Does the light influence astaxanthin production in

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Does the light influence astaxanthin production in Xanthophyllomyces dendrorhous? a

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A. Tropea , T. Gervasi , M.R. Melito , A. Lo Curto & R. Lo Curto

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Department of Food and Environmental Sciences, University of Messina, Viale F. S. d’Alcontres, Messina, Italy Available online: 22 May 2012

To cite this article: A. Tropea, T. Gervasi, M.R. Melito, A. Lo Curto & R. Lo Curto (2012): Does the light influence astaxanthin production in Xanthophyllomyces dendrorhous?, Natural Product Research: Formerly Natural Product Letters, DOI:10.1080/14786419.2012.688045 To link to this article: http://dx.doi.org/10.1080/14786419.2012.688045

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Natural Product Research 2012, 1–7, iFirst

Does the light influence astaxanthin production in Xanthophyllomyces dendrorhous? A. Tropea*, T. Gervasi, M.R. Melito, A. Lo Curto and R. Lo Curto

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Department of Food and Environmental Sciences, University of Messina, Viale F. S. d’Alcontres, Messina, Italy (Received 6 December 2011; final version received 13 March 2012) Astaxanthin (C40H52O4) is an important natural pigment that has considerable promising applications in human health. Until now, many efforts were made aimed to develop economically sustainable bioprocesses alternative to the chemical synthesis, to satisfy the increasing demand of this ketocarotenoid from feed, food and cosmetic industries. The extraction of natural astaxanthin from the yeast Xanthophyllomyces dendrorhous till now seems to be rather expensive if compared with chemically synthesized astaxanthin. In this article, astaxanthin production by Xanthophyllomyces dendrorhous under two different conditions was studied: a first effort was made using a conventional reactor while a second using an enlightened one. This research was aimed also to optimise astaxanthin production by testing the influence of the light and of some nutrient sources. From fermentation tests, an astaxanthin yield ranging about 970 mg g1 was obtained after fed batch cultivation in the conventional reactor. In the enlightened reactor lower values, about 930 mg g1, were found. Keywords: astaxanthin; carotenoids; yeast; fermentation; Xanthophyllomyces dendrorhous; fed batch

1. Introduction Astaxanthin (3,30 -dihydroxy- , -carotene-4,40 -dione; C40H52O4) is a xanthophyll. It belongs to the carotenoids, which are among the most common, naturally occurring terpenoid pigments. Their common structure is a C40 methyl-branched hydrocarbon backbone. Their colour, which ranges from yellow to orange or red, depends on the number of conjugated double bonds of the polyene chain and corresponds to their ability to absorb photons in the blue and near UV regions. Due to their physicochemical properties and their high-added values, carotenoids are widely used by industries as ‘natural’ food colorants: feed additives in aquaculture to give colour to salmon, to enhance colour of egg yolk in poultry breeding, in cosmetics and as active ingredients in pharmaceuticals (Boussiba, Fan, & Vonshak, 1992; Frankis, 2000; Fraser & Bramley, 2004; Guerin, Huntley, & Olaizola, 2003; Higuera-Ciapara, Felix-Valenzuela & Goycoolea, 2006; Johnson & An, 1991; Leeason & Caston, 2004; Tantillo, Storelli, Aprile & Matrella, 2000).

*Corresponding author. Email: [email protected] ISSN 1478–6419 print/ISSN 1478–6427 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2012.688045 http://www.tandfonline.com

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Astaxanthin, which exhibits a higher antioxidant activity than -carotene and -tocopherol (Guerin et al., 2003; Palozza & Krinsky, 1992; Terao, 1989), is among the most used carotenoids in food industries. Carotenoids play important roles in animal and human welfares, including immune response enhancement, conversion to vitamin A and scavenging of oxygen radicals (Bast, Haenen, R. Van den Berg, & H. Van den Berg, 1998; Hughes, 1999; Jimenez-Escrig, Jimenez-Jimenez, Sanchez-Moreno, & Saura-Calixto, 2000; Kiokias & Gordon, 2004; Lee, Ozcelik, & Min, 2003; Simpson, 1983). Epidemiological evidence and experimental results suggest that dietary carotenoids inhibit the onset of many diseases in which free radicals are thought to play a role in initiation, such as atherosclerosis, cataracts, multiple sclerosis and cancer (Baker & Guenther, 2004; Berset, 1999; Clark, Herron, Waters, & Fernandez, 2006; Forman, Hursting, Umar, & Barret, 2004; Goswami & Sharma, 2005; Henneckens, 1997; Hughes, 1999). Animals cannot synthesise carotenoids, so these pigments must therefore be added to the feeds of farmed species. Humans find carotenoids in their diet when they intake vegetables and fruits as well as animal products rich in carotenoids. The latter products may be further enriched in these compounds by specific additives. Carotenoids represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries, not only because they can act as vitamin A precursors, but also for their colouring, antioxidant and possible tumour-inhibiting activity. The scrutiny and negative assessment of synthetic food dyes by the modern consumer, gave rise to a strong interest in natural colouring alternatives. Despite of the availability of a variety of natural and synthetic carotenoids, there is currently renewed interest in microbial sources (Ausich, 1997; Johnson & Schroeder, 1995; Lee & Schmidt-Dannert, 2002; Nelis & De Leenheer, 1991). Compared with the extraction from vegetables (Coulson, 1980) or chemical synthesis (Counsell, 1980), the microbial production of carotenoids is of paramount interest, mainly because of the problems of seasonal and geographic variability linked to the production and marketing of several vegetal colorants (De Haan, Burke, & Bont, 1991), and because of the economic advantages of microbial processes when natural low-cost substrates as carbohydrate are used. Due to its increasing importance, biotechnological methods for astaxanthin production were developed with the green alga Haematococcus pluvialis and the hetero basidiomycetous yeast Xanthophyllomyces dendrorhous, the teleomorphic state of Phaffia rhodozyma (Baeza et al., 2009; Frengova & Beshkova, 2009; Golubev, 1995; Johnson & Lewis, 1979; Lemoine & Schoefs, 2010; Phaff, Miller, Yoneyama, & Soneda, 1972; Rodriguez-Saiz, de la Fuente, & Barredo, 2010), consider these microorganisms potential pigment sources. Generally, yeasts are more convenient than algae or moulds for large-scale production in fermenters, due to their unicellular nature and high growth rate. In this study, astaxanthin production by Xanthophyllomyces dendrorhous under two different conditions was studied: a first effort was made using a conventional reactor while a second by an enlighten one. This research was also aimed to optimise astaxanthin production by testing light and nutrient sources influence. 2. Results and discussion As shown in Figure 1 during fed batch cultivation in conventional fermenter, the highest astaxanthin production (969 mg g1) and biomass yield (5.1 g L1) were observed after (NH4)2SO4 and KNO3 addition (after 21 days). Glucose and peptone feeding had no relevant effects on pigment production. On the contrary, during fermentation carried out using enlightened fermenter (Figure 2), the highest astaxanthin production was obtained just after 7 days

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(930 mg g1), when no extra media were added. After glucose and ammonium feeding, the astaxanthin rate decreased while the biomass yield increased up to 23.8 g L1. 3. Experimental 3.1. Microorganism Xanthophyllomyces dendrorhous 5308 was provided by CBS (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands).

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The strain was maintained on glucose 20 g L1; (NH4)2SO4 5 g L1; KH2PO4 1 g L1; MgSO4  7H2O 0.5 g L1; CaCl2  2H2O 0.1 g L1; yeast extract 1 g L1 and agar 15 g L1. 3.2. Inoculum preparation and fermentation To carry-out the tests, Xanthophyllomyces dendrorhous was grown in 200 mL of malt extract (1 g L1) medium in a 2 mL baffled flask at 21 C for 3 days on a rotary shaker incubator at 80 rpm. Inoculum was then inoculated into a 25 L B. Braun Biotech International fermenter containing 10 L of culture medium. Culture medium was made of: glucose (20 g L1); (NH4)2SO4 (14 g L1); KH2PO4 (1 g L1); KNO3 (24 g L1); MgSO4  7H2O (0.5 g L1); CaCl2  2H2O (0.1 g L1); yeast extract (1 g L1) and antifoam (Sigma 298) (0.4 mL L1). Medium was sterilised at 121 C for 20 min. Fermentation was carried out in fed-batch mode, at 21 C; pH: 5.05 without any further correction; mixing: 400 rpm with two six-blade Rushton turbines set near air sparger (0.5 mm diameter holes); air flow: 4 L min1; air sterilisation: filtration through 0.2 mm Sartofluor filter and pressure: 200 mbar. Fermentation was followed also by monitoring, through a continuous IR monitor, CO2 concentration in exhaust air. Dissolved oxygen tension was monitored as well, by a pO2 probe. Enlightened fermentation was carried out in a 6 L Pyrex flask with 2.5 L of the same culture medium, assembled with a rubber stopper provided with steel tubes for inoculum, medium feeding and sampling, and air inlet sterilised through a 0.2 mm Millex FG filter. Illumination was made by six Radium NL fluorescent tubes giving a light intensity of 2500 lux cm2. Fermentation was carried out at 21 C; pH: 5.05; mixing: 1300 rpm; air flow: 4.5 L min1. To observe the nitrogen and carbon source influence, glucose, (NH4)2SO4, KNO3 and peptone were added separately during both the processes. Fermentation testes lasted 28 and 33 days, respectively, for conventional and enlightened fermentations. 3.3. Sampling Xanthophyllomyces dendrorhous culture samples (300 mL) were periodically collected by a Watson-Marlow 503U peristaltic pump, tested for sterility by spreading on PCA and MEA and finally centrifuged for 12 min at 9000 rpm at 15 C in a B. Braun DR 15 centrifuge. Cell pellet was washed twice with NaCl 0.8% and finally freeze-dried at 30 C for 24 h by a Heto Drywinner freeze-drier. 3.4. Pigment extraction Carotenoid pigment was extracted from 200 mg of dry yeast using 5 mL DMSO and shaked for 2 min at 55 C, then 0.5 mL of 0.01 M Na3PO4 and 4 mL hexane/ethyl acetate 1 : 1 (v/v) were added and agitated for further 2 min. Samples were centrifuged twice for 10 min at 3700 rpm at 25 C using an ALC4217 MKII centrifuge. The supernatant was harvested and evaporated to dryness under vacuum at 30 C using a Bu¨chi R-210 rotoevaporator. Residues were dissolved in 2 mL MTBE and, before HPLC-DAD-MS analysis, filtered through a 0.45 mm PTFE filter. Aliquots of 5 mL were used for injection in the HPLC-DAD-MS. Astaxanthin standard was provided by Extrasynthese (Genay, France) and a 30 mg L1 solution was prepared in chloroform/hexane 10 : 90 v/v.

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3.5. Astaxanthin analysis Astaxanthin was analysed by a Shimadzu liquid chromatography system equipped with two LC-10A-Vp pumps, a SCL-10 A-Vp controller and a SPD-M10Avp photodiode array detector. The system is coupled with a MS detector Shimadzu 2010 equipped with an APCI interface. UV and MS data were acquired and processed using an operating system Windows NT 4.0. Rheodyne injector valve (model 7725i) with a 5 mL loop was used and the compounds were separated on a YMC 30 C-30 column (250 mm  4.6 mm; 5 mm, YMC Europe, Schermbeck, Germany) at 25 C at a flow rate of 0.8 mL min1. The mobile phase consisted of MeOH (A)/MTBE (B). For a better separation, a gradient elution mode was used: 0.01–30.00 min, 5–95% B; 30.01–35.00 min 95–5% B. Peak purity control and identification both in a standard solution and in samples were performed with a HPLCDAD-MS system. The peaks were detected at 476 nm and the effluent from DAD was injected into the APCI-MS system. The APCI source was used in negative ionisation mode. Astaxanthin was identified according to its retention time and spectrum by photodiode array detection (Figures 3–5).

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Figure 5. Astaxanthin mass spectra (APCI-negative).

4. Conclusions In the present study it was observed how light has a real important role in astaxanthin production but just ammonium source feeding allowed us to obtain a greater pigment production. Moreover light seems to be an essential factor for biomass increase. As reported in literature (Dominguez-Bocanegra, Ponce-Noyola, & Torres-Munoz, 2007), the best results concerning astaxanthin accumulation were achieved using ultraviolet and white lights. The more reliable hypothesis for the induction of the astaxanthin biosynthesis after exposure to white light is the generation of active oxygen molecules, which may play a role in carotenogenesis stimulation. It is also possible to assume it as an antioxidative response protecting the cell against the reactive species generated by the light stress (De la Fuente et al., 2010). Further studies could better clarify the role played by some medium components in astaxanthin production in relationship with different light wavelength. Acknowledgements Authors thank the Department of Food and Environmental Sciences.

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