1. Introduction
African catfish (
Clarias gariepinus) occupy a large area in aquaculture in Africa [
1]. Recently it has spread in Europe and southern Asia for its great economic interest: faster growth rate, omnivorous feeding habit, and high resistance to environmental stress [
1–
3]. It is also considered one of the most important tropical catfish species for aquaculture [
4]. In Malaysia, it is called ikan keli and is the most preferred fresh water fish among Malaysians. Fish oils are rich sources of natural bioactive lipid components. These lipid components are commercially used in the pharmaceutical and food industries and as human health supplements. Moreover, fish is being considered an important diet due to its polyunsaturated fatty acids (PUFAs) content. Its curative and preventive effects are well recognized in treating cardiovascular diseases, autoimmune disorders, various kinds of inflammation [
5], cancers and their effect in the neurodevelopment of infants [
6]. Therefore, the consumption of fish and fish products is increasing day by day all over the world. Moreover, several food process industries supply their products under different brands throughout the year for those who are not habituated to the direct consumption of fish.
Hence, the by-product generation, including skin, viscera, head, scales and bones from the fish process industries has increased, mostly considered previously as worthless garbage and discarded without any attempt at recovery [
7,
8]. It is estimated that annually 20 million tonnes or equivalent to 25% of the total production of fish is discarded as by-products or waste materials [
9]. As a result, these huge amounts of by-products create both disposal as well as pollution problems [
10]. However, these materials can be a potential source of enzymes and fats [
8,
11,
12], protease producing bacteria [
13], lactic acid fermentation media [
14] as well as protein and bioactive lipid components. Depending on the species, food habit, geographical location, catch season and maturity, the oil content of fish waste lies between 1.4% and 40.1% [
15].
Many researchers have reported the extraction, fractionation and purification of fish oils using various conventional methods, such as hydraulic pressing, vacuum distillation, urea crystallization, hexane extraction, and conventional crystallization. The major disadvantages of these methods are the requirement of high temperatures that affect the nutritional quality of the fish oils, degradation of the heat sensitive labile natural compounds, and toxic solvent left in the final products, all of which have adverse human health effects [
16,
17]. Moreover, a large number of studies have been carried out on one salt water fish species for lipid extraction using various methods, while little attention has been paid to the extraction of lipid from fresh water fish species. Supercritical fluid extraction (SFE) is the method of choice for the extraction and fractionation of edible natural oils from various sources. To date, numerous studies have been carried out for the extraction of fish oil fatty acids using the SFE technique [
18–
23]. Over the last 20 years, SFE has been acknowledged as a promising alternative to the organic solvent extraction method in the field of natural fats and oils. The major merits of the SFE method is the lack of solvent residue left in the final products and better retention of valuable components [
24–
29]. Carbon dioxide is used as a solvent due to its nontoxic, non-flammable, inexpensive, and cleanness, which offer great opportunities for complex separation processes.
The waste materials from the African Catfish, mainly the viscera, can be a reliable source of raw material throughout the year for the extraction of lipid at an industrial scale as it has minimal/negligible seasonal variation regarding chemical composition [
4]. Therefore, the objective of this study was to optimize the SC-CO
2 extraction of fish oil from the wastes such as viscera.
3. Experimental Section
3.1. Materials
Fresh African Catfish (Clarias gariepinus) samples were collected from a local market in Malaysia. A cylinder of carbon dioxide with a purity of 99.99% was purchased from Malaysian Oxygen Ltd. Kuala Lumpur, Malaysia and all other solvents and chemicals used in this experiment were analytical grade and obtained in Malaysia.
3.2. Sample Preparation for Experiments
The samples were immediately de-headed and washed with copious amounts of fresh water to separate the viscera. The viscera were then stored overnight in a freezer at −18 °C, and then freeze dried (Model: LABCONCO, USA) at a constant drying temperature of −47 °C and vacuumed at 0.133 bar. The dried samples were ground using a blender and stored in an airtight glass bottle in a coldroom at 6 °C pending laboratory use.
3.3. Moisture Content Determination
The moisture content of the dried sample was determined by the oven dry method [
40]. Five grams of finely ground sample was place in pre-weighted ceramic crucibles before being put into the oven. The temperature of the oven was set at 105 °C, and the heating process continued until constant weights of the samples were achieved. Then, the crucibles were transferred to a desecrator to cool before reweighing. The difference of the two weights (initial and final) indicated the moisture content and it was found to be 3.95% in viscera.
3.5. Apparatus and Procedure of Supercritical Fluid Extraction
All the runs were carried out in a supercritical fluid apparatus (Model PU-1580, Jasco Corporation, Tokyo). For each trial, 5 g of dry sample was loaded into a 10 mL extraction vessel (model Ev-3, Jasco Corporation, Tokyo), and then placed into an external water bath at a temperature ranging from 35 °C to 80 °C. Then, the valve of the CO2 cylinder was opened and the CO2 allowed to circulate through the cooling jacket of the chiller to cool before reaching the extraction vessel at a constant flow rate ranging from 1 mL/min to 3 mL/min (Model 631 D, Tech-Lab Manufacturing sdn. Bhd., Selangor, Malaysia), where CO2 gas was converted to liquid form. After reaching the desired pressure in the extraction vessel, the CO2 valve was closed for a certain period of time to soak the sample in pure CO2: regarded as the “soaking time” for this experiment. A back pressure regulator (BPR) (model BP-1580-81, V, Jasco Corporation, Tokyo) was used to control the system pressure and separate the CO2 from the extract. Then, the CO2 valve was opened again during continuous extraction at constant pressure, temperature and flow rate. At each condition, experiments were conducted in duplicate, and each yield was the mean of duplicate measurements. Finally, the yield trap was collected and stored at −18 °C for further analysis.
3.6. Experimental Design
A central composite design consisting of 30 experimental runs with six replications at the central points were employed to optimize the extraction variables, namely temperature, pressure, flow rate and soaking time. The creation of design matrix, experimental data analysis and optimization were all undertaken using Minitab v.14 statistical software. The polynomial equation represents all possible combinations of the extracting variables (
X1,
X2,
X3 and
X4) of their main, quadratic as well as the interacting effects on the response variable of oil yield (
Y). A preliminary study was conducted to select the range values of the parameters (
Table 2). More emphasis was given for the selection of temperature level: the lower limit was 35 °C, just above the critical points of CO
2 (31.1 °C) and the upper limit was not more than 80 °C to save the thermally sensitive compounds from thermal degradation [
41]. All the design points were performed three times except the centre point. Experiments were run in randomized order to minimize the effect of unexplained variability induced by extraneous factors. The polynomial regression equation presented below was used for predicting the response variable (
Y).
Where, Y is the response (percentage of oil yield) β0 is a constant and βi, βii, βij are the linear, quadratic and interaction terms, respectively. Xi and Xj are the levels of independent variables.
4. Conclusions
At the optimized condition, the SFE extracted oil yield was 67.0% from the viscera on a dry weight basis, which was reasonable when compared with the yield extracted using the Soxhlet method. However, at the optimized conditions, all the individual variables e.g., pressure, temperature, flow rate and soaking time were the most significant linear terms and the system was very sensitive to minimal changes in those variables. By contrast, the quadratic terms: the flow rate and soaking time were the most significant whether positive or negative. On the other hand, in the interactions no (0) effect was found between pressure and flow rate; however, all the interaction terms had a positive effect except in the interaction between flow rate and soaking time.