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.
*miguel.rodri@uca.es

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

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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.

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