A review on column CO2 fractionation methods from fish oil to omega-3, 6 and 9

Commercial interest in getting polyunsaturated fatty acid concentrates, especially omega-3 fatty acids such as EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) was increasing. Unsaturated fatty acids could prevent cardiovascular disease and improved cognitive function of the brain. Fish oil was the main source of fatty acids, especially omega-3 fatty acids. The content of omega-3 fatty acids (24%), omega-6 (5.37%) and omega-9 (12.6%) fish oil were quite high. Fish oil concentrate was needed in the food and pharmaceutical industries. Degradation of thermolabile compounds cannot be avoided in conventional separation methods. Some conventional oil separation methods had weaknesses and strengths. Column chromatography: the concentrate was very difficult to separate so it required a lot of solvents; distillation: low oil yield due to a lot of yawning; centrifugation: the results were heterogeneous; membrane: easy to plug; using solvents: low cost, technology exists, could be at room temperature and atmospheric pressure, the results were homogeneous. The choice of solvent determined the level of yield and safety. CO2 as a solvent was non-polar, non-toxic, volatile, high diffusivity, low surface tension, low viscosity, good extract degradation, low critical temperature, (31 °C), could prevent thermal degradation, fast, cheap and efficient.


Introduction
Fish oil is one of the nutrients that contains fatty acids rich in benefits obtained from fish body tissues. Fish oil contains triglycerides from three fatty acids with phospholipids, glycerol ether and ester wax [1]. Fish oil contains about 25% of saturated fatty acids and 75% of unsaturated fatty acids. This is confirmed by [2] that omega-3 fatty acids from fish oil contains a little saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), but they contain a lot of polyunsaturated fatty acids (PUFA). The characteristics of fish oils are that they contain a variety of long chain fatty acids with a number of carbon atoms ranging from 14 to 22, and unsaturation levels ranging from one to six double bonds per molecule. In addition, the oil content and composition of fatty acids differ in each type of fish. Even differences are also found in similar fish that are influenced by diet, habitat, temperature, seasonal influences, age and sex [3,4,5].
The results of test conducted by [6] show that omega-3 (24%), omega-6 (5.37%) and omega-9 (12.6%) fatty acids are quite high. [7] confirms that omega-3 and omega-6 fatty acids are essential fatty acids. Essential fatty acids are fatty acids that cannot be produced by the body, but are needed so
Omega-3 is able to prevent cardiovascular disease, Alzheimer's, increase fetal development and better baby's immune response [9], maintain mental health [19], as an anti-inflammatory and antiarrhythmic agent that is beneficial for heart function [20]. Omega-3 fatty acids, especially EPA and DHA are already widely used in foods and supplements, such as cereal-based products, milk and juice drinks. The total daily recommended EPA and DHA intake is depended on age. Children aged 1-8 years can consume up to 1.5 g; 2.0 g for ages 9-13 years and 2.5 g for 14-18 years. The amount of consumption can reach up to 3.0 g/ ay for adults [17].

Omega-6 Fatty Acids
Omega-6 (PUFA) which is consumed in excess without being offset by consumption of omega 3 can reduce LDL (Low Density Lipoprotein) cholesterol. However, High Density Lipoprotein (HDL) cholesterol also decreased. In addition, the disturbed balance between omega 3 and omega 6 causes blood to clot easily. Both of these are unfavorable because the decreased LDL/HDL ratio and easy blood clotting cannot prevent the occurrence of coronary heart disease, it can even trigger coronary heart disease [21].

Omega-9 Fatty Acids
Omega-9 (MUFA) has a protective power that can reduce blood LDL cholesterol, increase HDL cholesterol which is greater than omega 3 and omega 6, more stable than PUFA. Omega 9 fatty acids can prevent coronary heart disease (laboratory-tested and epidemiology), where research is always done using oils with low saturated fatty acid levels (about 5%). The results showed that omega-6 in the single form has negative properties because it is associated with increased production of eicosanoids (stimulants for tumor growth in animal experiments). But the presence of omega-3 and omega-9 in suitable proportions will have the potential to block the product of the eicosanoids compound [22].

Fish oil refining
Fish oils are extracted require a purification process to achieve the well quality characteristics that acceptable for human and animal consumption [23], while they contain insoluble impurities, phospholipids, free fatty acids, moisture, primary oxidation products, minerals, pigments and even persistent organic pollutants (POP) so that must be eliminated to get highest quality [24]. The traditional refining process includes several stages, such as degumming, neutralization, bleaching, deodorization and winterization. Each of the stages is especially important to remove the different classes of compounds (Table 2) and is the most studied and industrially applied process for the refining of fish oils. Furthermore, each of refining technology has its principle work (Table 3) [25]. [23] states that the use high temperature such as at neutralization, drying and deodorization process can make increasing peroxide and acidity value so that oil is susceptibility to oxidation and the formation of peroxides.

Fractionation methods
Some conventional and advanced oil separation and purification methods, including chromatography, distillation, winterization, membranes, formation of complexes with urea, enzymatic methods and using solvents (fractionation by supercritical fluids) [14,25].

Using Chromatography
The separation process is based on the ability to absorb different compounds in the stationary phase. However, it is very difficult to separate, due to various polarities and most oxygenated compounds. As a result, it requires a lot of solvents [26]. Chromatographic methods can be used to purify PUFA concentrates with a high degree of purity (> 95%) [27]. Supercritical liquids have the density and capacity of certain liquid solvents. However, it has lower viscosity and better diffusion, so it can be used as a carrier of substances such as the mobile phase in gas chromatography or as a solvent as a solvent in HPLC. This technique is known as supercritical fluid chromatography (SCFC) [28]. SCFC is very suitable for separating high purity omega-3 PUFAs, because it combines high selectivity from supercritical fluids and stationary phases [29]. The results of a study conducted by [30] showed that the production rate of DHA ethyl esters with a purity of 90% of 0.85% g/(kg ODS * h) and EPA ethyl esters with 53% purity of 0.23 g/(kg ODS * h) where supercritical CO2 as the mobile phase and octadecylsilane (ODS) as the stationary phase.

Using Distillation
The process of splitting oil into different fractions based on its boiling point. However, some chemical compounds in oil are unstable and sensitive to high temperatures. This results in low oil yield, polymerization reaction and easy coke from the remaining fraction [31]. In addition, molecular distillation can be used for the separation and purification of substances that are sensitive to temperature, because they use high vacuum pressure (lower than absolute pressure of 1.000-500 kPa) [32,33], and are also capable of removing organic pollutants [34]. This technology can also be applied to fish oil to get its concentrate (PUFA or MUFA) and several studies have been successfully carried out. For example, by [35,36] EPA and DHA as ethyl esters. In addition, the results of a study conducted [37] that the total concentration of EPA and DHA from tuna oil has increased (from 32.11% to 82.23%). The same thing was reported by [36] that the concentration of acylglycerol from omega-3 increased in sardine oil (from 63% to 91%).

Using Winterization
Winterization is a process that involves the crystallization of a portion of the oil by controlling the cooling, followed by filtration. The aim is to separate saturated fatty acids from unsaturated fatty acids. The principle of separation is based on the melting point of fatty acids, which tends to the number of long chains and unsaturation. Unsaturated and monounsaturated fatty acids have higher melting points  6 and winterization so that they can be separated by filtration, whereas polyunsaturated fatty acids (PUFA) have lower melting points so that they remain liquid in the oil [38]. Organic solvents can be used in the winterization process to increase the rate of mass transfer and crystallization fraction of saturated fatty acids [39]. As the results of the study [40] showed that the best conditions when 40% hexane without freezing agitation were able to increase the concentration of unsaturated fatty acids by 9.2% and reduce saturated fatty acids by 13.3%. In addition, several other studies report that using hexane solvents can increase omega-3 concentrates compared without hexane solvents [41], using ethanol as a solvent and at -5°C gives the best results in sardine oil [42], DHA and EPA experience an increase of 69% and 51.6%, respectively.

Using Membranes
The separation process is based on the ability to absorb different compounds in the stationary phase. This technology has been used in degumming, recovery of solvents in the extraction process, pigment removal, acidity reduction, concentrations of minor components, removal of waxes and emulsion separation [43]. This technology has been used in PUFA [44,45]. As a result of studies conducted by [46] on salmon oil that saturated fatty acids have decreased (from 27.2% to 20.2%), while unsaturated fatty acids have increased (from 41.6% to 46.5%, with increasing DHA from 9.9% to 11.6%, and EPA from 3.6% to 5.6%). However, the method is easy to plug. As a result, it is necessary to clean, maintain and dispose of concentrates regularly [47].

Formation of Complexes with Urea
This technique is the simplest and most efficient technique for obtaining omega-3 PUFA concentrates as free fatty acids or triacylglycerol ethyl esters. That is, saturated and monounsaturated fatty acids are separated from polyunsaturated fatty acids found in urea solution. Saturated and monounsaturated fatty acids separate during the crystallization process, whereas PUFAs do not form complexes so they remain in the liquid fraction [48]. Furthermore, [49,50] examines the relationship of urea and fatty acids, temperature and crystallization time. The results of the study [49] showed that the content of EPA and DHA was higher than 85.02% in tuna oil with a ratio of urea and fatty acids is 15, a temperature of -5°C and a time period of 20 h. Also, [48] reported that EPA and DHA had increased, namely EPA from 15.39% to 19.76% and DHA from 17.45% to 29.61%.

Using Enzymatic Methods
Concentration by enzymatic method is based on the selectivity of several lipases for certain fatty acids or positions in the triacylglycerol molecule, catalyzing the hydrolysis reaction, alcoholysis or trans-esterification of triacylglycerol [51]. Some studies propose that this method is carried out before the application of other stages, such as distillation molecules or filtering with membranes, to obtain omega-3 fatty acid concentrates [25]. According to [52] that the use of supercritical CO2 as a solvent is very good in the process of alcoholysis, because CO2 is environmentally friendly, non-toxic and easily separated from the product so that it is suitable for use in enzymatic methods. Moreover it can be easily combined with other processes such as SCFE, FSCF, SCFC and encapsulation with supercritical CO2 [29]. Furthermore, a study conducted by [52] on Sardinella aurita oil showed that 40% increased than usual when using supercritical CO2.

Using Solvents
The separation process is based on differences in the solubility of different compounds to solvents, relatively low cost, available technology, can be at room temperature and at atmospheric pressure and is able to separate oil in different fractions, efficient, high yield [53].

Fractionation by Supercritical Fluids (FSCF).
Several studies have been successfully carried out using this method. [54] evaluated the fractionation of freshwater fish oil with quite low omega-3 PUFA content (10%). The best fractionation is obtained when isotherm 33 and 40°C at 20.000 kPa. [55] also studied this method in tuna oil by using ethanol as a joint solvent. The results show that it is very effective and can restore PUFA. Because the complexity of fish oil is hampered so that the formation of methyl esters and triacylglycerol ethyl esters from fish oil increases and is more stable than fatty acids. This is obtained by hydrolysis of triacylglycerol (TAG) and fatty acid alkylation before FSFC [54,29].

Types of Solvents
Ethanol is a common solvent and widely used by industry, has a low boiling point and tends to be safe to use. Ethanol has a boiling point of 70°C so that the extraction temperature used can attract all of components in the raw material [58]. According to [59], solvents which commonly used to extract fats are alcohol (methanol, ethanol, isopropanol, n-butanol), acetone, ether (diethyl ether, isopropyl ether, dioxane), halocarbons (chloroform, dichloromethane), hydrocarbons (hexane), benzene, cyclohexane, iso-octane), or mixtures of these solvents. [60] revealed that the greater the amount of organic solvents used in the extraction process, the higher the amount of dissolved components. The main factor in fractionation at low temperatures is the difference in melting point and solubility of the fat components to be separated. CO2 is one of the organic solvents, non-polar, non-toxic, volatile, high diffusivity, low surface tension, low viscosity, good extract degradation, low critical temperature, (31°C), can prevent thermal degradation, fast, cheap and efficient. In addition, supercritical CO2 is more beneficial compared to other organic solvents, because it is considered a safe and non-flammable solvent [61].

Supercritical Fluid
Supercritical fluid conditions are formed when the fluid conditions are above its critical temperature and pressure. Unlike gases, supercritical fluid cannot be condensed into a liquid-gas state by pressure regulation. Supercritical fluids provide a large density range. This can be used to control the solubility of the solvent. Supercritical fluids are characterized by high density, low viscosity, and medium diffusivity between gases and liquids. This unusual property, instead makes supercritical fluid as an ideal and potential solvent.

Viscosity and Diffusivity of Supercritical Fluid.
In supercritical conditions, the interaction force between molecules is relatively low. It causes high mobility of molecules and causes supercritical fluid to have a lower viscosity than liquid, so supercritical fluid has the ability to penetrate into the solute so that it extracts a better solute. The viscosity and diffusivity of the supercritical fluid approaches the gas as the pressure is raised. Diffusivity will increase with increasing temperature. Although not as large as gas, supercritical fluid diffusivity is greater than liquid diffusivity, resulting in a greater mass transfer rate. Low viscosity and high diffusivity will make it easier for the solvent to penetrate the extracted material. The main advantage of supercritical fluid compared to liquid is greater diffusivity. The ability of a solvent to dissolve a solute is expressed in terms of the number of solvents per unit volume. Supercritical fluid has a density that almost comparable to a liquid. As high density, many molecules can dissolve solutes. So that the ability of dissolving becomes greater.

Solubility of Supercritical Fluid.
Gas solubility in a solvent usually decreases with increasing temperature. But at high temperatures, close to the critical temperature of the solvent, the solubility of the gas generally rises in proportion to temperature. Most of solubility is expressed in units of mole fraction or Henry's constant. Many liquids are developed as solvents of supercritical states by heating and increasing pressure. The solvent that is often used for the supercritical fluid extraction process is CO2 solvent. This is caused the low critical temperature of CO2 allowing the experimental process to approach the ambient temperature. This condition is relatively easy to be achieved, because not too much energy is needed. Because it does not use non-organic solvents, so there is no solvent residue in the extract, besides that CO2 is non-toxic and non-flammable and odorless. However, although CO2 has some of these advantages, CO2 has a limited ability to dissolve polar substances. The supercritical CO2 characteristics can be increased by the addition a second component which is a polar substance and dissolves in supercritical CO2.
Guenther [62] states that the conditions for a good solvent are selective, low boiling point (so easily separated by extract), not easy to react (chemical inert), cheap and not dangerous. The use of chemicals as solvents, such as chloroform, methanol and hexane can have an impact on human health and the environment, because chemical solvents are toxic. Solvent selection is important to obtain optimum yield, good oil quality and good for health [61,63].

Conclusions
In this paper, the fractionation method and the selection of solvent type to separate fish oil content, especially the omega-3, omega-6 and omega-9 fatty acids have been discussed, and conclude that 1) using the solvent fractionation method is more profitable, because the low costs relatively, efficient, high yield, technology exists, can be at room temperature and at atmospheric pressure and able to separate oil in different fractions; 2) using type of CO2 as solvent is safer, healthier and cheaper.