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Most of the prawns are processed as frozen or cooked prawn meat. The remaining waste (heads, tails and shells) is used as a feed supplement or is directly discarded onto the land by food industries, seafood markets and capture fisheries. Disposal onto the land allows prawn waste to decompose easily in the open air and causes high environmental pollution. At the same time, many valuable compounds present in the waste are lost. It has been accepted that those from marine/brackish waters are considered ‘shrimps’ while their counterparts from fresh waters are considered ‘prawns’. There is a need to generate value-added products from these waste materials from an environmental and economical point of view. The recovery of value-added products from waste material is beneficial in two ways: firstly, to solve the waste disposal problem itself and secondly, to generate additional income. This research particularly focuses on the management of prawn waste and this small-scale research was carried out using the hand-peeled waste of school prawns. The major aim of this research is the recovery and characterization of one of the major valuable components of prawn waste called the ‘astaxanthin complex’ and its separation from the organic solvent using the antisolvent precipitation technique, which is an innovative approach.
*Address all correspondence to: renuevino@yahoo.com
1. Introduction
The major carotenoid present in prawn waste is the astaxanthin complex. A familiar example is that grey prawn becomes bright orange-red on cooking because the astaxanthin complex including astacene is liberated when cooking denatures the natural protein-pigment complex [1]. The total concentration of the astaxanthin complex in crustaceans including prawns varies with species and location [2]. Free astaxanthin (3, 3′-dihydroxy β, β′-carotene 4, 4′-dione) has two hydroxyl groups, one on each terminal ring. The hydroxyl group can react with fatty acid and form esterified astaxanthin. Astaxanthin monoester has one fatty acid attached to one of the hydroxyl groups and astaxanthin diester has one fatty acid attached to each of the hydroxyl groups. The monoesters and diesters are relatively non-polar, whereas free astaxanthin is relatively polar [3]. Thus, the polarity of the astaxanthin complex plays an important role in choosing the optimal solvent for its recovery. Natural sources such as krill, algae and prawn supply astaxanthin as a mixture of free and esterified forms [4, 5]. These extra-functional groups give astaxanthin an extraordinary antioxidant capability and properties unlike other carotenoids [6]. Astaxanthin esters function as powerful antioxidants under both hydrophobic and hydrophilic conditions during experimental in vitro studies [7]. However, the astaxanthin complex is sensitive to photo, thermal and oxidative degradation because of the presence of long chain-conjugated double bonds [8].
The astaxanthin complex has attracted considerable interest in recent years because of its powerful antioxidant activity [9]. Mostly the antioxidant properties of the astaxanthin complex are mainly focused on human health benefits [10, 11]. In food and aquaculture, the astaxanthin complex is mainly used as a colour enhancer [6]. In the United States, the astaxanthin complex is a permitted colour additive by the Food and Drug Administration in salmon feed to improve the colour of salmon during farming practices [12]. However, the antioxidant properties of the astaxanthin complex have not been paid much attention in relation to agricultural purposes. In aquaculture feed, the astaxanthin complex extracted from natural materials is preferable [1, 13]. However, the cost of the astaxanthin complex for its use in aquaculture feed is a major concern. Commercially produced synthetic astaxanthin costs about $1000/kg [14]. The high cost and consumer resistance to synthetic pigments have led to the exploitation of natural sources for obtaining the astaxanthin complex. Only a few species of microorganisms (eg: Haematococcus lacustris; Phaffia rhodozyma and Chlorella vulgaris) produce the astaxanthin complex in nature. The astaxanthin complex is also abundant in prawn waste [15]. Therefore, it may be worth recovering the astaxanthin complex from prawn waste and exploring its use in various applications.
Conventional recovery of the astaxanthin complex from natural sources is mainly carried out using solvents. However, there are two major problems associated with the solvent extraction of the astaxanthin complex. The first problem is that solvents also extract other compounds such as lipids present in the natural materials along with the astaxanthin complex. The second problem is that solvent extraction degrades this sensitive pigment [16]. To overcome the problems associated with solvent extraction, an effective way of recovering the astaxanthin complex from natural sources is necessary.
This research dealt with the extraction, characterization and recovery of the prawn pigment, the astaxanthin complex from prawn waste. The astaxanthin complex was extracted from prawn waste using two organic solvents, hexane and acetone. The better solvent for the maximum recovery of the astaxanthin complex was chosen. The compatibility of the solvent with the technique called supercritical antisolvent precipitation was also considered. Characterization of the astaxanthin complex was performed using chromatography techniques such as high-performance liquid chromatography (HPLC), and HPLC coupled with mass spectrometry (HPLC-MS). The astaxanthin complex was then precipitated from the organic solvent using environmentally friendly supercritical carbon dioxide (SCO2) instead of performing post-extraction steps such as purification or evaporation of the solvent. This application was the novelty of this research.
The astaxanthin complex is abundant in prawn waste; however, the concentration of the astaxanthin complex in prawn waste varies with species and location [17, 18, 19, 20, 21, 22, 23, 24]. Initially, the extraction of the astaxanthin complex from prawn waste was carried out using vegetable oils. The recovered red oil might then directly be incorporated into the commercial aqua feed [25, 26, 27, 28, 29]. The concentration of the astaxanthin complex in the pigmented oil could be further increased by reusing the pigmented oil to extract the astaxanthin complex from fresh waste [22, 26, 30].
Prawn waste is a highly perishable material, which deteriorates rapidly that in turn degrades the astaxanthin complex present in it [31]. Acid treatment and preliminary enzymatic digestion with and without antioxidants have been studied to stabilize the astaxanthin complex in prawn waste prior to oil extraction [17, 24, 31, 32, 33]. The recovery of the astaxanthin complex from prawn waste with the aid of preliminary enzymatic digestion depends on the prawn species, fermentation conditions and the type of enzyme used for digestion [24].
The use of organic solvents for the recovery of the astaxanthin complex from prawn waste is best limited to analytical investigations. The traditional solvent extraction processes described in the literature are poorly designed for the commercial recovery of the astaxanthin complex from prawn waste [16]. This is because either the presence of other compounds in the prawn waste, such as lipids, allows the recovery of only a very dilute material, or the solvents used in the extraction of astaxanthin are inefficient and cause degradation of the pigment. Various studies on the solvent extraction of the astaxanthin complex from prawn waste have been investigated [15, 20, 31, 34, 35, 36]. Solvent extraction of the astaxanthin complex from natural sources generates more solvent waste and consists of time-consuming complicated post-extraction steps such as evaporation or concentration and purification. The applied heat during extraction and post-extraction may also degrade the astaxanthin complex. To overcome all these possible difficulties associated with solvent extraction and the post-extraction of the astaxanthin complex, a more effective way of recovering the astaxanthin complex from natural sources is necessary.
2.2 Analysis and characterization of the astaxanthin complex from prawn waste
There are two general methods for analyzing the concentration of the astaxanthin complex in a sample: spectrophotometric analysis and HPLC. In both methods, the astaxanthin complex is extracted from a source into a suitable solvent. In spectrophotometric analysis, the light absorbance of the extraction solvent containing the astaxanthin complex is measured at a wavelength that corresponds to the maximum absorbance for the astaxanthin complex (usually between 470 and 480 nm). The concentration of the astaxanthin complex is then calculated using Eq. (1). The problem with the spectrophotometric assay method is that in addition to astaxanthin, other carotenoids or the degradation products of astaxanthin such as astacene will be falsely included as astaxanthin in the results [37, 38, 39].
[A]=XyE1cm1%E1
where, [A]—concentration of the astaxanthin complex (g); X—absorbance at a specific wavelength; y—amount of solvent used (mL) and E1cm1%—extinction coefficient of pure astaxanthin in the extraction solvent.
All those other compounds also absorb light at this wavelength, which in turn leads to an increase in the absorbance measurement. Consequently, this results in an overstatement of the astaxanthin complex concentration in a sample. This overstatement is usually minimized by reporting spectrophotometric analysis results as ‘the astaxanthin complex’ to indicate that the analysis includes other compounds as well [37]. This is the reason, ‘the astaxanthin complex’ is the term used in this research rather than ‘astaxanthin’. The most technically sound and accurate method for determining the astaxanthin complex concentration of a sample is HPLC, which separates the astaxanthin complex into individual esters, astaxanthin degradation products and other related compounds, to provide accurate concentrations of astaxanthin and its esters [37]. The separation of the astaxanthin complex is achieved by using both normal and reverse phases HPLC and HPLC-MS. This work characterizes the astaxanthin complex from prawn waste extract using normal phase HPLC coupled with atmospheric pressure chemical ionization mass spectrometry (APCI-MS)—negative mode.
2.3 Supercritical antisolvent processes
A supercritical fluid is defined as a substance above its critical temperature and critical pressure. The application of supercritical fluid extraction has been investigated in various industries [40, 41, 42, 43, 44]. These fluids can also be used as antisolvents to precipitate materials from conventional solvents where the particle of interest is not soluble in supercritical fluids [45, 46, 47]. In supercritical antisolvent processes, the supercritical fluid is used as an antisolvent to bring about precipitation of the substrate(s) dissolved initially in a liquid solvent. This research used carbon dioxide as a supercritical fluid. Figure 1 shows the working method of the supercritical antisolvent precipitation process. SCO2 and the organic solvent containing the sample are brought into contact by gradually adding compressed SCO2 to the solution containing the solute inside a pressure vessel (Figure 1 Step 1).
Figure 1.
(Steps 1, 2 and 3) Supercritical antisolvent precipitation technique using SCO2.
SCO2, which is used as an antisolvent for the solute, initially dissolves in the organic solvent. The organic solvent is completely miscible with carbon dioxide. Upon the addition of carbon dioxide to the organic solvent containing the solute, the antisolvent diffusion decreases the solubility of the solute within the organic phase (Figure 1 Step 2) and eventually the solute precipitates (Figure 1 Step 3). The mixture of the solvent and antisolvent can be separated when it is depressurized. Then the liquid solvent and gaseous antisolvent can be recycled [45, 48]. Supercritical antisolvent processes depend on the operating conditions and the affinity of the solute towards the liquid solvent [49]. The success of supercritical antisolvent precipitation relies on the selection of a suitable combination of organic solvent and a supercritical antisolvent for a specific compound of interest [45].
As the aim of this project was the recovery of the astaxanthin complex from prawn waste, the supercritical antisolvent precipitation technique was investigated to separate the pigment from the organic solvent. The use of supercritical fluid as an antisolvent is much simpler than organic liquid antisolvents, which need the application of complex purification processes. There are concerns with the stability of the astaxanthin complex during solvent evaporation. Using the supercritical antisolvent technique, it is possible to precipitate the pigment from the solvent at near ambient temperatures with the proper selection of the antisolvent, thus, avoiding the thermal degradation of the product. And also, supercritical antisolvent processes are carried out in an inert environment and reduced light. This reduces the possible photo- and oxidative degradation of the product [50]. Considering the advantages of the supercritical antisolvent process, we have chosen this technique in the recovery of the astaxanthin complex from prawn waste solvent extract to eliminate the problems associated with the solvent evaporation and purification, and to prevent the degradation of the astaxanthin complex during post-extraction steps.
At the time of this research, SCO2 was only used as a solvent to extract the astaxanthin complex from crustacean waste [14, 51, 52, 53]. The highest (98%) yield of astaxanthin is obtained when the extraction is carried out at low pressure and the highest temperature of 60°C [52]. Therefore, temperature and pressure play an important role in the recovery of astaxanthin from crustacean waste using SCO2. The SCO2 extraction process of the astaxanthin complex operates at nearly ambient or low temperatures, eliminating the possibility of heat damage to the pigment and leaving no solvent residue. However, the solubility of carotenoids in SCO2 is low because of the thick cell wall that resists greater mass transfer and, therefore, the technique may not be effective in extracting total the astaxanthin complex present in the source [14, 51]. The insolubility of the astaxanthin complex in SCO2 offers the possibility of using SCO2 as an antisolvent in the recovery of the astaxanthin complex from prawn waste. An examination of published studies on supercritical antisolvent precipitation indicates that there have been no previous studies carried out on the recovery of astaxanthin from prawn waste using SCO2 as an antisolvent. However, some of the studies indicate the application of SCO2 as an antisolvent in the precipitation of synthetic pigments such as red lake C, pigment yellow 1, pigment blue 15 [54], bronze red pigment [55] and lycopene [50] and grape pomace extract [56] from organic solvents. Therefore, an innovative application was studied in this research to apply SCO2 as an antisolvent on the recovery of the food and pharmaceutical valuable compound, the astaxanthin complex, present in prawn waste.
The phase behaviour of the supercritical antisolvent with the liquid solvent must be known before antisolvent precipitation. The study of volumetric expansion and vapour–liquid equilibrium data of hexane with SCO2 at different temperatures in this research offered an idea about the solubility behaviour of SCO2 in hexane before the supercritical antisolvent precipitation of the astaxanthin complex. The data was also helpful to choose the best operating conditions for the supercritical antisolvent precipitation of the astaxanthin complex.
Due to the difficulty in sourcing adequate commercial prawn waste, raw eastern school prawns (Metapenaeus macleayi, approximately 9 cm body length) were obtained from the Department of Primary Industries, Fisheries Conservation Technology Unit, NSW, Australia and hand-peeled to obtain prawn waste to conduct this research (moisture content 74%; Ash 23% and 11% chitin dry basis). This prawn waste was stored at –22°C until used for research. All solvents used were of HPLC grade and all chemicals were of AR grade.
3.2 Recovery of the astaxanthin complex from prawn waste
3.2.1 Solvent extraction of the astaxanthin complex
The astaxanthin complex was extracted from raw as well as freeze-dried prawn waste using hexane and acetone. Raw prawn waste (15 g) was mixed with 30 ml of hexane and acetone in a tightly capped flask separately. The mixture was shaken at 250 rpm under darkness at ambient temperature (23 ± 1°C) for 15 hours to achieve complete extraction of the astaxanthin complex into the solvents. The supernatant, called the pigment extract, was separated from the waste residue and centrifuged at 1100 rpm for 10 minutes. The supernatant was analysed using UV-spectrophotometer at 480 nm to measure the concentration of the astaxanthin complex (See Section 3.2.2).
In the second step, a further 15 g of prawn waste was added to the pigment extract and kept in the shaker under the same experimental conditions for further extraction of the astaxanthin complex. Successive removal of the waste residue from the pigment extract and further addition of prawn waste to the pigment extract was continued to obtain maximum pigment recovery. The concentration of the astaxanthin complex was measured before each addition of prawn waste to the pigment extract. Figure 1 shows the extraction of the astaxanthin complex from raw prawn waste.
Freeze-dried prawn waste (2.5 g) was mixed separately with 10 ml of hexane and acetone in a tightly capped flask. The pigment extraction was carried out as mentioned above. In the second step, a further 0.5 g of prawn waste was added to the pigment extract (4 ml) and kept in the shaker under the same experimental conditions for further extraction of the pigment. After 20 hours, the concentration of the astaxanthin complex in the pigment extract was measured using UV-spectrophotometer.
3.2.2 Analysis of the astaxanthin complex using UV-spectrophotometer
The synthetic astaxanthin obtained from Sigma-Aldrich is termed ‘standard astaxanthin’ in this work. UV-spectrophotometer analysis was carried out to measure the concentration of the astaxanthin complex in the pigment extract. The absorbance (A480) of the pigment extract recovered from prawn waste was measured at 480 nm. The concentration of the astaxanthin complex in the pigment extract was calculated using Eq. (1). The calculated specific extinction coefficient of astaxanthin in acetone was 1670. This value was used to calculate the concentration of the astaxanthin complex in the prawn pigment extract. However, the measured specific extinction coefficient of astaxanthin in acetone at a wavelength of 480 nm was different from the one published by [38], where the specific extinction coefficient of astaxanthin in acetone was found to be 2500 at the same wavelength. The specific extinction coefficient of astaxanthin in hexane was not measured because of the insolubility of standard astaxanthin in hexane and also because of the unavailability of standard astaxanthin esters. Therefore, the specific coefficient of astaxanthin in hexane used to calculate the concentration of the astaxanthin complex in the prawn pigment extract was 2100 at a wavelength of 480 nm as mentioned in [57]. The cholesterol and other steroids present in the pigment extract was removed by silica gel column chromatography before the HPLC analysis [8].
3.2.3 Characterization of the astaxanthin complex
3.2.3.1 HPLC analysis of prawn pigment extract
The astaxanthin complex in hexane after the removal of cholesterol and other steroids was analysed by normal phase HPLC to characterize the astaxanthin complex. This experiment was conducted using the same waters HPLC system equipped with a 996-photo diode array detector, which was used to measure the cholesterol content of the pigment extract. HPLC analysis was performed using a Lichrosorb silica gel column (150 mm × 2.1 mm, 5 μm) specially designed for mass spectrometry analytical purposes. Data analysis was performed using Millenium Chromatography Manager software.
The astaxanthin complex in hexane after the removal of cholesterol and other steroids was filtered using a 0.45 μm syringe filter before HPLC analysis. The sample injection volume was 20 μl. A mobile phase of different compositions of hexane: acetone (64:36; 65:35; 84:16; 88:12; 98:2; 99:1, v/v) was studied with a flow rate 0.5 ml/minute. Absorption spectra were taken in the range of 250–700 nm. Peaks were monitored by UV detection at a wavelength of 480 nm. The percentage of each discrete peak was calculated from the obtained peak area. The mobile phase that gave the better resolution of the astaxanthin complex peaks was chosen and the same mobile phase conditions were used for mass spectrometric analysis.
3.2.3.2 HPLC-APCI-MS analysis
Mass spectrometric study of the astaxanthin complex was performed by HPLC (ThermoFinnigan Surveyor) coupled with an ion trap mass spectrometer (ThermoFinigan LCQ Deca XP Plus) and atmospheric pressure chemical ionization (APCI) source (negative-ion mode). The data analysis was controlled using ThermoFinnigan Xcalibur software. The HPLC conditions and column were the same as the HPLC analysis.
The eluate from the HPLC column was delivered directly to the APCI source, which was set up with the following conditions: vaporizer temperature of 450°C; sheath gas flow rate of 80 ThermoFinnigan arbitrary units; 10 μA discharge current; the capillary temperature of 250°C; 15 V capillary voltage and 30 V tube lens offset. Spectra were acquired over the m/z range of 550–1300 Da. The analysis was controlled using ThermoFinnigan Xcalibur software.
Astaxanthin monoester and diesters standards were not available. Therefore, different possibilities of negatively charged astaxanthin mono [(A•–FA)–] and diester [(A•–FA–FA)–] ions were calculated manually from the obtained fatty acid profile. Mass spectra of the ester-derived ions were visualized by averaging the data acquired over individual peaks observed in the HPLC chromatograms. The observed m/z values were compared to the theoretical ones, facilitating the identification of the astaxanthin species present.
3.2.4 Vapour–liquid equilibrium study of hexane with CO2
The equilibrium cell consisted of a high-pressure sight gauge with an internal volume of approximately 70 mL. A syringe pump was used for the addition of high-pressure CO2 to the equilibrium cell. The temperature in the cell was monitored with a type K thermocouple. The equilibrium cell was immersed in a water bath for which the required temperature was maintained using a heater. The system pressure was measured with a pressure transducer with an uncertainty of ±0.035 MPa. The liquid phase was withdrawn from the bottom of the equilibrium cell and recirculated to the top using a metering pump. The solvent trap consisted of a 50 cm3 sample cylinder. The outlet of the solvent trap was connected to an inverted 5 ml burette (0.1 ml graduations) to determine the quantity of CO2 present in the liquid and vapour phase samples (Figure 2).
Vapour–liquid equilibrium experiments were performed at different temperatures (25°C, 35°C, 45°C and 55°C). During each experiment, the temperature was kept constant by maintaining a constant water bath temperature. Hexane was filled in the sight gauge to a required volume through V6. The system was kept under atmospheric pressure by opening V3 while loading the sample into the equilibrium cell. After sample loading, V3 was closed. The vessel and the connecting lines were purged with low pressure (approximately 5 bar) CO2 by subsequent opening and closing of V2 to displace any air present. After purging, the equilibrium cell was filled with CO2 to the desired pressure (approximately 2 bar) by V2 to operate the metering pump and then isolated by closing valves V2 and V3. The metering pump was kept in circulating mode by turning V6 and V4 in their suitable direction. The metering pump was turned on. The system was left for at least 30 minutes to get stable conditions of pressure and temperature. Then the initial pressure, the water bath and room temperature and the initial level of hexane in the sight gauge were noted. After that CO2 was added to the sight gauge at several stages by opening and closing V2.
During each stage, samples of the liquid phase were removed from the equilibrium cell to the solvent trap through a switching valve. The CO2 dissolved in a sample was separated from the hydrocarbon component using a solvent trap filled with a known quantity of isopropanol. The isopropanol in the solvent trap was saturated with CO2 before sampling. The gas evolved from the liquid phase and was measured in terms of displacement volume with an inverted graduated burette. The liquid component remaining in the connecting line between valve V6 and the solvent trap was recovered by rinsing the connecting line with isopropanol.
The rinse solution was combined with the solvent trap solution and transferred to a flask containing a known mass of internal standard (0.1 g of cumene). The combined solution was then analysed by gas chromatography to determine the total mass of hexane associated with CO2 from the sample. The use of an internal standard, in this case, cumene, eliminates the need to know the final volume of the combined solution when calculating the total mass of hexane. The mass of hexane in the collected sample was calculated using Eq. (2). Number of moles of hexane in the sample was calculated by dividing the mass of hexane by the molecular weight of hexane.
MH=1RF(AHAC×MC)E2
where, MH—mass of hexane; RF—response factor of hexane; AH—area of hexane; AC—area of cumene and MC—mass of cumene.
Since the solvent trap was operated at near atmospheric pressure, the actual quantity of CO2 collected from the sample was calculated using the ideal gas equation Eq. (3).
nc=(PfVf−PiVi)RTE3
where, nc—number of moles of CO2; P and V—pressure and gas volume in the solvent trap, respectively; R—universal gas constant and T—temperature. The subscripts ‘i’ and ‘f’ refer to the initial and final conditions in the solvent trap.
As can be seen from Figure 2, the gas space above the hexane in the solvent trap was directly connected to the gas space at the top of the burette. The pressure in the solvent trap was, therefore, deduced from the height of water in the burette. It is important to note that each gas volume term in Eq. (3) was the combined volume of gas in the solvent trap and the burette, corrected for the amount of hexane added initially to the solvent trap. In the case of Vf, a correction was also made for the amount of cumene collected in the solvent trap as well as the volume displacement of water in the burette. A small loss of hexane occurs during the initial saturation of the hexane in the solvent trap with CO2.
From the number of moles of hexane and CO2, the mole fraction of hexane and CO2 in percentage in the collected sample was calculated. The ratio of mole percentage of hexane to mole percentage of CO2 in the liquid phase was then determined. The composition of the liquid phase was determined from the average of at least three measurements, with a relative standard deviation of less than 5%. The relative standard deviation was calculated with respect to the ratio of mole percentage of hexane to mole percentage of CO2 in the sample. The drop in pressure in the equilibrium cell was less than 0.1 MPa during the sampling procedure. After each sampling, the desired pressure was restored in the equilibrium cell followed by recirculation of the liquid phase for at least 30 minutes.
3.2.5 Volumetric expansion of hexane using CO2
The measurement of the volumetric expansion of hexane using CO2 was carried out using the same apparatus shown in Figure 1 with some minor modifications. The vapour phase sampling line with the burette was removed from the switching valve. A scale, with 1 mm graduations, was fitted along the visible length of the sight gauge. The apparatus was then calibrated to determine the volume of liquid as a function of the scale length. Volumetric expansion runs were performed at different temperatures (25°C, 35°C, 40°C, 45°C and 55°C). The volumetric expansion data obtained at 25°C and 40°C was used to compare the results with the published data. The temperature was kept constant by maintaining a constant water bath temperature during each run.
Hexane was filled in the sight gauge to a required volume through V6. The system was stabilized for 30 minutes. After stabilization, the initial pressure, the water bath and room temperature and the initial level of hexane in the sight gauge were noted. Then CO2 was added to the sight gauge at several stages by opening and closing V2. The liquid phase was re-circulated and let come to equilibrium over approximately 30 minutes. For each stage of addition of CO2, the temperature, pressure and liquid level rose in the sight gauge were noted.
From the initial level of hexane and the level of hexane after the addition of CO2 in the equilibrium cell, the volumetric expansion of hexane in the equilibrium cell was calculated. The volumetric expansion of hexane at each stage of addition of CO2 at a given temperature and pressure was calculated using Eq. (4).
Expansion(%)=VF−VIVI×100E4
where, VI—initial volume of hexane in the equilibrium cell and VF—final volume of hexane after that addition of CO2
3.2.6 Supercritical antisolvent precipitation of the astaxanthin complex
Supercritical antisolvent precipitation of the astaxanthin complex was performed with the same experimental setup that was used for volumetric expansion experiments. Because the astaxanthin complex was sensitive to heat and light, the experiment was performed under reduced light and the lowest temperature possible (35°C). The experimental pressure and temperature were above the critical temperature and pressure of CO2. Therefore, CO2 used in this study was mentioned as SCO2. The solution containing the astaxanthin complex in hexane was made-up to a concentration of approximately 30 mg/100 mL after the removal of cholesterol and other steroids. This sample solution was filled in the equilibrium cell to the bottom of the graduated scale through V6. The volumetric expansion of hexane containing the astaxanthin complex was calculated at different pressure upon the addition of SCO2 to the equilibrium cell. This experiment was performed to evaluate the effect of the presence of the astaxanthin complex in hexane on the volumetric expansion using SCO2. The visual observations were recorded during the experiment.
The profile of the astaxanthin complex precipitated at different pressure during supercritical antisolvent precipitation was studied. The astaxanthin complex in hexane was filled in the equilibrium cell to the bottom of the graduated scale through V6. The pressure inside the cell was increased by adding SCO2 through V2 (Figure 2). The supernatant was collected at different pressure (5.0 MPa, 5.5 MPa, 6.0 MPa and 6.4 MPa) via V4 and analysed using HPLC. From the peak area of the HLPC chromatogram, the composition of the astaxanthin complex in the supernatant at respective pressure was calculated.
The yield of the astaxanthin complex during supercritical antisolvent precipitation was calculated. The astaxanthin complex in hexane was filled in the equilibrium cell to the bottom of the graduated scale through V6. The pressure inside the cell was increased by adding SCO2 through V2. The supernatant (200 μl) was collected at a pressure of 6.6 MPa into a sample holder containing a known volume of hexane through the switching valve. The concentration of the astaxanthin complex in the collected supernatant was analysed using UV-spectrophotometer. Then the concentration of the astaxanthin complex in the supernatant was calculated and corrected to a suitable dilution factor.
4.1 Solvent extraction of the astaxanthin complex from prawn waste
Figure 3 shows the extraction of the astaxanthin complex from raw as well as freeze-dried prawn waste using hexane and acetone. The concentration of the astaxanthin complex in the pigment extract increased with the extraction time when raw prawn waste was used in hexane. After 54 hours of extraction of raw prawn waste in hexane, the concentration of the astaxanthin complex increased to 4 mg/100 ml in hexane.
Figure 3.
The astaxanthin complex extracted from raw and freeze-dried prawn waste using hexane and acetone.
When acetone was used for the extraction of the astaxanthin complex from raw prawn waste, the concentration of the astaxanthin complex in the pigment extract was nearly the same as in hexane extraction. However, there was a reduction in the concentration of the astaxanthin complex in the pigment extract after the addition of the second batch of raw prawn waste into the pigment extract. For this reason, hexane was chosen as the better solvent to extract the astaxanthin complex from raw prawn waste.
When freeze-dried prawn waste was used for the recovery of the astaxanthin complex, acetone showed better recovery of the astaxanthin complex than hexane (Figure 3). Acetone was the better solvent to extract the astaxanthin complex from freeze-dried prawn waste than hexane; however, this method required freeze-drying of prawn waste.
The concentration of the astaxanthin complex recovered from prawn waste using hexane was nearly equal to the concentration of the astaxanthin complex recovered from freeze-dried prawn waste using acetone. For this research, hexane was chosen as the suitable solvent for the extraction of the astaxanthin complex from raw prawn waste. Using hexane, the astaxanthin complex can be recovered directly from raw prawn waste irrespective of its moisture content, thus eliminating freeze-drying of prawn waste. This solvent extraction method using hexane also provides the possibility of reusing the solvent.
HPLC analysis of the astaxanthin complex dissolved in hexane after silica gel chromatography showed the presence of a free astaxanthin peak [8, 17]. In contrast, experimental trials of this present work indicated that free astaxanthin was not soluble in hexane. Therefore, free astaxanthin peak was not expected during HPLC analysis of the astaxanthin complex dissolved in hexane and the same was confirmed by HPLC-APCI-MS.
4.2 Characterization of the astaxanthin complex by HPLC analysis
There was no separation of peaks when hexane: acetone (64:36 and 65:35, v/v) was used as a mobile phase. Both the solvent and the astaxanthin complex eluted out together without separation. When the polarity of the mobile phase was reduced slightly (hexane: acetone, 84:16 and 88:12 v/v), the solvent and the astaxanthin complex started to resolve into discrete peaks although with poor resolution (Figure 4A and B). When the polarity of the mobile phase was reduced considerably (hexane: acetone, 98:2 v/v), a good resolution of 12 discrete peaks of the astaxanthin complex was obtained (Figure 4C). The percentage composition of each peak was calculated from the peak area. When the composition of hexane in the mobile phase was increased further (hexane: acetone, 99:1, v/v), the retention time of the peaks was increased (result not shown). Therefore, the mobile phase containing hexane (98%) and acetone (2%) was chosen as the optimum condition for the analysis of the astaxanthin complex.
Figure 4.
HPLC analysis of the astaxanthin complex extracted from prawn waste using the mobile phase with different compositions of hexane and acetone (v/v). (A) 84:16 (B) 88:12 (C) 98:2.
Most of the diesters (peaks 1–6, 8) were eluted out first followed by monoesters (peaks 3, 9–11). The esters were classified into mono and diesters from the HPLC-APCI-MS results. It should be noted that peak 3 contained both mono- and diesters. The astaxanthin complex recovered from raw prawn waste contained about 71% of monoesters, 10% diesters, 5% impurities and 14% of the unidentified compound when analyzed using normal phase HPLC. In contrast, the pigment extract from prawn waste contained more diesters (76%) and fewer monoesters (18%) and free astaxanthin (6%) when analysed using reverse phase HPLC [17]. In addition, the reverse phase HPLC analysis of the sample dissolved in hexane after silica gel chromatography showed the presence of a free astaxanthin peak [8, 17]. In contrast, experimental trials of this present work indicated that free astaxanthin was not soluble in hexane. Therefore, free astaxanthin peak was not expected during HPLC analysis of the astaxanthin complex in hexane, which was confirmed by HPLC-APCI-MS. Thus, free astaxanthin was not extracted from prawn waste using hexane, and the astaxanthin complex extracted from prawn waste in hexane was esterified form. The detailed analysis of HPLC-APCI-MS results is not included in this article due to the page number constraints.
4.3 Vapour–liquid equilibrium study of hexane with CO2
The volumetric expansion of hexane with CO2 at different temperatures and pressure is shown in Figure 5. Each point in this figure represented a single determination of the volumetric expansion of hexane with CO2 at a given pressure. A smaller volume of liquid in the precipitation chamber allowed a greater degree of expansion. At low pressure, the data were almost a linear function of pressure where the temperature was kept constant. At high pressure, the volumetric expansion increased exponentially as a consequence of the considerable increase in the solubility of CO2 in this range of pressure at a constant temperature. At constant pressure, hexane showed more expansion at a lower temperature than at a higher temperature. This is attributed to the higher solubility of CO2 in hexane at lower temperatures [58].
Figure 5.
Expansion of hexane using CO2 at different temperatures: a binary system.
It was chosen that supercritical antisolvent precipitation of the astaxanthin complex was carried out at 35°C. The volumetric expansion of hexane with CO2 was started around 5 MPa at 35°C (Figure 5). Therefore, it was expected that the astaxanthin complex in hexane would precipitate around 5 MPa during antisolvent precipitation using SCO2. It was necessary to know the solubility data of CO2 in hexane before supercritical antisolvent precipitation of the astaxanthin complex recovered from raw prawn waste. The increase in the solubility of CO2 in hexane increases the precipitation of the astaxanthin complex from hexane. This is because the increase in solubility of CO2 in hexane reduces the affinity of hexane towards the astaxanthin complex, thereby facilitating the precipitation of the astaxanthin complex from hexane.
The relationship between volumetric expansion and the solubility of CO2 in hexane is shown in Figure 6. This indicated that although volumetric expansion was linear when the solubility of CO2 in hexane was low, the volumetric expansion increased exponentially to the increase in the solubility of CO2 in hexane. Volumetric expansion curves at different temperatures coincided in a single line when plotted as a function of the solubility of CO2 in hexane. The volumetric expansion of hexane with CO2 was initiated around 5 MPa when measured at 35°C (Figure 5). The solubility of CO2 at that pressure was about 0.6 (Figure 7), where the precipitation of the astaxanthin complex was started during supercritical antisolvent precipitation. An increase in pressure increased the solubility of CO2 in hexane, which in turn increased the volumetric expansion of hexane exponentially (Figure 6).
Figure 6.
Relationship between volumetric expansion and the solubility of CO2 in hexane.
Figure 7.
Solubility of CO2 mole fraction in hexane at different pressure and temperature.
Therefore, the rate of precipitation of the astaxanthin complex increased with an increase in pressure during supercritical antisolvent precipitation. At a pressure of 6.6 MPa, the solubility of CO2 hexane was over 0.8. Thus, the precipitation yield of 92.5% of the astaxanthin complex was obtained at this pressure range during supercritical antisolvent precipitation.
4.4 Supercritical antisolvent precipitation of the astaxanthin complex
The volumetric expansion of pure hexane and the astaxanthin complex in hexane using SCO2 was compared at 35°C (Figure 8). The volumetric expansion of pure hexane using SCO2 was a binary system. The astaxanthin complex in hexane was assumed to be a ternary system. In this case, the astaxanthin complex was assumed to be a solid phase as a whole even though the astaxanthin complex contained a mixture of astaxanthin esters, impurities and unidentified compound(s). The volumetric expansion of the ternary system (the astaxanthin complex, hexane and SCO2) was in close agreement with the volumetric expansion of the binary system (pure hexane and SCO2).
Figure 8.
Comparison of volumetric expansion of pure hexane (a binary system) and hexane containing the astaxanthin complex (a ternary system) using SCO2 at 35°C.
As expected from the volumetric expansion data of hexane with SCO2, the precipitation of the astaxanthin complex started at 5 MPa. For a solution, the volumetric expansion of the liquid phase should be independent of the initial volume of the solution once precipitation has occurred at a given temperature and pressure [59]. The data obtained from this work were consistent with this principle. This study also showed that the presence of the astaxanthin complex in hexane at a concentration of approximately 30 mg/100 mL did not affect the expansion behaviour of hexane with SCO2 significantly. This type of behaviour has been noted for other ternary systems such as dimethylsulfoxide containing yttrium acetate with SCO2 [60] while in some cases dimethylsulfoxide containing cefanoid with SCO2, an increase of the mixture critical pressure due to the presence of the solute has been evidenced [61].
Based on the HPLC-MS ionization analysis, the precipitation profile of the astaxanthin complex at different pressures during the supercritical antisolvent precipitation using SCO2 is given in Table 1. At lower pressure (5 MPa), most of the impurities and part of the unidentified compound(s) were precipitated. Impurities were fully precipitated at 5.5 MPa. The precipitation of astaxanthin monoesters was observed at the intermediate pressure (5.5 MPa). Eventually, diesters were precipitated at higher pressure (6.4 MPa).
Absolute pressure (MPa)
The precipitate obtained during supercritical antisolvent precipitation
Summary of the astaxanthin complex precipitation at different pressures during supercritical antisolvent precipitation.
Some proportions of the unidentified compounds remain in the supernatant throughout the precipitation process.
The polarity of the astaxanthin complex in hexane played a major role during supercritical antisolvent precipitation using SCO2. Non-polar material, the astaxanthin complex was dissolved in the non-polar solvent, hexane. As the polarity of impurities and unidentified compounds in hexane was relatively high compared to astaxanthin esters, these compounds were precipitated first at lower pressure. This is because non-polar hexane has less affinity toward polar compounds than non-polar compounds.
The polarity of astaxanthin monoesters was high compared to astaxanthin diesters because astaxanthin diesters are attached to two fatty acid end groups whereas astaxanthin monoesters have only one fatty acid end group. So, astaxanthin diesters have a stronger affinity towards hexane than astaxanthin monoesters. Monoesters were, therefore, precipitated first from the astaxanthin complex at intermediate pressure followed by astaxanthin diesters at high pressure. Non-polar hexane did not extract the unesterified astaxanthin or free astaxanthin because of its polar nature. The proportion of free astaxanthin present in the prawn waste could not be worked out using hexane as an extraction solvent.
During supercritical antisolvent precipitation using SCO2, some portion of the unidentified compound(s) remained in the sample even at higher pressure with no precipitation. The reason for this is still not very clear. However, this indicated that the unidentified compound(s) present in the prawn extract could be a mixture of several compounds. The yield study showed that 92.5% of the astaxanthin complex was precipitated at a pressure of 6.6 MPa. Further increase in pressure will increase the yield of the astaxanthin complex precipitation during supercritical antisolvent precipitation using SCO2. The yield of the astaxanthin complex obtained from published solvent extraction methods cannot be compared with this experiment because of the difference in the extraction method and prawn species used. Thus, an innovative method was developed in this research to recover the astaxanthin complex from prawn waste using SCO2. As the application is new, the results of this work cannot be compared with the published work.
Extraction of the astaxanthin complex from prawn waste using hexane offers an easy way of recovering the astaxanthin complex from prawn waste. The astaxanthin complex can be recovered directly from raw prawn waste irrespective of its moisture content eliminating the need for sample preparation steps such as freeze-drying. As the astaxanthin complex extraction in hexane is carried out at ambient temperature (23 ± 1°C), the energy required for heating is eliminated. This method also offers the possibility of reusing the solvent, hexane.
Characteristic study of the astaxanthin complex offers the separation of the astaxanthin complex into discrete fractions of either monoesters or diesters using normal-phase HPLC with a mobile phase consisting of hexane and acetone. Reliable identification of the type of astaxanthin ester in the astaxanthin complex can be achieved without prior derivatization by directly coupling HPLC with APCI-MS. This analytical method using normal phase HPLC-APCI-MS permits the isolation and identification of previously unreported impurities in the astaxanthin complex extracted from prawn waste.
The precipitation of the astaxanthin complex from hexane using environmental friendly SCO2 is the novelty in this research. The method is simple, easy and time-effective. This method eliminates the post-extraction steps such as purification, evaporation of the solvent and the associated time. Impurities can be precipitated at lower pressure during supercritical antisolvent precipitation of the astaxanthin complex instead of performing separate purification steps. It offers the recyclability of hexane as well as CO2. Heating hexane for the extraction process requires enormous amounts of energy that in turn produces greenhouse gas emissions. However, this method did not use a heating source. Therefore, this method saves energy and its associated costs.
This method may also reduce the degradation of the pigment as the precipitation of the astaxanthin complex occurs at low temperatures (35°C), with reduced light and an oxygen-free environment. The astaxanthin complex recovered from prawn waste can be used in aquaculture, food and pharmaceutical applications. Some of the processing steps can be omitted depending on the end use of the recovered astaxanthin complex. At last, this technique is not limited to recovering the astaxanthin complex from prawn waste only. The same technique can be used to recover the astaxanthin complex from other crustacean waste and other major producers of astaxanthin including microalgae.
A detailed characteristic study is required to identify the unidentified compound(s) present in the astaxanthin complex. Further improvement in the instrumental setup is required to effectively collect the pigment after precipitation with a view to the possible commercialization of this technique. The actual pressure that separates one form of the astaxanthin complex from others during supercritical antisolvent precipitation can be examined. This will offer an easy way to collect individual species of the astaxanthin complex and will facilitate the study of the stereoisomeric profile of each form of collected astaxanthin complex. These studies are best carried out while optimizing pilot-scale extraction and purification, a scale beyond the scope of this project.
I acknowledge Prof. Wilem F. Stevens, Mahidol University, Thailand, who guided me during this research, when required. I also acknowledge Dr Russel Pickford and Dr John Craske from UNSW for their support with the HPLC-MS analytical work.
Many Thanks to the Department of Primary Industries, Fisheries Conservation Technology Unit, NSW, Australia for supplying raw Eastern School prawns for the research. I also thank the Australian Government for the financial support by providing the International Postgraduate Research Scholarship to undertake this research.
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Written By
Renuka Vinothkumar, Frank Lucien and Janet Paterson
Submitted: 24 August 2022Reviewed: 29 August 2022Published: 24 September 2022
Open access peer-reviewed chapter
