solar UVB micosporina-glutaminol-glucósido (MGG). Por lo tanto, más estudios ambientales para dilucidar sus aspectos ecológicos y detectar nuevas cepas potenciales productoras de astaxantina y MGG son necesarios. Sin embargo, la obtención de nuevos aislamientos de P. rhodozyma y especies relacionadas no siempre es fácil debido a su baja abundancia y a la presencia de otras levaduras simpátricas y pigmentadas. En este trabajo se describe el desarrollo exitoso de un cebador especie-específico que tiene la capacidad de detectar rápidamente y con precisión cepas representativas de todos los linajes del género Phaffia previamente reportados (incluyendo nuevas especies del género) y excluir especies estrechamente relacionadas. Para ello, se diseñó un cebador de 20 nucleótidos (denominado PhR) para ser utilizado en combinación con los cebadores universales ITS3 y NL4 en una amplificación multiplex. El método propuesto tiene la sensibilidad y la especificidad requerida para la detección precisa de nuevos aislamientos y, por lo tanto, representa una importante herramienta para la búsqueda ambiental de nuevas levaduras productoras de astaxantina.

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

Background To study the relationship between intracellular anabolism and astaxanthin production, the influence of intracellular protein and fatty acids on astaxanthin production by four mutant Phaffia rhodozyma strains and their variations was investigated in this research. Results First, the content of astaxanthin in cells showed a reverse fluctuation in contrast to that of protein during the whole fermentation process. Moreover, compared with the three other strains, the astaxanthin-overproducing mutant strain of the yeast P. rhodozyma, called JMU-MVP14, had the highest specific productivity of astaxanthin as 6.8 mg/g, whereas its intracellular protein and fatty acid contents were the lowest. In addition, as a kind of sugar metabolic product, ethanol was only produced by P. rhodozyma JMU-VDL668 and JMU-7B12 during fermentation. Conclusions The results indicated that the accumulation of ethanol, intracellular protein, and fatty acids had competition effects on astaxanthin synthesis. This condition may explain why the P. rhodozyma strains JMU-VDL668 and JMU-7B12 achieved relatively lower astaxanthin production (1.7 and 1.2 mg/L) than the other two strains JMU-MVP14 and JMU-17W (20.4 and 3.9 mg/L).

Porcentagem de proteína em suplementos para ovinos mantidos em pasto de capim-aruana na época seca / Protein percentage in supplements for sheep grazing Aruana grass on dry season

Objetivou-se avaliar o consumo de forragem e o desempenho de ovinos mantidos em pastagem de capim-aruana, submetidos a porcentagens crescentes de proteína bruta (PB) no suplemento, na época seca. Vinte borregos da raça Santa Inês foram utilizados em delineamento inteiramente ao acaso, com cinco tratamentos e quatro repetições. Os suplementos foram fornecidos em 1,0% do peso corporal, nas porcentagens de 0, 15, 20, 25 e 30%. O aumento de proteína bruta influenciou o consumo total de matéria seca (kg/dia) e a porcentagem do peso vivo, com valores máximos estimados de 1.296g (3,2% de MS) com 21,48 e 21,89% de PB no suplemento, respectivamente. O consumo de forragem máximo, estimado de 893g/dia, ocorreu com a PB de 21,5%. O aumento de PB nos suplementos resultou em efeito quadrático sobre o ganho médio diário, com valor máximo de 104g/dia com a PB de 23% no suplemento. Recomenda-se o uso de suplementos múltiplos com 21 a 23% de PB fornecidos na proporção de 1% do peso corporal (PC) para ovinos mantidos em pastos de capim-aruana na época seca...

The aim of this study was to evaluate the forage intake and grazing sheep performance keep on Aruana grass subjected to increasing crude protein (CP) levels in the supplement on dry season. Twenty Santa Ines male lambs were used, with initial body weight of 31.80kg by a completely randomized design with five treatments and four replications. The supplements were provided daily at 1% of body weight, with protein levels of 0, 15, 20, 25 and 30%. The increase of the crude protein levels promoted a squarely effect on dry matter intake (kg/day and % of BW), with maximum estimated values of 1296g and 3.2% of DM in CP levels of 21.48 and 21.89, respectively. The maximum forage intake estimated of 893g/day occurred in CP level de 21.51%. The increased of crude protein level in supplements increased squarely the average daily gain, with a maximum of 104g/day, for the 23% crude protein in the supplement. Thus, the use of the multiple supplements supplied in 1% of body weight with CP levels ranged 21 a 23% is indicated for sheep grazing Aruana grass on dry season...

The effect of astaxanthin on resistance of juvenile prawns Macrobrachium nipponense (Decapoda: Palaemonidae) to physical and chemical stress / Efecto de la astaxantina en la resistencia al estrés físico y químico de los langostinos jóvenes de Macrobrachium nipponense (Decapoda: Palaemonidae)

In recent years, the use of new scientific techniques has effectively improved aquaculture production processes. Astaxanthin has various properties in aquacultureand its antioxidant benefits have been closely related to stress resistance; besides, it is an essential factor for growth in many crustaceans and fish. The objective of this study was to evaluate the resistance of prawn (Macrobrachium nipponense) fed diets containing different amounts of astaxanthin (AX) to the shock and stress of differentphysicochemical environments. A 70-day trial was conducted to evaluate the effect of supplementation of a source of astaxanthin (Carophyll Pink, 10% astaxanthin, w/w, Hoffman-La Roche, Switzerland) at various levels in the diet of M. nipponense juveniles. Four dry diets were prepared: AX0 without astaxanthin, AX50 with 50mg/kg, AX100 with 100mg/kg, and AX150 with 150mg/kg astaxanthin. The feeding trial was conducted in a recirculation water system consisting of 12 fiberglass tanks (1 000L) used for holding prawns. Three replicate aquaria were initially stocked with 36org/m² per tank. During the trial, prawns were maintained on a 12:12-h light:dark photoperiod with an ordinary incandescent lamp, and the water quality parameters were maintained as follows: water temperature, 25-26°C; salinity, 1g/L; pH, 8.5-8.8; dissolved oxygen, 6.0-6.5mg/L; and ammonia-nitrogen, 0.05mg/L. Incorporation of AX, production output, and physiological condition were recorded after 10 weeks of feeding. At the end of the growing period, the prawns were exposed to thermal shock (0°C), ammonia (0.75mg/L), and reduced oxygen (0.5mg/L). The time to lethargyand the time to complete death of the prawns were recorded. The results showed that control prawns had the shortest time to lethargy and death compared with prawns subjected to the other treatments. The results of this study have shown that the amount of muscle tissue and gill carotenoids in prawn fed with an AX150 diet showed greater reduction than those exposed to other treatments. It is possible that higher levels of astaxanthin in the body under oxygen reduction stress can be beneficial forprawns. These results suggest that male prawns showed lethargy earlier than females, and the percentage of carotenoid reduction in muscle and gill tissues was higher inmales. Rev. Biol. Trop. 62 (4): 1331-1341. Epub 2014 December 01.

En años recientes, la utilización de nuevas técnicas científicas ha tenido un efecto importante en mejorar los procesos de producción en acuicultura. La astaxantina tiene varias propiedades en la acuicultura y sus propiedades antioxidantes se encuentran estrechamente relacionadas con la resistencia al estrés. La astaxantina en muchos crustáceos y peces es un factor esencial para el crecimiento. El objetivo de este estudio fue evaluar la resistencia del langostino (Macrobrachium nipponense) alimentado con dietas conteniendo diferentes cantidades de astaxantina (AX), bajo diferentes condiciones de estrés ambiental. Un ensayo de 70 días fue llevado a cabo para evaluar el efecto de la suplementación de fuentes de astaxantina (Carophyll Pink, 10 % astaxanthin) en varios niveles en la dieta de jóvenes de M. nipponense. Cuatro dietas fueron preparadas: AX0 sin astaxantina, AX50 con 50mg/kg, AX100 con 100mg/kg y AX150 con 150mg/kg de astaxantina. Los ensayos de alimentación fueron conducidos en un sistema de recirculación de agua consistente en 12 estanques de fibra de vidrio (1 000L). Tres replicas fueron sembradas con 36org/m2 por tanque. Durante el experimento los langostinos fueron mantenidos con un fotoperiodo de 12:12 luz:oscuridad con lámparas incandescentes y los parámetros de la calidad del agua fueron mantenidos a 25-26°C la temperatura, 1 g/L la salinidad, 8.5-8.8 el pH, 6.0-6.5 mg/L el oxígeno y 0.05mg/L el nitrógeno amoniacal. La incorporación de la astaxantina, producción y condiciones fisicoquímicas fueron registradas después de 10 semanas de alimentación. Al final del periodo de crecimiento, los langostinos fueron expuestos a un shock térmico (0°C), amonio (0.75mg/L) y reducción de oxígeno 0.5 mg/L. El tiempo de letargia y el tiempo de muerte fueron registrados. Se encontró que la dieta con la mayor concentración de astaxantina (150mg/kg) presentó el mayor tiempo de letargia y la mayor concentración en branquias y músculo en el langostino M. nipponense.

Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2, 4-epibrassinolide (EBR)

The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1־ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.

Estudio del desarrollo, calidad de planta y acumulación de pigmentos en lígulas de tagetes erecta l: expuestas a dos intensidades de luz / Study of development, quality of plant and accumulation of pigments in ligules of tagetes erecta l: exposed to two light intensities

The carotenoids are photosensitive pigments during photosynthesis. The objective of this work was to study the effect on development and accumulation of carotenoids in ligules of Tagetes erecta exposed under two different lighting ambient (with mesh and without mesh of 50 percent). The plant development was evaluated measuring the height of the plant, number of floral buds, the ligules diameter. In adition, the quantification and identification of carotenoids from ligules was done by HPLC. The results showed significant differences (p<0.05) in the height of the plant, number of floral buds and ligules diameter of T. erecta. The group grown without mesh received greater UV radiation and different temperature, that under a mesh. The first conditions lead to a reduction of the ligules diameter and total content of xanthophylls (lutein and zeaxanthin). The plastids ultrastructure in the cells of T. erecta developed with mesh showed the greatest amount of thylakoid membranes and more conspicuous starch granules.

Los carotenoides son pigmentos fotosensibles frente a un exceso de intensidad luminosa durante el proceso de fotosíntesis. El objetivo de este trabajo fue el estudio del efecto en el desarrollo de la planta y la acumulación de carotenoides por la exposición a dos diferentes intensidades lumínicas (con y sin malla de sombra al 50 por ciento). Se evaluó el desarrollo de T. erecta en cuanto a la altura de la planta, número de botones florales y el diámetro de las lígulas. Adicionalmente, en las lígulas se cuantificaron e identificaron los carotenoides por HPLC. Los resultados mostraron diferencias significativas (p<0.05) en cuanto al desarrollo de las plantas expuestas a mayor radiación UV y temperatura, presentaron reducción del diámetro de las lígulas y disminución en el contenido de Xantófilas totales ( luteína y zeaxantina) con respecto a las cultivadas con malla,. La ultraestructura de los plastidios mostró mayor cantidad de membranas tilacoidales y gránulos de almidón más conspicuos en las células de las plantas de T erecta desarrolladas con malla.

Sexual stages of the female portion in the scallop Nodipecten nodosus (Linné, 1758) and astaxanthin quantity in each stage / Estágios sexuais da porção feminina da gônada da vieira Nodipecten nodosus (Linné, 1758) e a quantidade de astaxantina em cada estágio

This work describes the gametogenic cycle of the scallop Nodipecten nodosus kept in a culture system. To this end, during one year, samples were taken from the broodstocks every 30 days to be submitted to macroscopic and microscopic analyses and to measure the amount of astaxanthin. To perform the microscopic evaluation, 5 μ slices from the median portion of the female part of the gonad were submitted to the pattern methodology for histological analyses with paraffin and HE coloration. The remaining portion of the female gonad was lyophilised to extract and quantify the levels of astaxanthin using HPLC. The microscopic analyses revealed four well defined stages for the reproductive cycle. Analyses of data taken throughout the year indicated preferential spawning periods from December to January and from July to September. The astaxanthin analyses showed higher amounts of this carotenoid during the advanced pre-spawning and the initial spawning periods than during gametogenesis, initial pre-spawning, advanced spawning, and the spent stages. According to these results, it was possible to establish a descriptive table of the sexual stages of the female portion of the gonad and the amount of astaxanthin in the sexual stage of the scallop Nodipecten nodosus.

Este trabalho descreve o ciclo gametogênico da vieira Nodipecten nodosus mantida em ambiente de cultivo. Para isto, durante um ano, amostras de indivíduos reprodutores foram coletadas a cada 30 dias e submetidas à avaliação macroscópica e microscópica e à quantificação de astaxantina. Para a avaliação microscópica, secções de 5 μ da porção mediana feminina da gônada foram submetidas à metodologia de análise histológica padrão em parafina e coloração HE. O restante da porção feminina da gônada foi liofilizado para extração e quantificação de astaxantina em HPLC. A avaliação microscópica permitiu a descrição de quatro estágios bem definidos para o ciclo reprodutivo. Na análise ao longo do ano, foram observados períodos preferenciais de desova em dezembro e janeiro e de julho a setembro. A análise da quantidade de astaxantina, mostrou, nos estádios de pré-desova avançada e de desova inicial, uma maior quantidade desse carotenoide em comparação aos estádios de gametogênese, pré-desova inicial, desova avançada e repouso. Em função desses resultados, foi possível estabelecer um quadro descritivo dos estágios sexuais da porção feminina da gônada e quantidade de astaxantina em cada estágio sexual da vieira Nodipecten nodosus.

Pigmentos maculares / Macular pigments

A luteína e a zeaxantina são pigmentos amarelos que se localizam na mácula. Devido à sua localização, diminuem e filtram a quantidade de luz principalmente azul que chega aos fotorreceptores, atuam como antioxidantes e podem melhorar a qualidade visual. Esta é uma revisão do seu mecanismo de incorporação, ação, possíveis aplicações e conhecimento científico a respeito.

Lutein and Zeaxanthin are yellow pigments located at the macula. Because of your location macular pigments decrease and filter the amount of blue light that reach photoreceptors, protect the outer retina from oxidative stress and may improve the vision quality. This is a review regarding incorporation mechanism, function and knowledge update.

Carotenoid extraction from the gonad of the scallop Nodipecten nodosus (Linnaeus, 1758) (Bivalvia: Pectinidae) / Extração de carotenóides da gônada da vieira Nodipecten nodosus (Bivalvia: Pectinidae)

In marine bivalve mollusks, unsaturated molecules called carotenoids are present in the natural diet and play an important role in different biological process, especially in reproduction. In order to gain more insights into these compounds in Nodipecten nodosus it was necessary to develop a suitable protocol for extraction of carotenoids from the gonads. Female gonads of cultured scallops (75 mm length) were lyophilized and macerated in liquid N2. To verify the effect of composition in organosolvents on the extracting solutions, two organic solvents were tested: acetone and hexane (Ac = O:Hex) at four ratios, 1:1, 1:3, 1:5, and 2:3, in four static extraction times: 0, 5, 10, and 15 minutes. Total carotenoids and astaxanthin contents were determined in the crude extracts by UV-visible spectrophotometry and high performance liquid chromatography (HPLC), respectively. Triplicate aliquots of 50 mg were used for each treatment. The results indicated that the best single extraction (0.312 ± 0.016 µg carotenoids/mg) was attained with Ac = O: Hex 1:3, for 15 minutes. Through exhaustive extraction methodology (10x), a superior yield (0.41 ± 0.001 µg carotenoids/mg) was obtained from a gonad sample in comparison to the highest value found for a single extraction. Astaxanthin content was reduced by 8.6 percent in carotenoid extract preservation assay, i.e., -18 °C, 26 days incubation, under N2 atmosphere.

Em moluscos bivalves marinhos, carotenóides insaturados estão presentes na dieta natural, com um importante papel em diversos processos biológicos, em especial na reprodução. A elucidação dos efeitos destes compostos em Nodipecten nodosus requer o desenvolvimento de um protocolo adequado para a extração de carotenóides das gônadas desses animais. Para isso, gônadas de vieiras cultivadas (75 mm de comprimento) foram liofilizadas e maceradas em N2 líquido. Amostras em triplicata com 50 mg foram coletadas para a utilização em cada tratamento. Os conteúdos de carotenóides totais e astaxantina foram determinados via espectrofotometria de luz UV-visível e cromatografia líquida de alta eficiência (CLAE), respectivamente. O efeito da composição em organosolventes das soluções de extração foi testado utilizando-se acetona (Ac = O) e hexano (Hex) em quatro proporções (Ac = O:Hex): 1:1, 1:3, 1:5, e 2:3; em quatro tempos de extração: 0, 5, 10, e 15 minutos. Os resultados mostraram que o melhor rendimento de extração (0,312 ± 0,016 µg carotenóides/mg) foi obtido com Ac = O:Hex, 1:3, por 15 minutos. Com a utilização de protocolo de extração exaustiva (10x), uma quantidade superior (0,41 ± 0,001 µg de carotenóides/mg) foi obtida de amostras de gônada, comparativamente aos valores obtidos em extrações únicas. O conteúdo de astaxantina foi reduzido em 8,6 por cento em testes de preservação deste metabólito em extratos crus (-18 °C, 26 dias de incubação em atmosfera de N2).

Crecimiento y producción de metabolitos de la cianobacteria marina Synechococcus sp. (Chroococcales) en función de la irradiancia

Growth and metabolite production of the marine cyanobacterium Synechococcus sp. (Chroococcales) in function to irradiance. Changes in salinity, temperature and irradiance during wet and dry seasons have induced metabolic versatility in cyanobacteria from saline environments. Cyanobacteria from these environments have biotechnological potential for the production of metabolites with pharmaceutical and industrial interest. We studied the growth, dry mass and metabolite production of the cyanobacterium Synechococcus sp. MOF-03 in function of irradiance (78, 156 and 234 µmol q m-2 s-1). All batch cultures were maintained by triplicate in constant aeration, 12:12 h photoperiod, 30 ±2ºC and 35‰. Maximum values of protein, carbohydrates and lipids, of 530.19 ±11.16, 408.94 ±4.27 and 56.20 ±1.17 µg ml-1, respectively, were achieved at 78 µmol q m-2 s-1. Pigments, analyzed by HPLC, showed maximum values at 78 µmol q m-2 s-1 for chlorophyll a with 7.72 ±0.16 µg ml-1, and at 234 µmol q m-2 s-1 for ß-carotene and zeaxanthin with 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. Chlorophyll a:ß-carotene ratio decreased from 17.15 to 6.91 at 78 and 234 µmol q m-2 s-1; whereas ß-carotene:zeaxanthin ratio showed no changes between 78 and 156 µmol q m-2 s-1, around 1.21, and decreased at 234 µmol q m-2 s-1, to 1.04. Also, this cyanobacterium produced the greatest cell density and dry mass at 156 µmol q m-2 s-1, with 406.13 ±21.74 x106 cell ml-1 and 1.49 ±0.11 mg ml-1, respectively. Exopolysaccharide production was stable between 156 y 234 µmol q m-2 s-1, around 110 µg ml-1. This Synechococcus strain shows a great potential for the production of enriched biomass with high commercial value metabolites. Rev. Biol. Trop. 56 (2): 421-429. Epub 2008 June 30.

Las cianobacterias de ambientes salinos presentan una versatilidad metabólica inducida por los cambios de salinidad, temperatura e irradiancia, durante los períodos de sequía y lluvias. Por ello es importante la búsqueda en estos ambientes de cianobacterias con potencial biotecnológico para la producción de metabolitos de interés farmacéutico e industrial. Se reporta el crecimiento, masa seca y producción de metabolitos de la cianobacteria Synechococcus sp. MOF-03 en función de la irradiancia (78, 156 y 234 µmol q m-2 s-1). Los cultivos discontinuos por triplicado, fueron mantenidos con aireación constante, fotoperiodo 12:12 h, 30 ±2ºC y a 35‰. Los máximos valores de proteínas, carbohidratos y lípidos de 530.19 ±11.16, 408.94 ±4.27 y 56.20 ±1.17 µg ml-1 respectivamente, fueron obtenidos a 78 µmol q m-2 s-1. Los pigmentos, analizados por HPLC, mostraron los máximos a 78 µmol q m-2 s-1 para clorofila a con 7.72 ±0.16 µg ml-1; y a 234 µmol q m-2 s-1 para ß-caroteno y zeaxantina con 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. La relación clorofila a:ß-caroteno disminuyó de 17.15 hasta 6.91 a 78 y 234 µmol q m-2 s-1; mientras que la relación ß-caroteno:zeaxantina se mantuvo sin cambios entre 78 y 156 µmol q m-2 s-1, con cerca de 1.21 y disminuyó a 234 µmol q m-2 s-1 a 1.04. La cianobacteria produjo la mayor densidad celular y masa seca a 156 µmol q m-2 s-1, con 406.13 ±21.74 x106 cel ml-1 y 1.49 ±0.11 mg ml-1 respectivamente. La producción de exopolisacáridos se mantuvo alrededor de 110 µg ml-1 entre 156 y 234 µmol q m-2 s-1. Así, esta cepa de Synechococcus muestra un gran potencial para la producción de biomasa enriquecida con metabolitos de alto valor comercial.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

Expression of the carotenoid biosynthesis genes in Xanthophyllomyces dendrorhous

In the yeast Xanthophyllomyces dendrorhous the genes idi, crtE, crtYB, crtl and ast are involved in the biosynthesis of astaxanthin from isopentenyl pyrophosphate. The carotenoid production and the kinetics of mRNA expression of structural genes controlling the carotenogenesis in a wild-type ATCC 24230 and in carotenoid overproducer deregulated atxS2 strains were studied. The biosynthesis of carotenoid was induced at the late exponential growth phase in both strains. However, the cellular carotenoid concentration was four times higher in atxS2 than in the wild-type strain in the exponential growth phase, suggesting that carotenogenesis was deregulated in atxS2 at the beginning of growth. In addition, the maximum expression of the carotenogenesis genes at the mRNA level was observed during the induction period of carotenoid biosynthesis in the wild-type strain. The mRNA level of the crtYB, crtl, ast genes and to a lesser extent the idi gene, decayed at the end of the exponential growth phase. The mRNA levels of the crtE gene remained high along the whole growth curve of the yeast. In the atxS2 strain the mRNA levels of crtE gene were about two times higher than the wild-type strain in the early phase of the growth cycle.

The use of pigment "fingerprints" in the study of harmful algal blooms

Along the Mexican coast, harmful algae blooms (HAB) have become more frequent, and therefore, there is an urgent need to establish monitoring programs to avoid the undesired consequences of HAB in human and natural ecosystems. In this work, we analyzed the pigment signatures and the species composition from phytoplankton samples to evaluate the utility of the specific pigment "fingerprints" in HAB monitoring programs. Vertical profiles from a coastal lagoon and temporal samples of a red tide occurring in a shrimp-culture pond and in a coastal zone were taken into consideration. Between 76% and 84% of dinoflagellate and diatom cell density was explained by their specific signature variation, in both vertical and temporal samples. Only the variation of zeaxanthin and the cyanobacteria Anabaena sp. showed a poor relationship, probably from difficulties in counting other cyanobacteria present in the samples examined with the microscopic method. These results suggest that inclusion of pigment analysis in the study and monitoring programs dealing with harmful algae would be very useful

A lo largo de las costas mexicanas, los florecimientos algales nocivos (FAN) se han vuelto cada vez mas frecuentes y por lo tanto, existe una necesidad urgente de establecer programas de monitoreo para evitar las consecuencias no deseadas por su desarrollo, sobre los ecosistemas naturales y el ser humano. En este trabajo, nosotros analizamos las huellas pigmentarias y la composición de especies de diversas muestras de fitoplancton para evaluar la utilidad que pueden representar estos pigmentos específicos o "huellas pigmentarias" en programas de monitoreo de florecimientos algales nocivos. Los perfiles verticales de muestras de fitoplancton de una laguna costera y muestras de mareas rojas que ocurrieron en un estanque de cultivo de camarón y en una laguna costera, fueron considerados en este estudio. Tanto en muestras verticales como en temporales, entre el 76% y 84% de la densidad celular de dinoflagelados y diatomeas fueron explicados por la variación de su huella específica, mientras que la variación de zeaxantina y la densidad de la cianobacteria Anabaena sp. mostró una relación pobre, la cual fue debida probablemente a la dificultad en el conteo que presenta este grupo al ser analizadas mediante un microscópico invertido. Estos resultados sugieren que la inclusión del análisis de las huellas pigmentarias en los programas del estudio y monitoreo de las algas nocivas sería de gran utilidad