DETERMINATION OF FREE AMINO ACIDS FROM MICROALGAL BIOMASS

Transcription

1 DETERMINATION OF FREE AMINO ACIDS FROM MICROALGAL BIOMASS Fagundes, M. B. 1, Vendruscolo, R. G. 1, Maroneze, M. M. 1, Jacob-Lopes, E. 1, Wagner, R. 1, Zepka, L. Q. 1 1 Department of Technology and Food Science, Federal University of Santa Maria- RS, Brazil ( rogerwag@gmail.com ).

2 DETERMINATION OF FREE AMINO ACIDS FROM MICROALGAL BIOMASS Fagundes, M. B. 1, Vendruscolo, R. G. 1, Maroneze, M. M. 1, Jacob-Lopes, E. 1, Wagner, R. 1, Zepka, L. Q. 1 1 Department of Technology and Food Science, Federal University of Santa Maria- RS, Brazil ( rogerwag@gmail.com ). Abstract The microalgal biomass can be exploited in several ways and they are considered a source of high-value products. In this work the free amino acids were identified present on two distinct intra-cell microalgal biomass Chlorella vulgaris and Phormidium autumnale. The biomasses obtained from photo bioreactors were subjected to Bligh & Dyer extraction, later was done a derivatization and free amino acids derivates were analyzed by GC-FID and were positively identified by spectra comparison with their standards by GC/MS. The amino acids profile presented as 38.1% of essential amino acids (EAA) and non-essential amino acids (NEAA) 61.9% for P. autumnale and for the microalgae C. vulgaris was found 12.8% EAA and 87.2% of NEAA. These values demonstrate the high-value biomass that can be produced and proving their nutritional importance. Keywords: microalgae, protein, intracellular compounds. INTRODUCTION Microalgae are unicellular microorganisms, prokaryotes or eukaryotes. The organelles are the major difference between prokaryotes and eukaryotes. Prokaryotes do not possess chloroplast, mitochondria and nucleus but they contain chlorophyll and high protein contents (RASHID, 2014). The microalgae are the basis of food chain, citric components in many habitats and are the major oxygen producing. Additionally, they present a commercial importance in the food industry, pharmaceutical, aquaculture and energy as a natural fount of high-value products such as, lipids, fatty acids and pigments (WILLIAMS & LAURENS, 2010). However, among these bioproducts produced by microalgal metabolic pathways are present several polar compounds, for example the free amino acids. Amino acids (AA) are defined as organic substances containing both amino and acid groups (LI, et al., 2011), except for glycine, all AA have an asymmetric carbon and exhibit optical activity and the absolute configuration of AA (L- or D-isomers) is defined with reference to glyceraldehydes. Except for proline, all protein AA have a primary amino group and a carboxyl group linked to the α-carbon atom (hence a-aa). Because of variations in their side chains, AA have remarkably different biochemical properties and functions (SUENAGA, TOMONAGA, YAMANE, 2008).Adequate provision of amino acids is essential for the health, growth, development and survival of animals and humans (WU, et al., 2013). Based on growth or nitrogen balance, AA have traditionally been classified as nutritionally essential or non-essential (LE PLE NIER, et al., 2012). Therefore, the aim of this study was to identify the free amino acids (FAA) present in the biomasses of the microalgae Chlorella vulgaris and Phormidium autumnale. MATERIALS AND METHODS

3 Sample The experiments for biomass production were set up in a bubble column bioreactor (height/ diameter (h/d) ratio equal to 10 and a nominal working volume of 2 L), operating at 25 C, a ph adjusted at 7.6, photon flux density of 15 µmol m -2 s -1, constant aeration of 1 VVM (volume of air per volume of culture per minute) and an inoculum of 100 mg L - ¹ of C. vulgaris and P. autumnale in the exponential growth phase. Sample preparation In order to obtain the FAA, approximately 150 mg of dried biomass were weighted and transferred to 15 ml Falcon tube, and were added 3 ml of methanol, 1.5 ml of chloroform and 1.2 ml of mili-q water according to Bligh & Dyer (1959). The cell rupture was assisted by an ultrasound microprobe for 30 min (Sonifier 250, Branson USA) using 20 khz of frequency, 150 W of output and 70% of amplitude in the pulsed mode. After that was add 1.5 ml of Mili-Q water. The polar phase containing FAA were partitioned in methanolic/aqueous solution. 150 µl of this fraction were transferred, dried and heated 60 C in a block with the assistance of hydro- vacuum pump for solvent evaporation. FAA derivatization procedure The FAA derivatization was achieved according with OMS-OLILU, et al. (2011). The residue was re-dissolved in 50 μl of 20 mg ml - ¹ methoxyamine hydrochloride in pyridine and derivatized at 40 C for 90 min followed by a 30 min treatment with 50 μl MTBSTFA (N-methyl-N(tert-butyldimethylsilyl) trifluoroacetamine) at 40 C. FAA determination The FAA derivatives were analyzed in a gas chromatography equipped with a flame ionization detector (GC-FID) Varian star 3400 (CA, USA). 1 μl of samples were introduced into injection port operating in splitless mode (splitter port off for 1 min; 30:1) at 320 C. The separation was performed in a RTX-5MS (Restek Corporation, Bellefonte, PA, USA) (30 m 0.25 mm id 0.25 μm). The programming temperature of the oven column was initially 100 C for 2 min, then increased to 180 C with a rate of 6 C min - ¹, than increased for 200 C for 1 min and was up to 320 C with a rate 15 C min - ¹, maintaining isothermal for 5 min. Hydrogen was the carrier gas used at constant pressure of 15 psi. The temperature detector was maintained at 280 C. The identification was held in a gas chromatography coupled to a mass spectrometer (GC/MS) Shimadzu, QP-2010 Plus (Tokyo, Japan) at same chromatographic conditions described for GC/FID. GC/MS interface and ion source were held at 280 C. The results were expressed in percentages of the total chromatographic area from GC-FID and the compounds were positively identified by the comparison spectra of standards and experimental data acquired by GC/MS. RESULTS AND DISCUSSION The free amino acids profiles present in two distinct microalgae biomass are shown on Table 1. Intra-cell microalgae content were identified 13 amino acids in the P. autumnale biomass from total FAA about 38.1% were identified as essential amino acids and 61.9% are shown as non-essential amino acids. For C. vulgaris were

4 identified 12 FAA. The profile of EAA and NEAA were 12.8% and 87.2%, respectively. It was detected the alanine in higher abundance of 26.4% for P. autumnale and 35.3% for C. vulgaris. The pathways for alanine synthesis has nutritional importance, physiological significance (WU, 2010) and cellular osmoregulation (YOKOYAMA, et al., 2005). On the other hand, asparagine was found only in C. vulgaris biomass with 19.3%. This amino acid is related with the cell metabolism and physiology (WU, 2009). The amino acids valine and leucine were also found in distinct values 10.2% and 10.9% for P. autumnale and 3.4 and 3.0% for C. vulgaris. Table 1. Free amino acids profile from two different microalgal species Amino acids P. autumnale (%) C. vulgaris (%) Alanine Glycine Valine Leucine Isoleucine Proline Methionine Serine Threonine Phenylalanine Aspartic acid Cysteine Glutamic acid Asparagine Lysine Total EAA Total NEAA The differences found in the profile and also in the FAA percentages can be attributed, initially, to the stage that the cells were found in the moment of the analysis, since the microalgae are biologic systems with high synthesis power (WIJFELS, KRUSE & HELLINGWERF, 2013). Furthermore, they have different cell structures, P. autumnale is a prokaryote organism, whereas C. vulgaris is eukaryote. All the unicellular microorganisms show amino acids detection mechanisms, in order to absorb them from the extra-cell environment or synthetize new ones when those amino acids are missing, due to nutrient drastic changes that can occur in the place they live. However, the prokaryotes have specific proteins to detect those compounds, which allow them to have an increased nutrient availability (EFEYAN, ZONCU & SABATINI, 2012). In relation to the nutritional classification for the profile, the values found in EAA were lower than for NEAA in both species of microalgae. However, it was tactically assumed, that animals or humans could synthesize sufficient amounts of all NEAA and did not need them in diets for optimal nutrition or health. However, growing evidence from cell culture and animal studies shows that some of the traditionally classified NEAA play important roles in multiple signaling pathways, thereby

5 regulating gene expression, intracellular protein turnover, nutrient metabolism, and oxidative defense (WU, 2010). CONCLUSION The free amino acids profile from the different species of microalgal biomass showed the abundance and variability of essential and non-essential for this reason can be comproved their nutritional importance. ACKNOWLEDGEMENT: CAPES, CNPq, FAPERGS. BIBLIOGRAPHIC REFERENCES BLIGH, E. G.; DYER, W. J A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys., 37: 7. EFEYAN, A., ZONCU, R., SABATINI, D. M Amino acids and mtorc1: from lysosomes to disease. Trends Mol. Med., 18: LE PLE NIER, S. et al Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway Amino Acids, 43: LI, G. et al Development of a pair of differential H/D isotope-coded derivatization reagents d0/d3-4-(1-methyl-1h-phenanthro[9,10-d]imidazol-2-yl) phenlamine and its application for determination of aldehydes in selected aquatic products by liquid chromatography tandem mass spectrometry. Biomed Chromatogr., 6: OMS-OLILU, G.; HERTOG, M. L. A. T.; VAN de POEL, B.; AMPOFO-ASIAMA, J.; GEERAERD, A. H.; NICOLAI, B. M Metabolic characterization of tomato fruit during preharvest development, ripening and postharvest shelf-life. Postharvest Biol. Tec., 62: RASHID, N. et al Current status, issues and developments in microalgae derived biodiesel production. Renew. Sust. Energ. Rev., 40: SUENAGA, R. et al Intracerebroventricular injection of L-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks. Amino Acids, 35: WIJFFELS, R. H.; KRUSE, O.; HELLINGWERF, K. J Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae. Curr. Opin. Biotechnol., 24: WILLIAMS, P. J. L. B.; LAURENS, L. M. L Microalgae as biodiesel & biomass feedstocks: review and analysis of the biochemistry, energetics and economics. Energy Environ. Sci., 3: WU, G Amino acids: metabolism, functions, and nutrition. Amino acids, 37: WU, G Functional amino acids in growth, reproduction and healthe. Adv Nutr., 1: WU, G. et al Dietary requirements of nutritionally non-essential amino acids by animals and humans. Amino Acids, 44: YOKOYAMA, et al Alanine racemase activity in the microalga Thalassiosira sp. Fisheries Sci., 71: