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.

HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

An improved and fast UHPLC-PDA methodology for determination of L-ascorbic and dehydroascorbic acids in fruits and vegetables: evaluation of degradation rate during storage

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultrahigh-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm×2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intraday precision. Validation experiments revealed very good recovery rate of 96.6±4.4% for L-AA and 103.1±4.8 % for total vitamin C, good linearity with r2-values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C18 column (150 mm × 3.9 mm, 4 μm) at 35 °C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.