Enrichment of Omega-3 from Anchovy (Stolephorus sp.) Fish Oil by Enzymatic Hydrolysis

Article history: Anchovy (Stolephorus sp.) is an economically important fish in Indonesia. Anchovy contains Omega-3 that is important to maintain the health of the heart and brain. This study aimed to enrich the omega-3 content of anchovy oil from the North Sea of West Java. The extraction of anchovy oil was carried out by the soxhlet method. Enrichment of omega-3 from anchovy fish oil is carried out by hydrolysis with a commercial lipase enzyme at a concentration of 500, 1000, 1500, and 2000 unit/0.6 g fish oil for 5, 10, 15 and 20 hours. Before enzymatic hydrolysis by lipase, fish oil was added with water and organic solvent (ethanol, toluene, n-hexane). The Omega-3 content of fish oil products were analyzed by using Gas Chromatography (GC) with FID detector with retention time 14.068 min and 15.506 min for α-Linolenat (ALA) and eicosapentaenoic (EPA), respectively. The results showed that the highest omega-3 content of ALA and EPA were 0.54 and 1.103%, respectively that produced by the addition of nhexane was 0.5 mL and lipase activity of 1000 units for 15 hours of hydrolysis. Received date : 14 November 2019


INTRODUCTION
Indonesia was awarded a vast ocean with various of fish resources. Indonesia is the largest archipelago country in the world because it has a large sea area and a large number of islands. million Km 2 and 2.55 million Km 2 exclusive economic zone total territorial area of 7,81 million Km 2 [1].
Indonesia as a country endowed with large marine resources, including the greatest richness of marine biodiversity. Anchovy is widely available in Indramayu's market. In this research, enrichment of omega-3 content is carried out by enzymatic reaction using commercial lipase and anchovy oil obtained from fishermen of Indramayu beach.
Omega fatty acids (omega-3 and omega-6) are types of long-chain unsaturated fatty acids, Poly Unsaturated Fatty Acids (PUFA), which are essential or cannot be produced by a human body. Therefore, the intake of omega-3 and omega-6 nutrients derived from the food is needed. Adequate omega-3 and omega-6 intakes can optimize the growth of brain cells related to children's intelligence. The lack of omega-3 in the human body can cause fatigue, weak memory, depression, Alzheimer's, and schizophrenia [2,3]. In addition to omega-3, the intake of omega-6 is also important, although the amount needed is less than the amount of omega-3 [4]. The lack of omega-6 fatty acids can cause a decrease in growth rates, hair fall, infertility, fat infiltration in the liver, and scaly skin [5].
Oleic acid (OA) is the major monounsaturated fatty acid (MUFA) present in salmon oil and belongs to the family of omega-9 fatty acids, which correlates with the high MUFA content of fish oil with cardioprotective effects [12].
On the contrary, Palmitic acid (PA; C16:0) and OA affect cholesterol and fatty acid metabolism oppositely than oleic acid [13]. Saturated fatty acids (SFAs) affect involved factors in cholesterol metabolism [14]. For the aforementioned reasons, an in-depth knowledge of the presence and concentration of lipids in fish oil is mandatory.
DHA and EPA are important to prevent some diseases, including coronary heart disease, inflammation, hypotriglyceridemic effect, and diabetes [16,17].
Omega-3 PUFAs can be obtained from fish oils and marine microalgae and they have been purified using several procedures: urea complexation, chromatography, distillation, low-temperature crystallization, supercritical fluid extraction and enzymatic methods [18,19]. The main advantage of the enzymatic method is that low temperatures and non-aggressive reagents are used, which allows to maintain the PUFA structure unchanged. However, stable lipases, which are not deactivated in the reaction conditions, are essential for these processes to be economically viable [20].
During the last decade, several studies have shown positive effects of fish oils on cognitive development and vision enhancement in newborns, as well as in young children [21]. The aim of this study was to enrich the Omega-3 (EPA and ALA) content of anchovy oil by the enzymatic hydrolysis method.

Methods
Drying anchovy and extraction of fish oil was done one time, while the measurement of enzymatic reaction results was duplicated using GC.

Anchovy flour Preparation [22]
Anchovy sample was dried at 50 o C, for 48 hours. The dried sample was then crushed with a chopper to become anchovy flour. Fish oil was extracted from anchovy flour by using the soxhlet method.

Anchovy Oil Extraction
The extraction of fish oil was carried out by the soxhlet method. 80 grams of fish flour was wrapped in filter paper, then the wrapped flour was placed in the Soxhlet, and 300 mL of nhexane was added. This process was carried out for 8 hours, and n-hexane was evaporated. [23] 0.3 grams of fish oil was added with 10 ml of carbon tetrachloride and 20 ml of iodinebromide. The solution was placed in a dark room for 30 minutes and shaked for each 10 minutes. 10 mL of 15% KI and 100 mL of distilled water free of CO2 were added to the solution and then titrated with 0.1 N Na2S2O3 until the solution was pale yellow. Furthermore, into these solution was added 1 mL of starch solution and titrated until the blue color disappears. Iodine numbers can be calculated using the following equation:

GC and GC-MS Sample Preparation (Sample Esterification)
Firstly 0.125 g of fish oil was put in a test tube. Then, 0.5 ml of boron trifluoride (BF3) in MeOH (14%) was added to the test tube. Next, the test tube containing the fish oil and boron trifluoride (BF3) in MeOH (14%) was incubated in an incubator shaker at 55 o C for 1.5 hour. Then, 0.5 ml of saturated sodium hydrogen carbonate (NaCHO3) and 1.0 mL of n-hexane was then added to the test tube. The mixture was mixed and shaken well using a vortex for about 30 second. Then, the mixture was stored at freezer for 10 minutes, so that it will form two layers. Lastly, 0.5 ml of upper layer contain hexane was carefully transferred into a vial for GC and GCMS analysis.

Gas Chromatography (GC) analysis
Fatty acids composition of fish oil samples were analyzed using gas chromatography (GC) Agilent Technologies 7890B equipped with split injector, detector flame ionization detection (FID) system to separate and quantify each FAMEs components. FAMEs were separated using HP-5 column (30 m x 0.32 mm i.d, 0.25). Oven temperature was held at 100 o C, 2 min, then increased to 240 o C at 10 o C/min, held for 1 min. Temperatures for injector and detector were set at 250 o C and 300 o C. 1 µl of sample was injected with ratio 100:1 at column temperature 100 o C. Carrier gases that were used for the system are helium gas 3.0 mL/min controlled at 15.726 psi, hydrogen and air used for FID was held at 30 and 400 mL/min.

RESULT AND DISCUSSION
Enrichment of omega-3 content in Anchovy oil has been carried out by enzymatic hydrolysis method using commercial lipase. Anchovy oil has been extracted from anchovy flour with the yield 5.09%. The iodine number of Anchovy oil was 95.35g I2 / 100 g According to SNI proper oil consumed > 45-46 mg / g. The higher the iodine number of oil, the more double bonds it has, the better its quality for consumption [24]. The fatty acid content of anchovies oil has been analyzed using GC-MS and the chromatogram was presented in Figure 1. Based on the data from GSMS chromatogram, The types of fatty acids profile derived from anchovy oil was presented in Table  1.
In this study, the effect of the organic solvent in enzymatic process was evaluated by using some solvent such as water, toluene, ethanol and hexane. The fatty acids content linolenic acid (ALA) and eicosapentaenoic acid (EPA) of the hydrolysis products was then analyzed by using GC-FID. The effect of the organic solvent in enzymatic process on ALA and EPA content was presented in Table 2. The data on Table 2 showed that the highest EPA (0.16%) and ALA (0.44%), was obtained when n-hexane is used in enzymatic reaction. Enzymatic hydrolysis with the addition of hexane, heptane and toluene solvents can increase the catalytic activity of enzymes [25]. The study was continued to find the optimum concentration of n-hexane in hydrolysis of Omega-3 in Anchovy fish oil and the results is presented in Figure 2. The data on Figure 2 showed that the optimum addition of n-hexane was 1.25 mL for EPA and 0.5 mL for ALA (total volume of reaction 3 mL). The addition of n-hexane volume affected the EPA and ALA content. The addition of organic solvent can increase the thermostability of lipase. Hydrophobic properties of organic solvent will depend on some parameters such as dielectric constant, dipole moment and log P value. Hexane is known has a log P value (3.5) higher than toluene (2.5). [26] To optimize the enzymatic hydrolysis of anchovy oil by lipase, in this study was evaluated the effect of incubation time and enzyme concentration to Omega-3 content. The results was presented in Figure 3. Reaction time is one of parameters for an enzyme to catalyze and generate the product. Long reaction time is not effective and likely to suffer from substrate reformation due to reversible property of an enzyme. This phenomenon can be observed in Figure 4 as Lipase is used to hydrolyze triglycerides: Fig. 4. The mechanism of the triacylglycerol hydrolysis reaction by the lipase enzyme [28].
The data on Figure 3 showed that the highest of EPA (1.75%) and ALA (0.54%) content was reached after 15 hours incubation time.
The study was continued to evaluate the optimum of enzyme concentration in enzymatic hydrolysis process.  Figure 5 showed that the optimum enzyme concentration was 1000 units per 0.6 g of fish oil with the concentration of EPA and ALA are respectively 1.103% and 0.520%. Lipase was concentrated the polyunsaturated fatty acids such as EPA and DHA. The ester bond between glycerol and monounsaturated fatty acid and saturated fatty acid was cleaved by lipase [27]. Based on the data on Figure 5, the increasing of EPA and ALA concentration are between 30-50%, after hydrolysis with lipase (1000 unit) for 15 hours. A probable explanation for the low lipase activity is the structure of enzyme, which do not accommodate the esters of PUFA from Anchovy oil in their active sites.

CONCLUSION
Enrichment of Omega-3 anchovy oil by hydrolysis using the commercial lipase enzyme was successfully carried out. The addition of 0.5 mL n-hexane solvent, lipase activity of 1000 units / 0.6 gram fish oil and 15 hours incubation time were the most optimum conditions to produce EPA and ALA with concentration of 1.103 and 0.520%, respectively.