Bioresource Technology 101 (2010) 7598–7604

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Extraction, purification and concentration of partially saturated canthaxanthin from Aspergillus carbonarius
D. Krupa a,b,1, E. Nakkeeran a,1, N. Kumaresan c, G. Vijayalakshmi c, R. Subramanian a,*

Department of Food Engineering, Central Food Technological Research Institute, CSIR, Mysore 570 020, India Department of Biotechnology, PSG College of Technology, Coimbatore 641 004, India c Department of Food Microbiology, Central Food Technological Research Institute, CSIR, Mysore 570 020, India

a r t i c l e

i n f o

a b s t r a c t
A mutant Aspergillus carbonarius produces partially saturated canthaxanthin (PSC; C40H62O2) during submerged fermentation. The pigment was extracted from dried biomass using various organic solvents and purified using nanofiltration (NF) and nonporous membranes. Particle size had a great influence; PSC extractability from fines fraction of biomass (75–105 lm) was 1.5-fold higher compared to the coarse fraction (850–920 lm) in ethanol. Among the four solvents, hexane exhibited the highest PSC extractability of 5.83 mg/g and purity of 32 mg/g. On a relative scale, the extraction performance of hexane, acetone, methanol and ethanol were in the order 100, 16.1, 7.5 and 5.4. An assessment based on enrichment factor and permeate flux revealed notable performance with NF-250 membrane in ethanol extract followed by NF-200 and NF-GKSS membranes in methanol extract. These results suggested the suitability of hexane for extraction followed by alcohol phase purification and concentration employing NF. Accordingly, a PSC purity of 206 mg/g was achieved. Ó 2010 Elsevier Ltd. All rights reserved.

Article history: Received 30 December 2009 Received in revised form 23 April 2010 Accepted 27 April 2010 Available online 26 May 2010 Keywords: Aspergillus carbonarius Partially saturated canthaxanthin Extraction Nanofiltration Purification

1. Introduction Colours are one of the important aesthetic properties of food and colouring of foods has been an age old practice. This practice has increased many folds with the invention of synthetic colourants largely owing to their good stability and colouring ability (Pattnaik et al., 1997). Though synthetic colours dominate the market, even among the permitted synthetic pigments, some of them may be toxic, carcinogenic or may cause severe damage to vital organs (Duran et al., 2002). This has given rise to a strong interest in natural colouring alternatives. Carotenoids are a group of pigments of varying tints from red to yellow and possess significant biological activities. Irrespective of their biological functions, selected carotenoids are widely used as food colourants and additives in feeds (Pandey and Pandey, 2008). Industrial interest is gradually shifting away from the yellow carotenoids such as b-carotene and lutein towards more valuable orange-red keto-carotenoids owing to the beneficial effects these compounds exert on various disease conditions (Bhosale and Bernstein, 2005). Canthaxanthin and astaxanthin are the two major keto-carotenoids and canthaxanthin is the precursor of asta-

* Corresponding author. Tel.: +91 821 251 3910; fax: +91 821 251 7233. E-mail address: (R. Subramanian). 1 These authors contributed equally and are listed in alphabetical order. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.093

xanthin. Canthaxanthin has been reported to be an effective antioxidant (Palozza and Krinsky, 1992) and has important applications in nutraceutical, cosmetic, food and feed industries (Zhu et al., 2009). Canthaxanthin is abundant in marine sources such as crustaceans, fishes and microalgae, and other biological sources include fungi and bacteria (Krupa, 2009). However, canthaxanthin is currently produced only synthetically on an industrial scale (Bhosale and Bernstein, 2005), for which at present no cheap commercially exploitable plant or animal sources exist. Even though this carotenoid can be synthesized chemically, synthesis of nature identical enantiopure compound is difficult (Ernst, 2002). In this context, microorganisms have an advantage over other natural sources, as the fermentation process is highly manipulable to achieve higher growth rate and greater cell density (Das et al., 2007) without posing any serious production limitations in terms of space and time. In our earlier studies attempts were made towards production optimization of partially saturated canthaxanthin (PSC; C40H62O2) from Aspergillus carbonarius (Sanjay et al., 2007) and its characterization as 11, 12, 13, 14, 15, 110 , 120 , 130 , 140 , 150 decahydro b, b carotene-4,40 dione (Kumaresan et al., 2008). Canthaxanthin is lipophilic, and so is accumulated in the cell membrane, lipid bilayer, or in lipid deposits accumulated within some oleaginous organisms, hence requires purification before their indented applications. Pigment extraction from microbial sources is generally carried out employing organic solvents. A wide range of polar and non-po-

2002. acetone and hexane).5% ammonium dihydrogen phosphate. 250.04  10À3 lg) was dissolved in a known volume (1 ml) of specific solvent (methanol. Organism A mutant A. Papaioannou et al. Potassium dihydrogen phosphate. 20 ml of sol- . Nafion) were used. India. Chemicals Peptone. ethanol.. Purification of PSC by thin layer chromatography Pigment from biomass was extracted by mixing 2 g of biomass with 100 ml absolute ethanol under stirring at 250 rpm for 1 h at room temperature (28 ± 2 °C).D. Two types of NF membranes of $1 nm pore size. Japan). yeast extract and agar-agar were purchased from M/s Hi-Media. In order to enhance aeration.2. carbonarius in polar and non-polar solvents used as extractants.4. Ultrasonic. India. 2008). deposited at the culture collection centre of the Food Microbiology Department. v/v) at 1 ml/ min) (Kumaresan et al.and HCl-assisted extractions. The harvested culture broth was filtered through muslin cloth and the biomass obtained was dried in a lyophilizer (Lyodryer LT5B. namely. carbonarius. as well as supercritical CO2 extraction have also been attempted for carotenoids extraction from microbial sources (Gu et al.0 with 250 mM citrate–phosphate buffer. 2. Thana et al. there are only a few attempts towards processing carotenoids extracts using membranes. Determination of percentage extinction coefficient of PSC in different solvents 2. Methods 2. 350–710. whereas b-carotene rejection was only 18% under similar conditions. eluted into ethanol and centrifuged at 2200Âg for 5 min to remove silica gel. Determination of percentage extinction coefficient A known amount of PSC (9. The kmax and corresponding absorbance values of PSC in the above solvents were determined after suitable dilution using a UV–vis spectrophotometer (UV-160A. Shimadzu Scientific Instruments. 150. 105–150 and 75–105 lm) were used in the extraction experiments. Krupa et al. Singh et al. Darmstadt. 2007.. These studies revealed that the method of extraction and solvent to be employed depends on the nature of carotenoid as well as biomass produced by the typical microbial source. acetone and hexane. In the present investigation. 0. the membrane rejected lutein by 60% in the absence of added surfactants. In the present study. 500 W.1. (1998) employed supercritical CO2 extraction followed by NF to extract and purify b-carotene from a model carrot oil (sunflower oil and b-carotene) and natural extract of carrot seed. Kyoto. 2008.4.2. 2008). 180–250. the influence of particle size of ground biomass and type of solvent were studied towards greater extraction of PSC. Krichnavaruk et al. 2. 1999). was used in this study. carbonarius was carried out using various organic solvents to improve the extractability as well as purity. Corn flour was procured from the local market.. Membrane processing offers several advantages over conventional downstream processing methods such as chromatographic separations and it has been demonstrated that they are convenient and easy to scale-up in the purification and concentration of pectinase (Devi and Appu Rao. Five different size fractions (850–920. extraction of PSC produced by A. Bangkok. In model systems. The PSC obtained was concentrated in the flash evaporator and subjected to further purification by repeated TLC. Sieving The lyophilized biomass was pulverized in a mixer grinder (Slimline. The ethanol extract obtained was concentrated in a flash evaporator at 40 °C (Rotavapor R205. To 1 g of dried biomass. These membranes rejected 96% of chlorophyll from model oil containing added chlorophyll (Kondal Reddy et al.. 2001). 710. Büchi Labortechnik. The results indicated that the membrane selectivity for b-carotene could vastly differ depending on the nature of the active surface of the membrane as well as the individual system.4. inorganic membrane-T (TiO2) and organic membrane-TN (TiO2 coated with an organic layer. / Bioresource Technology 101 (2010) 7598–7604 7599 lar solvents either individually.. However. fungi and bacteria with and without pretreatment to biomass (Meckel and Kester. UF eliminated ethanol-soluble proteins (zein) and other large solutes from the extract. Lyophilsation Systems. 2008. Valsad. The ground biomass was then subjected to successive sieving starting from large to small sieves with 920. Further. (2001) made attempts to understand the actual rejection mechanism of carotenoids in vegetable oils by a nonporous membrane (NP). Park et al.3. carbohydrates and total acids were of analytical grade and purchased from M/s Sisco Research Laboratories.000 rpm. baffled Erlenmeyer flasks were used which were incubated for 48 h at 30 °C on a rotary shaker (200 rpm). M/s Chhaya Industries. CFTRI under the accession number UV 10046. Tsui and Cheryan (2007) employed ultrafiltration (UF) and NF for the purification and concentration of xanthophylls in ethanol extracts of corn. 1996. 2008).1. ethanol. Extraction of PSC PSC extraction from biomass was carried out using methanol. Subramanian et al. The concentrate was then spotted on a preparative thin layer chromatography (TLC) silica plate (Merck. The major yellow spot (PSC) from the TLC plate was scrapped. L-dextrose and oxalic acid used for the estimation of inorganic phosphates.5% diammonium hydrogen phosphate and 0. The hydroxyl groups present in lutein make it more polar compared to b-carotene which explained the higher rejections of lutein in the model system and oxygenated carotenoids in the crude vegetable oils by the hydrophobic membrane.5) for 48 h at 30 °C and transferred to the production medium that had an identical composition as that of inoculum medium while maintaining pH at 3. 2.5 l capacity. 0. 1980.. Germany) and developed in a chamber saturated with isooctane:acetone:diethyl ether (3:1:1. The inoculum was developed in corn flour medium (4% corn flour. Postfach. Thailand). All the other analytical and laboratory grade chemicals and solvents were procured from reputed companies in the country.. 105 and 75 lm openings. Mumbai. Then the percentage extinction coefficient of PSC in each of the solvent was determined from the following relation. to get various defined range of size fractions. 850. Hejazi et al. The purity of the PSC was established by High Performance Liquid Chromatography on a C18 Supelco Discovery Column (isocratic elution using methanol and acetone (95:5. NF membranes with 200–300 Da MWCO exhibited almost total rejection of xanthophylls and a 300 Da NF membrane displayed greater solvent stability indicating its suitability for concentration and solvent recycle in the process. Switzerland).5. 180.. India) for 2 min. 2. 350. various NF and NP membranes were evaluated for the purification and concentration of PSC in biomass extracts of A. sequentially or in combination have been employed for optimal extraction of carotenoids from algae. pH 5. Percentage extinction coefficient ð1=ðg=100 mlÞ=cmÞ ¼ Absorbance at kmax  10 Concentration of PSC ðmg=mlÞ Â Path length ðcmÞ 2. v/v/v) at room temperature. 18. Mumbai.. 2. Sarrade et al.

Germany was also used. The corresponding recoveries with the fines fraction were 1. It clearly showed that smaller the particle size or in other words greater the surface area.5. a laboratorymade NF membrane (NF-GKSS) developed by GKSS Forschungszentrum. The MPF-34 (NF-200) is a hydrophilic membrane and not recommended to exposure to organic solvents. 1998). The cumulative improvement in PSC extraction during second.10. Other performance parameters of membrane process (NF and NP) were determined as follows.694 mg by 28% after four successive steps with 50 ml ethanol.5 and 410 nm) in the UV–vis spectrophotometer and the PSC concentration was calculated using the respective percentage extinction coefficient. Further. 1). Tsukuba. Influence of particle size on PSC extractability from biomass The yield of target compound is very important in any extraction process. respectively. 2. Performance parameters The performance of the membrane process was expressed as percentage observed rejection (Ro) of each component. 1).9. Therefore percentage extinction coefficient of PSC in methanol. 10% and 6%. 2. These results revealed that particle size had an inverse influence on PSC recovery. The partially extracted biomass (centrifuged solids) was subjected to repeated extraction (with 10 ml of respective solvent each time) to maximize extraction till the concentration of PSC in the extract (supernatant) reached to 610% of the first extract. the amount of PSC extracted increased for all the five size fractions used in the study (Fig. Results and discussion 3. 3. by the manufacturer.6.7. In addition. The NF and NP membranes were cut into circular discs (7. respectively. respectively. Total acids were determined as titrable acidity using 0. 2003). Massachusetts. USA. ethanol. . 1989). 2. These results revealed that recovery could be improved by $85% by optimizing the extraction conditions. as the volume of solvent employed increased. 412. permeate and retentate (ml). The amount of PSC recovered from these five size fractions is plotted against various stages of extraction and the volume of solvent used (Fig. ethanol. Membranes were cleaned with the respective solvents for 1 h after the experimental run.8. The unit was operated in batch mode by charging the cell with 100 ml of PSC extract. Inorganic phosphate content was determined using potassium dihydrogen phosphate as standard (Ames.7600 D. To minimize concentration polarization effect. This membrane too is hydrophilic in nature and resistant to various organic solvents (acetone. Japan. Ro was determined. Hydrophobic NP membrane (NTGS 2200) with silicon as active layer and polyimide as support layer (Nitto Denko. Japan) is resistant to hexane and was obtained from the National Food Research Institute. permeate and retentate. 3. the extract was separated from biomass by centrifugation at 2200Âg for 15 min and PSC concentration in the supernatant was determined spectrophotometrically. The stepwise extraction basically indicated the extent to which the extraction process should be carried out. Percentage extinction coefficient of PSC in extraction solvents The extractability of PSC varies with different solvents. Osaka.2.1. Flux corresponded to the actual measurement for the experimental run. After stirring. Pure solvent fluxes were also measured before processing each of the PSC extract. the contents in the cell were stirred on a magnetic stirrer (800 rpm). VP and VR are the volume of feed. Membrane filtration system Experiments were conducted at room temperature (28 ± 2 °C) and 2 MPa pressure under nitrogen atmosphere. Krupa et al.861 and 2. more so in the case of valuable compounds such as carotenoids because it has a direct bearing on the production economics.1 N sodium hydroxide (Ranganna. It also helped to optimize the volume of solvent required in the extraction process. The near complete recovery of original solvent flux was the criterion followed for membrane reuse after cleaning. inorganic phosphate and total acids Total carbohydrates were determined by the phenol–sulphuric acid method using dextrose as standard (Rao and Pattabiraman. Ro ð%Þ ¼ 2. The recovery of PSC was 1. 1966). The percentage extinction coefficient and absorbance at kmax at a particular concentration of PSC were greater in polar solvents compared to the most non-polar sol- 2. The various defined range of size fractions of dried biomass were subjected to stepwise extraction with ethanol until the concentration of PSC in the extract reached to 610% of the first extract. acetone and hexane were measured at their respective kmax (414. The experimental run was stopped upon achieving the desired volume concentration ratio (VCR) of 5. Membranes The composite NF membranes (MPF-34 and MPF-44) of MWCO 200 and 250 Da were procured from M/s Koch Membrane Systems. 100 ln ðC R =C F Þ ln ðV F =V R Þ VF VR ð1Þ ð2Þ ð3Þ ð4Þ Volume concentration ratio ðVCRÞ ¼ Recovery ð%Þ ¼ ð CR Â V R Þ Â 100 CF Â V F   ðC F Â V F Þ À ðC R Â V R Þ Â 100 Elimination ð%Þ ¼ CF Â V F where CF. acetone and hexane were determined to quantify the actual amount of PSC extracted (Table 1). 415. Estimation of PSC The optical density of PSC extracts obtained with methanol. CP and CR are the PSC concentration (lg/ml) or carbohydrate content (mg/ml) or phosphate salts (mg/ml) or total acids (N) in feed.261 mg. greater was the extractability for a given volume of solvent. acetone and alcohols) and water.223 mg from 1 g of coarse fraction of dried biomass during the first step of extraction with 20 ml ethanol which improved to 1. The MPF-44 (NF-250) is also a hydrophilic membrane but claimed to be resistant to various organic solvents (hexane. Subsequent studies were made only with very fine (75– 105 lm) and coarse (850–920 lm) fractions of ground biomass to assess the comparative extraction performance with polar and non-polar solvents. / Bioresource Technology 101 (2010) 7598–7604 vent was added and stirred at 250 rpm for 1 h at room temperature (28 ± 2 °C). respectively. assuming that it was constant for each batch experiment (Cheryan.5 cm diameter with 32 cm2 effective area) and fitted in the selfstirred membrane cell in such a way that active surface comes into contact with the feed material. third and fourth steps were 4%. All extraction experiments were carried out in triplicate and membrane runs in duplicate and the mean values are reported. Estimation of total carbohydrates. Geesthacht. alcohols and ether) and water. VF.

CHO: carbohydrates.044 ± 0. hexane extract is likely to contain substantial amounts of membrane lipids owing to its hydrophobic nature.389 ± 0.412 ± 0. 3. 1:50 extract (1 g biomass and 50 ml solvent). Table 1 Percentage extinction coefficient of PSC in various organic solvents.3. polar solvents contain higher amounts of hydrophilic impurities while extracts of non-polar solvent would contain substantial amounts of lipophilic impurities. Solvent Methanol Ethanol Acetone Hexane kmax (nm) 414.0003 0.0003 0. The extractability of hexane was 2. Amount of PSC extracted in ethanol from various size fractions of ground biomass.0000 Coarse fraction 0. salts and acids were found in polar solvent extracts owing to their solubility in the order of solvent polarity (Table 2). acetone and hexane as described earlier with ethanol.047 0.0003 0.D.0003 0.003 ± 0.0088 ± 0. Accordingly the normalized performance with hexane was 6–19-fold higher than polar solvents (Table 3). The extraction performance was expressed as a product of extractability and extraction purity of PSC. However. Extracts of 3.3.0096 ± 0. Krupa et al.002 Total acids (N) Fines fraction 0.0 Amount of PSC (mg/g of dry biomass) 2.013 0.5 105-150 µm 75-105 µm 0.063 ± 0.5 412.0 1.0 350-710 µm 180-250 µm 0.0 15 20 25 30 35 40 45 50 55 Volume of ethanol (ml) Fig.044 0. / Bioresource Technology 101 (2010) 7598–7604 7601 3.5 850-920 µm 1.094 ± 0. 2 and Table 3). The extractability of methanol and acetone fell between ethanol and hexane.006 0.236 Percentage extinction coefficient (1/(g/100 ml)/cm) 781 891 793 261 Concentration of PSC used for estimation was 9.825 mg/g of fines fraction of biomass while the maximum amount extracted with the conventionally employed ethanol was only 2. fines fraction: 75–105 lm.417 ± 0. These impurities were co-extracted along with PSC during extraction with polar and non-polar solvents. Purity of biomass extracts The biomass contains the media components such as carbohydrates.014 Inorganic phosphates (g/l) Fines fraction 0.0025 ± 0.017 0. methanol being closer to ethanol (Fig.093 0.0566 ± 0.485 ± 0. 3. Step-wise extractions were carried out with methanol. Solvent extraction of PSC Extractability of PSC was studied with fines and coarse fractions of biomass using polar and non-polar solvents with a view to enhance its recovery. Membrane lipids would also potentially be extracted by more non-polar solvents. 1.0664 ± 0.008 Coarse fraction 0.591 ± 0.0000 All the experiments were carried out in triplicate (n = 3) and samples were analyzed in triplicate (n = 3).036 0.1. amount of PSC extracted with hexane was 3.000 Coarse fraction 0. 2008). salts and acids besides the target compound.04 Â 10À3 lg/ml.002 ± 0.551 ± 0. relatively larger amounts of carbohydrates. salt and acid contents in various solvent extracts of biomass from A.806 0. PSC. hexane (Table 1) suggesting that the solubility of PSC was greater in polar solvents which is rather expected considering the molecular structure of PSC (Kumaresan et al.0017 ± 0.706 0. Comparatively these impurities were present in much lower amounts in the hexane extract leading to a higher degree of purity of extracted PSC (32 mg/g of total soluble solids).0 415.261 mg/g of biomass.020 ± 0.0003 0.0003 0. Table 2 Carbohydrate. Accordingly.367 ± 0.042 0. The increase in extractability was higher in subsequent steps with increased volume of solvent employed. Solvent CHO (g/l) Fines fraction Methanol Ethanol Acetone Hexane 0.007 0. Solvent selection for PSC extraction Hexane exhibited the highest PSC extractability among the various solvents studied (Fig.033 ± 0. coarse fraction: 850–920 lm.1fold higher than ethanol in the first step of extraction with coarse fraction of biomass.0 Absorbance at kmax (–) 0.717 0.5 410. The maximum amount of PSC extracted with hexane was 5.2-fold higher than ethanol with coarse fraction after four steps of extraction.069 0.5 2.018 0.0296 ± 0. vent.3.0326 ± 0.. carbonarius.315 ± 0.134 0.2. . 2).048 ± 0.

2007). 2002). This membrane gave the highest rejection of PSC in methanol. higher extractability in hexane could be attributed to the greater permeabilizing ability of hexane in disrupting the cell walls and membrane (lipid) layers to effectively release the PSC from the biomass.. 2003.084 3.5 5.4. (2002) reported the solute permeation characteristics of Sudan IV (384 Da Extraction purity = Ratio of PSC concentration to total soluble solids. Solvent compatibility of these membranes was tested before the experimental runs. In addition. ðExtractabilityÂExtraction purityÞany solvent Normalized extraction performance ¼ ðExtractability ÂExtraction purity  100: Þ Max Max All the experiments were carried out in triplicate (n = 3) and samples were analyzed in triplicate (n = 3). alcohols and ether) (Zwijnenberg et al. membranes were chosen to aid in concentrating the target compound in the product stream (retentate).13 9. Amount of PSC extracted from coarse and fines fractions of biomass in various organic solvents. acetone and hexane extracts compared to other membranes while NF-250 membrane gave the highest rejection with ethanol extract. 3. Table 3 Extraction performance of solvents for PSC from biomass (fines fraction) of A. The solubility of PSC is expected to be greater in polar solvents compared to hexane as reflected in the differences in percentage extinction coefficients (Table 1). Although fineness of particle generally increases the extractability it could lead to other operational problems such as fouling and flooding of the extractor. / Bioresource Technology 101 (2010) 7598–7604 7 Methanol -CF Methanol -FF Amount of PSC (mg/g of dry biomass) 6 Ethanol -CF Ethanol -FF Acetone -CF Acetone -FF Hexane -CF Hexane -FF 5 4 3 2 1 0 10 20 30 40 50 60 70 Volume of solvent (ml) Fig. Krupa et al. The NF-250 membrane is claimed to be resistant to various organic solvents by the manufacturer but was found to be not stable with hexane.. Park et al. Extractability = Amount of PSC extracted (mg of PSC from 1 g of dried biomass) with 50 ml of solvent.170 ± 0. 3. suggesting elimination of size reduction step in the process (Fig.6-fold higher compared to the polar solvents used in the study.825 ± 0. FF – fines fraction (75-105 lm).095 2. The NF-200 membrane is not recommended to exposure to organic solvents but remained stable with methanol and ethanol.261 ± 0.15 4. The experimental runs with three different NF membranes with ethanol and methanol extracts revealed their contrasting behaviour in terms of PSC rejection. there was no significant difference between the extractability of PSC with fines and coarse fractions of biomass with hexane.7602 D. size reduction greatly increases the amount of energy used for the process. 1999) and was actually resistant to all the solvents including hexane used in the study. CF – coarse fraction (850–920 lm). carbonarius. The laboratory-made NF-GKSS membrane was claimed to be resistant to various organic solvents (acetone. Interestingly. Solvent Methanol Ethanol Acetone Hexane Extractability (mg/g) 2. Selectivity for PSC The selectivity of NF and NP membranes was assessed for PSC and other known impurities in four different solvent phases and the results are presented in Table 4.35 31.28 ± 0. Therefore. Extractability depends on the solvating power and ability of the solvent to permeabilize the cell wall (Leshchev and Sin’kevich. The NTGS 2200 membrane was stable only with hexane.4 16.605 ± 0. as was also reported by other researchers (Van der Bruggen et al.29 ± 0. NF-250 and NF-GKSS) and a hydrophobic NP membrane (NTGS 2200) to improve the purity of PSC while simultaneously aiming for higher recovery.. These results suggest that the penetrating power of hexane played a more important role during the .4. For instance lutein containing two hydroxyl groups is more polar than b-carotene while PSC with two keto (carbonyl) groups in its structure is less polar than lutein but more polar than b-carotene. The chemical formula of a specific carotenoid influences its relative polarity.59 ± 0. Bhanushali et al.1 100 extraction of PSC than the particle size and surface area as well as the matching polarity of solvent.40 Normalized extraction performance (–) 7. The maximum extractability of PSC was obtained with hexane which was nearly 2. Also. NF-GKSS membrane exhibited the highest rejection of 89% and recovery of 84% PSC in methanol extract among the various combinations of membrane and extract studied. 2).96 ± 0.1. 2. Membrane processing of biomass extracts Membrane processing of various solvent extracts of biomass was attempted with three different hydrophilic NF membranes (NF-200.096 5.099 Extraction purity (mg/g) 5.2–2. The purity of PSC obtained with hexane extract was 32 mg/g of soluble solids and attempts were made to improve the purity further by employing membrane processing.

0 71.9 45. Membrane selectivity of PSC with respect to CHO.12 NS Hexane EF (–) NS NS 2. These selection parameters have a direct bearing on the process economics.7 22. volume concentration ratio (VCR): five-fold. (1999) reported good selectivity for deacidification by NF-GKSS membrane while processing model systems containing triglycerides. In the case of hexane extract.2 NS Flux (l/m2Áh) NS 52.01 0. their solubility differed to varying degrees in different solvents.1 61. demonstrated that only NF-250 membrane in ethanol extract.27 4. Performance assessment in solvent phase The purity as well as recovery of the target compound and the process productivity determines the selection of a process step during downstream processing.6 1.8 5.26 0.59 NS Saltsc 1.87 NS Acidsd NS 0.3.1 4.4 Flux (l/m2Áh) NS NS 1. FFA and acetone.53 16.1 NS NS ND ND Saltsc NS NS ND ND Acidsd NS NS 0.46 0. Though the purity was higher in hexane and acetone extracts. 3. All the three combinations exhibited greater improvement in purity (Table 6) compared to processing in a single solvent system (extraction and purification in the same solvent phase) (Table 5).5. the membrane performance. Krupa et al. ND – not determined.0 NS MP (–) 23. NS – not stable.7 16. Membrane selectivity of PSC with respect to phosphate salts.0 NS 1.29 1. values are expressed as mean of duplicate (n = 2) measurements.0 MP (–) NS NS 2.0 38. Values are expressed as mean of duplicate (n = 2) measurements. process flux and purity for the three select process combinations are presented in Table 6. EF – enrichment factor = Ratio of PSC concentration in retentate to feed.84 16.40 0. . Taking this into consideration along with the superior extraction performance of hexane.2 NS Flux (l/m2Áh) 33. VCR: five-fold. Solvent Membrane Methanol EF (–) 2. the enrichment factor or flux was much lower.31 NS Saltsc NS 0. The rejection of Sudan IV in n-hexane medium was $25% while in methanol it was $10% with the hydrophobic membrane. 3.2-fold). / Bioresource Technology 101 (2010) 7598–7604 Table 4 Rejection of PSC and impurities during membrane processing of A. 3.21 1. Purity – Ratio of PSC concentration to total soluble solids (retentate).88 NF-200 NF-250 NF-GKSS NP-NTGS 2200 MP – membrane performance (notional) = EF Â Flux.88 6.6 68.2 2. NS – not stable.8 NS Purity (mg/g) 3.7 3.44 1.8 NS NS 1.55 NS Acidsd 0.2.59 1.5 50.4. The selectivity of NF process employing an organic solvent is rather complicated with several factors such as solubility and diffusivity of permeating components and their solubility in solvent and membrane material playing vital role. NF-200 membrane in methanol extract and NF-GKSS membrane in methanol extract could be considered (Table 5) for further evaluation.0 1. Removal of impurities The membrane selectivity towards PSC with respect to removal of impurities such as carbohydrates.66 0.15 2.5 Purity (mg/g) NS NS 46. The estimation of acids includes citric acid (from the media) and other organic acids.D.54 0. Overall performance of extraction and membrane purification Hexane extract of PSC was desolventized and reconstituted in methanol and ethanol for further processing with NF membranes.3 19.4.8 2.3 83.49 2. but actual values could not be determined.2-fold).0 NS MP (–) 94.03 NS Ro – Rejection of PSC. organic dye) in polar and non-polar solvent medium using hydrophobic (PDMS) and hydrophilic (aromatic polyamide) NF membranes. A very high selectivity for the removal of phosphate salts was observed only with the NF-250 membrane.38 12.4 NS Flux (l/m2Áh) 20.88 38.4 20.7 NS Selectivity CHOb 0.5 60. carbonarius biomass extractsa. Membrane selectivity of PSC with respect to total acids. as well as hydrolyzed fatty acids of membrane lipids.97 NS 8. but only with the ethanol extract (Table 4).5 NS Purity (mg/g) 4. Therefore. The direction of separation reversed with the hydrophilic membrane (86% in methanol and 43% in n-hexane).6 73. NF-GKSS membrane exhibited an excellent selectivity towards removal of acids during processing acetone extract (Table 4).36 1. the highest selectivity towards removal of carbohydrates was exhibited by NF-250 membrane with both ethanol and methanol extracts. (2005) and Zwijnenberg et al.98 NS Acetone EF (–) NS 1.0 101. Solvent Membrane Methanol Ro (%) Selectivity CHOb NF-200 NF-250 NF-GKSS NP-NTGS 2200 a b c d 7603 Ethanol Ro (%) Acidsd 1. Bhosle et al.20 NS Hexane Ro (%) Selectivity CHOb NS NS 42. However.79 Saltsc 1. All the other combinations were virtually ineffective towards removal of acids.3 89.11 NS Ethanol EF (–) 1. The overall extraction and purification performance in terms of enrichment factor. salts and acids during membrane processing of various solvent extracts are shown in Table 4. Membrane processing with various extracts exhibited that the PSC enrichment was highest with NF-GKSS membrane in methanol extract (4.10 3. followed by the same membrane in acetone extract (3.09 1.2 NS Purity (mg/g) NS 22. NF-200 membrane exhibited the highest purity of 206 mg/g of total soluble solids followed by NF-GKSS membrane (167 mg/g of total soluble solids) in Table 5 Membrane performance during processing biomass extracts.06 NS 64. Factors such as solute-membrane interactions and solute charge/conformation could play a role in the actual rejection behaviour of specific solute in various solvent systems (varying in polarity) by different membranes (hydrophobic and hydrophilic). carbohydrate and phosphate salt contents were below detectable level suggesting very high membrane selectivity. taking into account both membrane productivity and enrichment factor.08 11. extraction with hexane followed by reconstitution in a polar solvent before membrane processing was examined to improve the overall performance.69 NS Acetone Ro (%) Selectivity CHOb NS 32.3 35. Among the three polar solvent extracts. The acids present in the hexane extract could be mainly the free fatty acids that were extracted due to their polarity (Table 2).0 50. NF200 membrane in methanol extract and NF-250 membrane in ethanol extract (Table 5).0 NS MP (–) NS 88.

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