Bowen, J .

Morais, R.

Pinto, M.  M . Mendes

Raposo, M.  .F de J.

Young, A .

English

Repositório Institucional da Universidade Católica Portuguesa

Evaluation of different disruption processes efficiency on encysted Haematococcus pluvialis cells for astaxanthin recovery and bioavailabilityM M Mendes-Pinto1, M F de J-Raposo1, J Bowen2, A Young2 and RMorais11- Escola Superior de Biotecnologia - Universidade Católica PortuguesaRua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal2- John Moores University, Liverpool L3 3AF, UKMATERIAL AND METHODSMicroalgae: Haematococcus pluvialis (34/7 strain of CCAP - Culture Collection of Algae andProtozoa, Cumbria - UK) was grown and carotenised in Bold Basal Medium mod., and the biomass was firstobtained by sedimentation of the culture.Physical and chemical treatments: After sedimentation and further centrifugation, thecarotenised biomass was submitted to the following processes:  autoclave 30 min, 121 C, 1 atm;  HCl 0.1M, 15 min and 30 min;  NaOH 0.1 M, 15 min and 30 min;  enzymatic treatment with a mixture of 0.1%protease K and 0.5 % driselase in a phosphate buffer, pH 5.8, 30 C, during one hour;  spray drying, inlet180 C, outlet 115 C; and mechanical disruption, with a cell homogeniser developed for this purpose.After the treatments, all samples (30 mg each, in triplicate) including the control, were submitted to theastaxanthin extraction procedure.Astaxanthin extraction: The astaxanthin extraction was performed using a solvent mixture ofethanol:diethylether:n-hexane, 1:1:1 (v/v/v), all laboratory grade (Merck).After the addition of some glass beads to the test tubes, triplicates of 30 mg samples were submitted to acombination of vigorous vortex for 1 min and ultra-sounds for 15 min. After this, all samples were centrifugedfor 5 min at 5000 rpm and evaporated under a steady stream of nitrogen.Comparative bioavailability into acetone: Simple extraction of astaxanthin, fromH. pluvialis cells previously centrifuged, into 5ml acetone, was carried out overnight (16 hours) at roomtemperature, for all the treatments, including the control.Spectrophotometry: The spectra were recorded in a double beam UV-Vis spectrophotometer(Shimadzu 1601). For quantification of astaxanthin, the extracts originated from the solvent mixture extractionwere redissolved in 5ml n-hexane (extinction coefficient 2100, Abs max 470nm). Those obtained from theextraction of astaxanthin into acetone, were analysed directly in aceton (extinction coefficient 2100, Abs max476nm).Thin-layer chromatography: Precoated silica gel H60 plates 20x20 cm (Merck,Darmstadt, FRG) were used for the separation of carotenoids. Solvent system: acetone:n-hexane, 3:7 (v/v)(Kobayashi et al., 1991), and a pre-run in 2.5% (w/v) solution of citric acid in methanol (Andrew J. Young,1999). After the pre-run, the TLC plates were dried under warm air, at 40ºC, during 10 min.The qualitative analysis of TLC plates was performed using an imaging densitometer (Model GS-700, BIO-RAD).HPLC: Samples were analysed by normal phase HPLC (Hewlett Packard) using a UV detector (450.8,470.8 nm) and a Lichrosorb column (12.5 cm x 4.6 cm), 5 m, pre-coated with 1% H3PO4 in methanol (v/v)before used. Mobile phase n-hexane:acetone, 1:1 (v/v).SEM: Haematococcus biomass, submitted to the different treatments, was analysed by Scanning Electron Microscopy  (Hitachi, Ltd), after being lyophilised and coated with a thin gold-palladium film (10-15nm).INTRODUCTIONHaematococcus pluvialis is a unicellular microalga, and one of the most important natural sources ofastaxanthin (Sommer et al., 1991), which is produced when cultured in nutrient deficient media and / or underhigh light intensities or high temperatures (Fan et al., 1994; Droop, 1954).Astaxanthin (3,3’-dihydroxy- , ’-carotene-4,4’-dione) is a reddish pigment with antioxidant activity, being animportant component in the aquaculture industry (Choubert & Heinrich, 1993). Haematococcus canaccumulate up to 1% of dry weight astaxanthin, mainly in the form of the 3S,3’S enantiometer, mono and di-esters (Grung et al., 1990)However, due to the very hard cellulosic cell wall, astaxanthin extraction and its bioavailability in intactencysted cells is low.The purpose of this work was to evaluate different physical and chemical processes which allow the disruptionof H. pluvialis encysted cells, in terms of astaxanthin recovery and the facility of its extraction from the cells.The astaxanthin quantification, employing spectrophotometry and chromatographic techniques (TLC andHPLC), was used as an indirect method to evaluate the efficiency of each process.The direct effect of those processes on the cell wall disruption was analysed by scanning electron microscopy(SEM).REFERENCES Young A J (1999). Personal communication Choubert G and Heinrich O (1993). Carotenoid pigments of the green alga Haematococcus pluvialis: assay on rainbow trout, Oncorhynchusmykiss, pigmentation in comparison with synthetic astaxanthin and canthaxanthin. Aquaculture 112: 217-226 Droop M R (1954). Conditions governing haematochrome formation and loss in the alga Haematococcus pluvialis. Arch. Mikrobiol. 20: 391-397 Fan L, Vonshak A, Boussiba S (1994). Effect of temperature and irradiance on growth of Haematococcus pluvialis (Chlorophyceae). Journal ofPhycology 30(5): 829-833 Grung M, Bjerkeng B, Borowitzka M A, Skulberg O, Liaaen-Jensen S (1990). Alternative sources of astaxanthin, including secondarycarotenoids of microalgae. Proc. 9th Internat. Symp. Carotenoids, Kyoto Kobayashi M, Kakizono T, Nagai S (1991). Astaxanthin production by a green alga, Haematococcus pluvialis accompanied with morphologicalchanges in acetate media. Journal of Fermentation and Bioengineering 71(5): 335-339 Sommer T R, Potts W T, Morrisey N M (1991). Utilization of microalgae astaxanthin by rainbow trout (Oncorhynchus mykiss). Aquaculture94: 79-88ABSTRACTThe microalga Haematococcus pluvialis is one of the most important sources of astaxanthin for theaquaculture industry. However, due to a very hard cellulosic cell wall, astaxanthin extraction and itsbioavailability in intact encysted cells is low.Different physical and chemical processes, which allow the disruption of the encysted cells of H. pluvialis interms of astaxanthin recovery, were evaluated as well as the facility of its extraction from the cells.Key words: Haematococcus, microalgae, processing, bioavailability, astaxanthin.ACKNOWLEDGMENTSThis work was supported by project FAIR CT-1518.RESULTS AND DISCUSSION when the appropriate solvent mixture is used, the best results were obtained with autoclave, spray-dryer andmechanical disruption processing (figure 1). This was confirmed by HPLC results (not shown) and TLC analysis(TLC I) the highest recovery of astaxanthin (into acetone) from intact cells was observed with autoclave andmechanical disruption processing. These results were supported by TLC analysis (TLC II). However, thepigment amount in all treatments was lower than the one obtained with the solvent mixture;Figure 1. Astaxanthin content (mg/g dw biomass)with solvent mixture extraction and bioavailabilityinto acetone.TLC I. Chromatogram of solvent mixture extract,ressuspended in 500 l n-hexane. Applied volume of sample 15 l;standards: -carotene (2.5 g) and astaxanthin (3S,3’S - 2.5 g).For both the plates, tracks correspond to : 1- -carotene 5- HCl 0.1M, 15 min 9- enzymes2- astaxanthin 6- HCl 0.1M, 30 min 10- spray-dryer3- control 7- NaOH 0.1M, 15 min 11- mechanical disruption (control) 4- autoclave 8- NaOH 0.1M, 30 min 12 - mechanical disruption under a stream of nitrogen.TLC II. Chromatogram of bioavailability into acetoneextracts, ressuspended in 500 l n-hexane. Applied volume ofsample 15 l; standards: -carotene (2.5 g) and astaxanthin(3S,3’S - 2.5 g).1         2         3        4         5        6         7         8         9        10       11       121        2        3         4         5        6        7        8         9        10      11      12Figure 2. Scanning Electron Micrographs of Haematococcus pluvialis cells, which suffered no treatment(control/a), autoclave (b), spray-drying (c ) and mechanical disruption (d) (x2000).a bc d The micrographs (figure 2) show us the different effects of each treatment on the cells, like the cell walldisruption in (d), the clustering (c), or the roughening (b) of the cells, allowing the way out of all thecontents and, mainly, the carotenoids.CONCLUSION We can conclude that the best extraction and so far the most effective process is the mechanicaldisruption.

Evaluation of different disruption processes efficiency on encysted Haematococcus pluvialis cells for astaxanthin recovery and bioavailability

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Evaluation of different disruption processes efficiency on encysted Haematococcus pluvialis cells for astaxanthin recovery and bioavailability

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