co2 scf extraction of chlorophyll

11
ISSN: 1579-4377 SUPERCRITICAL EXTRACTION OF FOOD PIGMENTS WITH ANTIOXIDANT ACTIVITY Miguel Rodríguez*, Casimiro Mantell, María-Dolores Macías-Sánchez and Enrique Martínez de la Ossa. Department of Chemical Engineering, Food Technology and Environmental Technologies, Science Faculty, University of Cadiz, Avda. República Saharaui, s/n, 11510 - Puerto Real (Cádiz), Spain. *[email protected] ABSTRACT This study presents some of the most significant results obtained from research conducted on processes for the extraction of colorants with antioxidant properties, in particular, antocyans, from red grape marc, and carotenoids and chlorophylls, from microalgae of marine origin Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina, using supercritical carbon dioxide, either alone or mixed with co-solvents (water, methanol, ethanol). KEYWORDS Microalgae, colorants, antioxidants, supercritical fluids

Upload: charles

Post on 25-Nov-2014

124 views

Category:

Documents


4 download

TRANSCRIPT

Page 1: CO2 SCF extraction of Chlorophyll

ISSN: 1579-4377

SUPERCRITICAL EXTRACTION OF FOOD PIGMENTS WITH ANTIOXIDANT ACTIVITY

Miguel Rodríguez*, Casimiro Mantell, María-Dolores Macías-Sánchez and Enrique Martínez de la Ossa.

Department of Chemical Engineering, Food Technology and Environmental Technologies, Science Faculty,

University of Cadiz, Avda. República Saharaui, s/n, 11510 - Puerto Real (Cádiz), Spain. *[email protected]

ABSTRACT This study presents some of the most significant results obtained from research conducted on processes for the extraction of colorants with antioxidant properties, in particular, antocyans, from red grape marc, and carotenoids and chlorophylls, from microalgae of marine origin Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina, using supercritical carbon dioxide, either alone or mixed with co-solvents (water, methanol, ethanol). KEYWORDS Microalgae, colorants, antioxidants, supercritical fluids

Page 2: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3260

INTRODUCTION

Antioxidant compounds of natural origin are being employed in significantly increased quantities in the food, pharmaceuticals and cosmetics industries. In this context, the enormous advantages presented by the family of antocyan compounds have been the subject of much research in recent decades, essentially for their colorant properties and because of their wide availability. Antocyans are present in numerous plants, and are responsible for the colouring displayed by many of them. However, the principal raw material that has traditionally been utilised to obtain this colorant on an industrial scale is red grape marc - more specifically, the byproduct obtained from the red wine vinification process. Recent studies have demonstrated that, in addition to their colorant properties, antocyans present antioxidant and anticarcinogenic characteristics that make them a very attractive additive for use in food and drink products [1].

Another colorant, beta-carotene, has been identified as an antioxidant of considerable importance for human health [2]. Beta-carotene is found in several plants and marine organisms, and plays a key role as provitamin A [3]. For its properties, it is used as both a colorant and a food ingredient. It is used in the formulation of skin creams, as blockers of free radicals, and as an antioxidant element in sun protection creams, associated with tocopherols; it is also an ingredient of protective creams applied after skin exfoliation treatment.

The epidemiological studies carried out to date have demonstrated a correlation between the increasing ingestion of carotenoids and a reduction in the incidence of cardiovascular diseases and certain types of cancers, together with a greater resistance to viral and bacteriological infections [4-7]. The anticarcinogenic effects of beta-carotene can be attributed to its antioxidant activity [8-12].

Finally, with respect to the relevance of chlorophylls in food technology, studies have been centred on preventing their degradation during food processing and storage, since they are present naturally in many foods [13]. In addition, their use as a colorant is authorised in the production of soft drinks, ice cream and other food products [14-15].

Marine algae constitute a raw material of great interest due to their content in carotenoids and chlorophylls. Currently, there are numerous commercial applications of microalgae due to their chemical composition [16-17]; notable among these are applications in which they are utilised to increase the nutritional value of foods and animal feedstuffs, in aquaculture [18], and in the cosmetics industry [19].

The methods employed for the extraction of the pigments studied, using conventional organic solvents, are not only limited by the ruling legislation but also require various stages of purification; their utilisation is diminishing progressively for environmental, health and safety reasons.

Extraction with carbon dioxide in supercritical conditions constitutes a technology of low environmental impact; carbon dioxide possesses many undisputed advantages as a solvent, among which the more significant are its low toxicity, low cost and facility for subsequent separation from the product extracted [20]. Moreover, an extra dimension of quality is added to the products obtained by this technique since they are not subjected to any kind of excessive heating, which usually has negative effects on thermolabile compounds. The choice of ethanol as co-solvent is based on the bibliographical references consulted [21-23], in which it is considered an effective co-solvent in the supercritical extraction of hydroxycarotenoids originating from various matrices. Its presence, in trace

Page 3: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3261

quantities, in the final extract does not compromise the use of the carotenoids obtained in nutritional or pharmaceutical applications [24].

The research work already carried out has demonstrated that extraction with supercritical carbon dioxide of pigments, such as carotenoids, from carrots [25], cabbage [26] and microalgae [27-29] enables good extraction yields to be obtained. However, previous studies dedicated to the extraction of chlorophylls are scarce.

Experimental set-up The experimental work for this research study has been carried out using a micro-scale supercritical extraction plant made by Isco (Nebraska), model SFX 220.

This plant comprises an extractor, a SFX 200 controller, a restrictor and two syringe pumps, models 260D and 100DX, to supply the carbon dioxide and the ethanol, respectively Figure 1. The flow rate is regulated by a micrometric valve, thermostatically controlled to 50°C, to maintain the flow rate constant at 4.5 mmol/min set in the experiments with microalgae, and at 22.5 mmol/min for the extraction of anthocyans. The extraction times employed are 3 hours for the microalgae and 2 hours for the anthocyans. The samples extracted are collected in glass tubes that contain ethanol, and are stored at 4°C in darkness, until their subsequent analysis.

With the object of having a reference value with which to compare the experimental results obtained in the supercritical extraction process, experiments were conducted utilising as solvent methanol and N, N-dimethyl formamide at atmospheric pressure. The total concentrations of antocyans, carotenoids and chlorophylls have been determined by measuring the absorbance of the various samples in a model U-2010 Spectrophotometer of Hitachi (Japan).

Figure 1.- Flow diagram of the extraction process with supercritical carbon dioxide and co-solvent.

Page 4: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3262

RESULTS

Figure 2 presents the extraction yield results for anthocyans obtained from red grape marc utilising liquid mixtures of supercritical carbon dioxide and methanol or water. The tests have been conducted at pressures of 100 and 500 bar, and at temperatures of 40 and 60ºC, with an extraction time of 120 minutes. In substances of this type, it is not feasible to utilise carbon dioxide without the addition of a polarity modifier, given the high polarity of the compounds to be extracted. However, it can be seen that the yields obtained when methanol is utilised as additive are better than those obtained with water, given the better extractive properties of methanol compared with water, as solvents; but the possibility of utilising water together with the carbon dioxide should not be discounted, since in the food industry there are undoubted benefits in using water as a solvent. Furthermore, increasing the percentage of co-solvent significantly increases the extraction yield in all the cases. This behaviour indicates that, in the case of the anthocyans, the addition of the polarity modifier is necessary to increase the extraction yields [30-31].

Figure 2.- Extraction yields of anthocyans with supercritical carbon dioxide and methanol or water, from red grape marc, after 120 minutes of extraction.

With respect to the effect of the pressure and temperature, from the analysis of this figure it can be deduced that, in general, there is a positive effect from increasing the temperature and, conversely, a negative effect from increasing the pressure. The reason for this effect is that an increase of pressure increases the solubility of other co-solutes to a greater extent than it increases the solubility of the anthocyans, thus resulting in a significant reduction in the capacity of the solvent to extract anthocyans.

Page 5: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3263

Shown in Figure 3 are the experimental results for the extraction yields of carotenoids obtained with Nannochloropsis gaditana for the pressure conditions of 200, 300, 400 and 500 bar, and temperature conditions of 40, 50 and 60 ºC, employing as the solvent system supercritical carbon dioxide and 5% molar of ethanol as co-solvent, and an extraction time of 180 minutes.

From the analysis of this figure it can be seen that, for each temperature, the extraction yields generally increase with the pressure. The trend followed by the yield values is due to the density of the solvent system increasing with the pressure, at the temperatures studied, at which the diffusivity of the system is very high.

Figure 3.- Extraction yields of carotenoids with supercritical carbon dioxide and 5% molar ethanol from Nannochloropsis gaditana, after 180 minutes of extraction.

The same trend is observed when the effect of temperature is analysed. For each

pressure assayed, as the temperature is increased, the extraction yields of carotenoids also increase. This trend is similar to that observed in the extraction processes with supercritical CO2 without co-solvent, employing the same algal biomass.

At the pressures of 300, 400 and 500 bar, the density of the supercritical carbon dioxide, and its capacity for dissolving, are greater, and these properties show less variation than at 200 bar. Consequently, when the temperature is increased, the vapour pressure of the solute, and the coefficient of diffusion of the solvent, both increase; these increases compensate for the decrease in the density as a result of the increase of the temperature; thus a higher extraction yield is obtained. Similar trends can be observed in the case of the yields obtained from Synechococcus sp. and Dunaliella salina [32-33].

With respect to the extraction of chlorophylls, Figure 4 gives the experimental results for the extraction yields of chlorophylls obtained from Dunaliella salina using supercritical carbon dioxide mixed with 5% molar ethanol in the conditions of pressure and temperature indicated previously, for an extraction time of 180 minutes. This microalga contains chlorophyll a and b and so the extraction yields given in the figure are the sum of the two types of chlorophyll.

Page 6: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3264

Figure 4.- Extraction yields of chlorophyll with supercritical carbon dioxide and 5% molar ethanol from Dunaliella salina, after 180 minutes of extraction.

On analysing the effect of the pressure, it can be observed that an increase up to 500

bar improves the extraction yield of chlorophyll when the temperature is maintained at 50ºC. For a temperature of 60ºC, the maximum extraction yield is obtained at 400 bar, while at a temperature of 40ºC, the yield only increases when the pressure is increased from 300 to 400 bar.

Further analysis of figure 4 indicates that at 200 bar an increase of the temperature from 40ºC to 50ºC reduces the extraction yield of chlorophyll, while at a pressure of 300 bar, the yields increase progressively as the temperature is raised to 60ºC. At 400 bar also the maximum yield of chlorophyll is obtained at 60 ºC; however at 500 bar the maximum extraction yield is obtained at 50 ºC, with a slight fall in yield observed as 60 ºC is reached.

Comparison of the conventional and supercritical extraction processes With the object of having a reference value with which to compare the experimental results obtained in the supercritical extraction process, experiments were conducted utilising as solvent methanol and N, N-dimethylformamide at atmospheric pressure. Figure 5 presents the results obtained in the extraction process with liquid methanol at atmospheric pressure in continuous operation, after 120 min of extraction, at three different temperatures (40, 50 and 60 ºC), together with a reference value for the extraction process at high pressure, in order to compare the yields obtained. From analysis of this graph, it can be concluded that the yield of the extraction process increases with the temperature, giving values that are generally somewhat higher than those for the high pressure process. In this case, the purity of the extract obtained and its other characteristics would allow one process to be differentiated from the other.

Page 7: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3265

Figure 5.- Extraction yield of antocyans in the conventional extraction process with liquid methanol from red

grape marc.

Figures 6 and 7 show the yields per unit obtained for one of the pigments using methanol and supercritical carbon dioxide + ethanol, in comparison with the values of the yields obtained with N, N-dimethylformamide, for three marine microalgae, Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina.

It can be deduced from Figure 6 that the addition of co-solvent to the supercritical carbon dioxide enables the extraction yield of carotenoids to be increased, compared with the yield obtained with methanol. Conversely, the extraction yield of chlorophyll with supercritical carbon dioxide + ethanol is lower, as shown in Figure 7.

Figure 6.- Yields of total carotenoids per unit, compared with extraction with N, N-dimethylformamide.

Page 8: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3266

With respect to the three microalgae studied, Figures 8 and 9 show the maximum values obtained for the extraction yields of carotenoids and chlorophyll, respectively. It can be deduced from figure 8 that the largest quantity of carotenoids is extracted from the microalga Dunaliella salina when supercritical CO2, mixed with 5% ethanol as co-solvent, is utilised. Lastly, with respect to the extraction of chlorophyll, in Figure 9 it can be observed that the largest quantity of this pigment is obtained from biomass of Synechococcus sp.

Figure 7.- Yields per unit of chlorophyll with respect to extraction with N, N-dimethylformamide.

Figure 8.- Maximum yields of carotenoids employing CO2 + 5% ethanol.

Page 9: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3267

Figure 9.- Maximum yields of chlorophylls employing CO2 + 5% ethanol.

FINAL CONSIDERATIONS

The food industry has a need for new processes to obtain valuable additives of natural origin. From the work undertaken in this study, supercritical extraction can be considered a viable alternative to the extraction with conventional solvents, the utilisation of which is being progressively limited in the food industry and other sectors, by the imposition of increasingly demanding regulations for the processes permitted for obtaining the additives needed for many food products. The possible utilisation of additives that not only serve as colorants but also present antioxidant characteristics, which provide health benefits to consumers, contributes to the added value of the final product. If these additives can be obtained by processing methods that are more respectful of the natural environment, as is the case of extraction with fluids in supercritical conditions, this represents a further improvement of the sales prospects of the final product by adding another feature to its intrinsic quality.

In the experimental work described, two types of additive have been studied that have these characteristics and are already being utilised in food and drink products. Both were obtained from raw materials that have a very low cost of production: antocyans from red grape marc, which constitutes a byproduct of the vinification process for red wines; and carotenoids and chlorophylls from the lyophilized biomass of marine microalgae. The relatively low cost of the raw material means that the product obtained has a greater added value, which should compensate for the higher initial cost of the supercritical extraction technology in comparison with other extraction technologies.

In addition, the high degree of purity of the pigments obtained by means of supercritical extraction is one of the factors that should be studied over the medium to long term, since it constitutes a factor of considerable importance, in the context of the possible implementation of this technology on an industrial scale. In this respect, the degree of

Page 10: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3268

selectivity presented by carbon dioxide as a supercritical solvent contributes to reducing the number of substances co-extracted, thus increasing the purity of the principal extracts.

Finally, although the mixing of certain co-solvents with the carbon dioxide significantly improves the yields obtained, these substances have a series of limitations as co-solvents under the strict regulations in force for food production. REFERENCES

[1] Shenoy, V. R. Anthocyanins - prospective food colors. Current Science., 1993 64, 8, 575-579

[2] Funazukuri, T.; Kong, C.Y.; Kagei, S. Binary diffusion coefficients, partition ratios and partial molar volumes at infinite dilution for β -carotene and α -tocopherol in supercritical carbon dioxide. J. Supercrit. Fluids., 2003, 27, 85-96.

[3] Murthy, K. N. C.; Vanitha, A.; Rajesha, J.; Swamy, M. M.; Sowmya, P. R.; Ravishankar, G. A. In vivo antioxidant activity of carotenoids from Dunaliella salina - a green microalga. Life Sci., 2005, 76, 1381–1390.

[4] Bendich, A.; Olson, J.A. Biological actions of carotenoids. FASEB J., 1989, 3, 1927-1932.

[5] Bendich, A. In: Carotenoids and the immune system, Chemistry and Biology. Eds. N. I. Krinsky, M. M. Mathews-Roth, R. F. Taylor. New York: Plenum Press., 1990, pp 323-335.

[6] N.I. Krinsky. The biological properties of carotenoids. Pure Appl. Chem., 1994, 66, 1002-1010.

[7] Tapiero, H.; Townsend, D. M.; Tew, K. D. The role of carotenoids in the prevention of human pathologies. Biomedicine & Pharmacotherapy., 2004, 58, 100-110.

[8] Poppel, G. V.; Goldbohm, R. A. Epidemiologic evidence for β-carotene and cancer prevention. Am. J. Clin. Nutr., 1995, 62, 1393S–1402S.

[9] Ziegler, R. G.; Colativo, E. A.; Hartge, P.; McAdams, M. J.; Schoenberg, J. B.; Mason, T. J.; Fraumeni, J. F. Jr. Importance of alpha-carotene, beta-carotene, and other phytochemicals in the etiology of lung cancer. J. Natl. Cancer Inst., 1996, 88, 612-615.

[10] Kumaris, S. S. Beta-carotene in health and disease. Biomedicine., 2000, 20(4), 225-235.

[11] Young, A. J.; Lowe, G. M. Antioxidant and prooxidant properties of carotenoids. Arch. Biochem. Biophys., 2001, 385, 20-27.

[12] Stahal, W.; Sies, H. Antioxidant activity of carotenoids. Molecular Aspects of Medicine., 2003, 24, 345-351.

[13] Schwartz, S. J.; Lorenzo, T. V. Chlorophylls in foods. Crit. Rev. Food Sci. Technol. 1990, 29, 1-17

[14] Madrid, R.; Madrid, J. M. Food colourings. Alimentación, Equipos y Tecnología., 1990, 9, 185–191

[15] Directive 94/36/CE of the European Parliament and the Council, 30th June of 1994, about dyes that can be used in food products.

[16] Liang, S.; Xueming, L.; Chen, F.; Chen, Z. Current microalgal health food R&D activities in China. Hydrobiologia, 2004, 512, 45-48

[17] Becker, W. Microalgae for aquaculture. The nutritional value of microalgae for aquaculture. In: Richmond, A. (Eds.), Handbook of microalgal culture, Blackwell, Oxford., 2004, pp. 380-391

[18] Muller-Feuga, A. Microalgae for aquaculture. The current global situation and future trends. In: Richmond, A. (Eds.), Handbook of microalgal culture. Blackwell, Oxford., 2004, pp. 352-364

Page 11: CO2 SCF extraction of Chlorophyll

Rodríguez et al. EJEAFChe, 7 (8), 2008. [3259-3269]

3269

[19] Stolz, P.; Obermayer, B. Manufacturing microalgae for skin care. Cosmetics Toiletries, 2005, 120, 99-106.

[20] Hawthorne, S. Analytical-scale supercritical fluid extraction. Anal. Chem. 1990, 62, 633A–642A

[21] Careri, M.; Furlattinia, L.; Mangiaa, A.; Muscia, M.; Anklamb, E.; Theobaldb, A.; von Hols, C. Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina pacifica algae: a chemometric approach. J. Chromatogr. A 2001, 912, 61–71.

[22] Lim, G. B.; Lee, S.Y.; Lee, E. K.; Haam, S. J.; Kim, W. S. Separation of astaxanthin from red yeast Phaffia rhodozyma by supercritical carbon dioxide extraction. Biochem. Eng. J. 2002, 11, 181-187.

[23] Sovová, H.; Sajfrtová, M.; Bártlová M.; Opletal, L. Near-critical extraction of pigments and oleoresin from stinging nettle leaves. J. Supercrit. Fluids 2004, 30, 213–224.

[24] Reverchon, E.; De Marco, I. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit. Fluids 2006, 38, 146–166.

[25] Barth, M. M.; Zhou, C.; Kute, K. M.; Rosenthal, G. A. Determination of optimum conditions for Supercritical Fluid Extraction of carotenoids from carrot (Daucus carota L.) tissue. J. Agric. Food Chem. 1995, 43, 2876-2878.

[26] Albino, A. M. S.; Penteado, M. D. V. C.; Lanòcas, F. M.; Vilegas, J. H. Y. First International Congress on Pigments in Food –“Pigments in Food Technology” (Seville, Spain), 1999, pp. 65.

[27] Mendes, R. L.; Fernandes, H. L.; Coelho, J. P.; Reis, E. C.; Cabral, J. M. S.; Novais, J. M.; Palavra, A. F. Supercritical CO2 extraction of carotenoids and other lipids from Chlorella vulgaris. Food Chem. 1995, 53, 99–103

[28] Mendes, R. L.; Nobre, B. P.; Cardoso, M. T.; Pereira, A. P.; Palavra, A. F. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorg. Chim. Acta 2003, 356, 328-334

[29] Macías-Sánchez, M. D.; Mantell, C.; Rodríguez, M.; Martínez de la Ossa, E. M.; Lubián, L. M.; Montero, O. Supercritical fluid extraction of carotenoids and chlorophyll a from Nannochloropsis gaditana. J. Food Eng. 2005, 66, 245–251.

[30] Mantell, C.; Rodríguez, M.; Martínez de la Ossa, E. A screening analysis of the high-pressure extraction of anthocyanins from red grape pomace with carbon dioxide and cosolvent. Eng. Life Sci. 2003, 3, 38-42.

[31] Mantell, C.; Rodríguez, M.; Martínez de la Ossa, E. Kinetics and mathematical modeling of anthocyanin extraction with carbon dioxide and methanol at high pressure. Sep. Sci. Tecnol. 2003, 38, 3689-3712.

[32] Montero, O., Macías-Sánchez, M. D., Lama, C. M., Lubián, L., Mantell, C., Rodríguez, M., Martínez de la Ossa, E. Supercritical CO2 extraction of β-carotene from a marine strain of the cyanobacterium Synechococcus species. J. Agric. Food Chem. 2005, 53, 9701-9707.

[33] Macías-Sánchez, M. D.; Mantell, C.; Rodríguez, M.; Martínez de la Ossa, E.; Lubián, L. M., Montero, O. Extraction of carotenoids and chlorophyll from microalgae with supercritical carbon dioxide and ethanol as cosolvent. J. Sep. Sci. 2008, 31, 1352-62.