Microalgal Extracts as Functional Food Supplements: Experimental Approaches for the Evaluation of Antioxidant Activity

Background of the study

Clinical trials and epidemiological studies habe established an inverse correlation between the intake of fruits and vegetables and the occurrence of diseases such as inflammation, cardiovascular disease, cancer, and agingrelated disorders [Willet, 2001]. One hypothesis that has been advanced is that the protection provided by fruits, vegetables, and drinks such as tea or red wine, can be attributed to a large class of antioxidant phytochemicals contained in these foods and drinks [Prior, 1999]. Beside fruits and vegetables also microalgae such as Chlorella or Spirulina are a known source of antioxidants [Wu, 2005]. Their selective cultivation under highly standardized conditions in photobioreactors represents a major technological advantage. Biochemical antioxidant activity assays: The ABTS assay was conducted according to Reet al., 1999 and Wu et al., 2005 with some minor modifications. The DPPH assay was conducted according to Milleret al., 2001 and Wu et al., 2005 with some minor modifications. The ORAC assay was conducted according to Prior et al., 2003 with some minor modifications. All tests were performed in 96-well plates. Voltammetry (VM): Voltammetry has first been introduced in 2001 by Buratti and coworkers as a novel electrochemical method to evaluate the antioxidant activity of lipophilic compounds in crude vegetable extracts. Up to now, this method has not been widely used for this application. Nevertheless, unlike nearly all other methods for measuring antioxidant activity, it does not employ a spectrometric endpoint and, therefore, works well with turbid and coloured samples. In the study presented here, a Computrace 797 VA instrument from Metrohm was used according to the manufacturers instructions with a glassy carbon working electrode, a Pt counter electrode and a Ag/AgCl reference electrode. Voltage range was set to 0.3 1.2 Volts in order to exclude the influence of metals present in the microalgal extracts. Data Analysis: An Area Under Curve approach was used in all four assays. Results obtained with different microalgae species or culture conditions respectively were ranked according to their corresponding Area Under Curve value. Finally, rankings were plotted as spider charts on axes starting from the same origin. Fig. 4: Voltammograms of extracts (ethanol/toluol/H2SO4) from 1: Haematococcus pluvialis, 2: Synechocystis sp., 3: Anabaena sp., 4: Spirulina sp., 5: Nannochloropsis salina, 6: Porphyridium purpureum.

Antioxidants
A biologically relevant definition of antioxidants is synthetic or natural substances added to products to prevent or delay their deterioration by action of oxygen in air. In biochemistry and medicine, antioxidants are organic substances, , that are capable of counteracting the damaging effects of oxidation in animal tissues [Huang, 2005]. Physically, they can be classified by their solubility into (i) hydrophilic antioxidants such as vitamin C and the majority of polyphenolic compounds and (ii) lipophilic antioxidants, mainly including vitamin E and carotenoids. Both types of antioxidants play an important role in a wide spectrum of biochemical and physiological processes. Of primary interest is their optimal antioxidant activity in vitro and in vivo. Lipophilic antioxidants penetrate the lipoprotein cell membrane more easily and therefore can reach a high level of bioavailability. In contrast, hydrophilic antioxidants do not accumulate in the body but are excreted rapidly [Huang, 2002]. However, they are important as functional additives in beverage industry.

Results

Porphyridium purpureum:

Spirulina sp.:

Synechocystis sp.:

Antioxidant activity assays

There are a number of established methods available for measuring antioxidant activity in vitro, such as the ABTS, ORAC, and DPPH assay [Huang, 2005]. However, it is not a trivial task to accurately measure antioxidant activity since crude extracts of microalgae generally present a complex composition of different compounds acting as antioxidants. Depending on the reactions involved, antioxidant activity assays can roughly be classified into two different types: assays based on hydrogen atom transfer (HAT) and those based on electron transfer (ET). Moreover, some assays are conducted in aqueous systems, others in organic solvents. In view of this, the application of just a single antioxidant activity assay using one chemical reaction and solvent seems to be rather unrealistic and may give rise to misleading results.

% antioxidant activity

extract concentration [g/L]

Fig. 1: Antioxidant activity in the DPPH assay (mechanistically ET based) of methanolic extracts from Anabaena sp., Isochrysis galbana, Nannochloropsis salina, Phaeodactylum tricornutum, Porphyridium purpureum, Spirulina sp. and Synechocystis sp.

Fig. 5: Antioxidant activity of extracts from 3 different microalgae species cultivated in full medium (F medium [Guillard and Ryther, 1962]) obtained with the ABTS, DPPH, ORAC assay and the voltammetric approach (VM). Synechocystis (right) and Spirulina both displayed a significantly higher antioxidant activity in all assays as compared to Porphyridium (left).

Objectives of this study

to establish a battery of (bio-)chemical antioxidant assays covering different reaction mechanisms and being applicable to extracts in water as well as organic solvents to establish an integrative data analysis scheme summarizing the results of the different assays to test the feasibility of this approach by employing it for: i. the selection of microalgae species with superior antioxidant activity ii. testing culture conditions favoring high antioxidant activity

% antioxidant activity

extract concentration [g/L]

Fig. 6: Antioxidant activity of extracts from Nannochloropsis salina cultivated in F medium (left) and F medium containing only 1/4 of regular phosphate (mid) and F medium containing only 1/8 of regular nitrate concentration (right) respectively. Obviously, a reduction in nitrate or phospate leads to a drop of overall antioxidant activity in Nannochloropsis.

Materials & Methods

Microalgae extracts: Dried algal powder was resuspended in different solvents depending on the assay and homogenized with an T-25 UltraTurrax on ice for 30 sec. Crude extracts were centrifuged for 10 min at 4000 g. The supernatants were filtered (0,2 m PTFE filter), stored on ice and used for testing on the same day.

Fig. 2: Antioxidant activity in the ABTS assay (HAT based) of aqueous extracts from Isochrysis galbana, Nannochloropsis salina, Phaeodactylum tricornutum, Porphyridium purpureum, Spirulina sp. and Synechocystis sp.

Conclusions
applying a battery of antioxidant assays covering different reaction mechanisms and solvents seems to be a more reliable approach individual results can clearly be summarized in spider diagrams for comparative evaluations of different algae species or culture conditions compared to the other assays investigated, the ORAC assay showed a rather narrow dynamic range in its response to samples differing in antioxidant activity. However, this might be overcome by further improvements. the voltammetric approach seems to be rather promising, since it gave results similar to the standard assays without any interference with turbid or coloured algal extracts.

Authors: Marion Justen, Michael Schrder, Dieter Pollet, University of Applied Sciences, Darmstadt; Maren Hoffmann, Research and Technology Centre, Kiel University, Bsum; Kai Marxen, Sebastian Lippemeier, BlueBioTech GmbH, Bsum

Contact: Prof. Dr. D. Pollet University of Applied Sciences Darmstadt Department of Chemical Engineering and Biotechnology Schnittspahnstrasse 12 D-64287 Darmstadt Germany Phone +49 (0)6151 168226 Fax +49 (0)6151 168404 Email: pollet@h-da.de

area under curve

extract concentration [g/L]

Fig. 3: Antioxidant activity in the ORAC assay (HAT based) of extracts (acetone/water, 1+1) from Anabaena sp., Isochrysis galbana, Nannochloropsis salina, Phaeodactylum tricornutum, Porphyridium purpureum, Spirulina sp. and Synechocystis sp.