Malays used material that comes from plant, animal and natural resources as a traditional medicine. The eel, scientifically known as Monopterus Albus, is included in the fish genus. The body is long and its head is rounded, with the presence of gills. According to traditional medicine practitioners, regular consuming of eels helps to boost the body's immune system, stabilizes the blood pressure, smoothens the skin texture, prevents hepatitis and enhances the memory power. However, many people are quite reluctant to eat the eel.
According to Rout et al. (2007) extraction is
one of the key processing steps in recovering and purifying active ingredients
contained in bio materials. Several types of extraction methods such as hydro
distillation, soxhlet and supercritical can be used to extract the oil (Reverchon
and Marco, 2006; Quitain et al., 2006; Li
et al., 2000; Suslick and Price, 1999; Raghuram
et al., 1992). Trusheva et al. (2007),
reported that ultrasonic extraction is the most efficient method based on yield,
extraction time and selectivity. Other than that, the extraction process can
be done at room temperature. Ultrasonic extraction process also reported as
a fast, inexpensive and efficient alternative compared to other extraction process
(Kimbaris et al., 2006). The sonication of liquids
will generate sound waves that propagate into the liquid media resulting in
alternating high-pressure and low pressure cycles.
The high-pressure cycles of the ultrasonic waves support the diffusion of solvents,
such as hexane into the cell structure. As ultrasound breaks the cell wall mechanically
by the cavitations shear forces, it facilitates the transfer of lipids from
the cell into the solvent (Adeniyi and Bawa, 2002).
Therefore, attempt was made in this research to extract oil from the eel. This
research focussed on the preliminary study to identify the best operating condition
in extracting oil from the eel using ultrasonic extraction method. The extracted
oil has a great market value especially in pharmaceutical industries.
MATERIALS AND METHODS
Materials: Fresh eels (Monopterus albus) purchased from Kuantan wet market were used in this research. Ethanol with 99% purity was used as solvent for extraction process. Hexane, potassium hydroxide and methanol also with 99% purity were used during samples analyses using Gases Chromatography Mass Spectrometer (GC-MS).
Sample preparation: Fresh eels purchased from the wet market were washed using fresh water. The internal organs were removed. Then, the Eels were cut into fillets and dried at temperature of 60°C using an oven. After that, the dried fillet was grinded into powder form by using a dry blender. Finally it was stored in a sealed plastic container and placed in a refrigerator until used.
Extraction method: The extraction process was dome using ultrasonic extraction unit. This apparatus consist of 500 mL extraction beaker, ultrasonic bath and ultrasonic generator. The ultrasonic bath has frequency of 25 kHz while the power can be varied up to 500W. The extraction beaker was immersed in the ultrasonic bath. Ethanol was used as solvent during the extraction process. Extraction process was initially done in the absence of ultrasonic wave. For example, 10 g of dried eel was mixed with 300 mL of ethanol and placed in a 500 mL beaker. The beaker was left for 20 min at ambient condition without sonication. After that, the sample was filtered to remove the powder and evaporated by using rotary evaporator to get oil. The amount of extracted oil was recorded. The extracted oil was analyzed using 785 DMP Titrino and Gas Chromatography Mass Spectrometer (GCMS). Then, the same procedure was repeated using different ultrasonic power of 100, 200, 300 and 450 Watts to determine the most suitable operating power. Next, the same procedure was repeated using different solvent volume of 50, 100, 200 and 500 mL to determine the best solvent volume. Finally, the experiment was run at 20, 30, 50, and 60 min to determine the best sonication time.
Free fatty acid and acid value determination: Free Fatty Acids (FFA)
values were used as the quality indicator of oil. It can be determined by using
free fatty analyzer model 785 DMP Titrino. This equipment can also determine
acid value of the oil without using conventional method which is titration method.
The Acid Value (AV), which is defined as the number of milligrams of KOH required
to neutralize the free fatty acids in 1 g of sample, is a measure of FFA content
or a measure of the amount of free acids present in a given amount of fat. Five
milliliter of fish oil was diluted with 50 mL of ethanol and the free fatty
acid content and acid value was detected by this equipment.
Chemical composition determination: Agilent 6890 GCMS equipped with a Flame Ionization Detector (FID) and automated split injection 7683 auto sampler was used to determine the fatty acid composition in the extracted oil. The inlet temperature and the detector temperature for GCMS were set at 250 and 280°C, respectively. The injection volume was set at 1 μL. Hydrogen gases were used as detector.
Yields determination: The extraction yield was calculated using Eq. 1:
where, Wo denotes the weight of extracted oil in grams and Ws denotes the weight of eel powder used in grams.
RESULTS AND DISCUSSION
Influence of ultrasonic power on oil yield: The oil yields at different
ultrasonic powers are shown in Fig. 1. The yields for ultrasonic
power of 100, 200, 300 and 50 Watts were 2.20, 2.50, 2.40 and 2.30%, respectively.
The results show that the best ultrasonic power was obtained at 200 Watts. Improved
of oil extracts from ell at ultrasonic power less than 200 Watts may be explained
in terms of cavitational effects caused by the application of the ultrasonic
waves. Cavitation normally takes place in liquid medium once the media is subjected
to rapid, alternating high pressure. Voids containing small micro bubbles are
created when the differences between amplitude pressure of ultrasonic waves
and the hydrostatic pressure in the liquid is large enough to exceed the local
tensile strength of the liquid medium. These bubbles expand during negative
part of pressure cycle or rarefaction cycle; reach the maximum radius and then
collapse at the onset of positive pressure cycle or compression cycle (Ensminger,
1998). Bubble collapse may cause strong shear forces to be exerted that
can cause micro fractures to be formed in biological tissues (Vinatorua
et al., 1996).
According to Mason (1990), an increase in ultrasonic
intensity will contribute to an increase in cavitation effect. Larger ultrasonic
intensity indicates greater ultrasonic energy entering the liquid system, thereby
producing more cavitation micro bubbles. This consequently enhances stronger
shear forces to be exerted during the bubble collapse and can cause more microfractures
to be formed in biological tissues. This will ease the penetration of lipids
or oil from the eels powder.
However, as the ultrasonic power increased beyond 200 Watts, the extraction
yields were decreased. This may due to the formation of a large amount of cavitation
micro bubbles at intensity above 200 Watts. When a large amount of cavitation
bubbles are present inside the liquid medium, the tendency of the bubbles to
collide becomes higher.
||Oil yields vs. ultrasonic power in 300 mL ethanol and 20 min
||Oil yields vs. solvent volume in 200 Watts ultrasonic power
and 20 min sonication time
Upon collision, bigger micro bubbles are created at ultrasonic power higher
than 200 Watts. Since, the time available for the bubbles to collapse is insufficient,
the bubbles will form a bubble cushion at the radiating face of the ultrasonic
transducer, thereby reducing the effects of coupling sound energy to the liquid
system. Such phenomenon tends to reduce the amount of ultrasonic energy being
transmitted to the liquid medium and produces less cavitational effects. This
ultimately results in reduced formation of micro fractures in the biological
tissues. This phenomenon explains why further increase in ultrasonic power beyond
200 Watts decreases the extraction yields.
Influence of solvent volume on oil yield: Figure 2
shows that the oil yields started at 0.80% and increased to 1.50% and 2.60%
when the amount of solvent are increased. The highest extraction yields which
were 5.00% were determined at solvent volume of 500 mL. As ultrasound breaks
the cell wall mechanically by the cavitation shear forces, it also facilitates
the lipid transfer from the cell in to the solvent. Larger solvent volume promotes
an increasing concentration gradient between solvent and solid samples. As a
consequence, a larger mass transfer between solid and solvent occurs. This finding
is aligned with those reported by Franco et al. (2007).
Influence of sonication time on oil yield: Figure 3 shows the percentage of oil yield as a function of sonication time. The oil yields are increases with the increased of sonication time. The amount of extraction yields without sonication is 4.20%. The highest extraction yields, which were 7.20% was obtained at 60 min sonication time. The increase of sonication time, increased the duration of cavitation process occurs in the extraction process. Therefore, increase the oil yields.
Free fatty acid contents in oil yield: The amount of Free Fatty Acid (FFA) contents in the oil yields are tabulated in Table 1-3.
||Oil yields vs. sonication time in 500 mL of ethanol and 200
Watts ultrasonic power
||Amount of free fatty acid in the oil yields using 300 mL of
ethanol and 20 min sonication time with different ultrasonic power
||Amount of free fatty acid in the oil yields using 200 Watts
of ultrasonic power and 20 min sonication time with different solvent volume
||Amount of free fatty acid in the oil yields using 200 Watts
of ultrasonic power and 500 mL solvent volume with different sonication
The results particularly show that FFA volume increased with increased sonication
time as well as ultrasonic power. Solvent volume also plays an important role
in increasing the yield of FFA volume. As what has been claimed by Boran
et al. (2005) acid value is generally associated with lipase activity
originating from microorganisms or biological tissue. The maximum acid value
obtained through this study is 0.22 g/100 g and are within the acceptable limit
as to compare to what have been reported by Bimbo (1998).
The effects of ultrasonic power, solvent to solid ratio and sonication time on extraction yields were investigated. The best parameter to extract the oil was ultrasonic power of 200 Watts, 500 mL of ethanol and 60 min sonication time. The amount of oil extracted was 7.2% with FFA contents of 0.22 (g/100 g).
The authors would like to express their heartiest gratitude to the members of separation pyramid in the Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang.