The aliquots were centrifuged at 4000 × g, and the supernatants w

The aliquots were centrifuged at 4000 × g, and the supernatants were subsequently discarded. Each cell pellet was suspended in 2 ml of an acetone:water mixture (1:1), and 500 μl of 0.5-mm glass beads were then added. After 10 min of vortex shaking,

the mixture was centrifuged at 4000 × g for 3 min. Next, the supernatant was transferred to a clean test tube, and 2 ml of acetone was added learn more to the pellet. The tube containing the pellet was then vortex stirred for 3 min and centrifuged at 4000 × g for 3 min, after which the supernatant was collected and mixed with the supernatant that had been previously set aside. These steps were repeated until the recovered supernatant was completely colorless. The collected supernatants were then treated with 0.25 volumes of water and 0.25 volumes of petroleum ether; this mixture was mixed and centrifuged for 3 min at 4000 × g. Subsequently, the petroleum ether (top) phase was recovered, and its absorbance at 465 nm was determined. The pigment concentrations were quantified using the average of the molar extinction coefficients of astaxanthin and β-carotene (2346 cm-1/M). The pigment composition was determined by RP-HPLC using a LiChrospher RP18 125-4 (Merck) column and an acetonitrile:methanol:isopropanol (85:10:5) mobile phase with a 1 ml/min flow BKM120 concentration rate under isocratic conditions.

Each pigment was identified by comparison with specific standards (Sigma) based on their retention time and absorption Montelukast Sodium spectra [40] using a Shimadzu SPD-M10A diode array detector. Quantification of glucose in the extracellular medium The glucose present in the extracellular medium was quantified by determining the increase in absorbance at 340 nm due to the production of NADPH as a product of the oxidation of the glucose present, using the D-Glucose/D-fructose kit (Megazyme). Acknowledgements This work was supported by Fondecyt 1100324, Deutscher Akademischer Austanschdienst (DAAD) through a graduate scholarship to AW, Fundación María Ghilardi Venegas through graduate scholarships to CL and AM and MECESUP UCH0106 through graduate scholarships

to MN and JA. References 1. Baker RTM, Pfeiffer AM, Schöner F-J, Smith-Lemmon L: Pigmenting efficacy of astaxanthin and canthaxanthin in fresh-water reared Atlantic salmon, Salmo salar . Animal Feed Science and Technology 2002, 99:97–106.CrossRef 2. Bjerkeng B, Peisker M, Von Schwartzenberg K, Ytrestøyl T, Åsgård T: Digestibility and muscle retention of astaxanthin in Atlantic salmon, Salmo salar , fed diets with the red yeast Phaffia rhodozyma in comparison with synthetic formulated astaxanthin. Aquaculture 2007, 269:476–489.CrossRef 3. Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H: Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006, 69:443–449.PubMedCrossRef 4. Schroeder WA, Johnson EA: Antioxidant role of carotenoids in Phaffia rhodozyma . J Gen Microbiol 1993, 139:907–912. 5.

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