Abbreviations
AA Arachidonic Acid
ACR Aseton Cristallization fraction
ALA α-Linolenic Acid
Cis-FA cis-fatty acids
DHA Docosahexanoic acid (22:6n3)
DPA Docosapentaenoic acid (22:5n3)
EPA Eicosapentaenoic acid (20:5n3)
EA Elaidic Acid
FAME Fatty acid methyl ester(s)
GC Gas Chromatography
HDL High Density Lipoprotein
HCR Hexane Cristallization fraction
LA Linoleic Acid
LDL low density lipoprotein
MAM Minyak Ala Muncar
MUFA Monounsaturated fatty acid(s)
n-3 omega-3
NUCF Non-urea complexing fraction
OA Oleic Acid
PUFA Polyunsaturated fatty acid(s)
SFA Saturated fatty acid(s)
SDA Stearidonic acid (18:4n3)
Trans-FA trans-fatty acids
UCR Urea Cristallization fraction
INTRODUCTION
Muncar Banyuwangi is one of the biggest fish canning industry centers in Indonesia. Both large industries and small fish processing factories operated in Muncar. One of the serious impacts resulted from this situation is oil waste that is circulated throughout Indonesia by the local community used as animal feed supplements for fish, chicken, goats, and cows. The local communities give the term waste oil as ”Minyak Ala Muncar (MAM)” which means oil typically of Muncar. they treated the waste by heating at high temperatures accompanied by the addition of caustic soda so that the oil and residue are separated. This process is known as saponification in oil purification [1], [2].
GCMS assay showed that the MAM rich in a variety of fatty acids namely LA, AA, ALA, EPA, DHA, palmitic acid, stearic acid, arachidic acid, myristic acid, Palmitoleic acid, and EA [3]. EPA, DHA, ALA are a type of n-3 PUFA that have a good influence on the cardiovascular system [4] including preventing sudden death [5]. EPA and DHA also could accelerate the healing process of keloids [6], increased HDL levels, reduced LDL, reduced platelet aggregation [7], and reduced the growth of breast cancer cells [8]. The combination of n-3 and n-6 groups in oil also has an impact on health. The ratio between n-3 and n-6 in the body greatly affects somebody’s health [9]. Greenland Eskimos have the lowest death rate from coronary heart disease compared to the other races. it is because of the ratio between n-3 against n-6 in Eskimo’s dietary nutrition is highest compared to other races. This ratio greatly affects the increase or decrease in LDL in the body [10].
The presence of EA in MAM should be an important concern because not only its large amount reaching 26.8% in MAM [3] but also EA caused many health problems. EA appears in MAM was allegedly due to the high-temperature of heating during processing, where heating was known affecting of fatty acid content [11] and increasing of oil toxicity [12]. Under conditions of high heating temperatures repeatedly and continuously, cis-FA such as OA could turn into trans-FA such as EA [2], [13].
The recycling process that could be conducted on MAM was to separate the content of EA and all trans-FA from n-3. The most possible method to be applied to MAM recycling is crystallization. This method is easy to apply because it uses very simple equipment [14] and can be applied to samples in large quantities. Crystallization was known able to separate PUFA from SFA and trans-FA [15].
The crystallization methods applied in this recycled process were crystallization with n-hexane solvent, acetone solvent, and urea. Saturated fatty acids and EA were known to have low solubility in acetone and n-hexane solvents so that resulted in the best separation [1]. Urea crystallization method was the most efficient, reproducible, fast, and environmentally friendly method [16] and could be applied to waste [17] both to a large size or a little. The three crystallization methods are carried out at freezer temperature (under 0 OC). Crystallization at zero or below zero was an effective step in the process of fractionating fatty acids and their derivatives [18], because the solubility of both fatty acids also decreases along with the decrease in temperature [19].
In this research, three crystallization methods consisting of crystallization with n-hexane solvent, acetone solvent, and urea complexation were applied in MAM recycling which aims to produce oil fractions that rich in n-3 PUFA especially EPA and DHA and low in trans-FA especially EA.
Experimental Procedures
Materials
MAM waste material was obtained from one of the producers in the Muncar Region, Banyuwangi Indonesia. The MAM had been processed before by the producer with heating and adding caustic soda. The fatty acid standard used was the FAME Mix 37 comp (SIGMA).
Methods
Hydrolysis of oil became free fatty acids
MAM was firstly analyzed its fatty acid content by the GCMS method. The oil was then hydrolyzed with a saponification technique using a solution of 15% Potassium Hidroxide in a mixture of water and methanol (1:1). The mixture was then heated at 60 OC accompanied by constant agitation. The mixture was then separated from the compound that not soaped compound by using a n-hexane solvent so that it obtained a compound that soaped. The mixture was then acidified to pH 1 using HCl, then n-hexane was added. the n-hexane layer was taken and then evaporated so that it obtained free fatty acids [20].
Winterisation with acetone and n-hexane
5 grams of fatty acids were added to 50 mL acetone in an Erlenmeyer, then shaken until the fatty acids dissolve completely. The mixture was then stored in the freezer with the temperature set below 0OC for 24 hours. The mixture was then filtered with a vacuum filter, then the resulting filtrate was evaporated until a fatty acid fraction was obtained [21]. The same procedure was also carried out using n-hexane solvents.
Urea crystallization
5 grams of fatty acids were added with 20 grams of urea (in methanol). The mixture was then shaken until the fatty acids dissolve completely. As with crystallization with acetone, the mixture was stored in the freezer for 24 hours. The mixture was then filtered with a vacuum filter, and the filtrate was put back in the freezer for 3 hours. The mixture was refiltrated and the filtrate obtained was acidified with 6N HCl, then 20 mL of n-hexane was added. the n-hexane layer was separated and then evaporated to produce a fatty acid fraction.
Esterification of fatty acids
Fatty acids obtained from the three crystallization methods were then esterified by the method carried out by Guil-Guerrero and Belarbi [20]. Fatty acids were put into a three-hole flask, then added absolute methanol at a ratio of 1:20 (w/v). the mixture was then stirred until it dissolved completely. The acetyl chloride catalyst was then added to the mixture carefully then the mixture was refluxed at 70OC with a paraffin bath under N2atmospheric conditions [20] for 3 hours until FAME was obtained. Subsequent FAME was taken using n-hexane solvent. The mixture was then evaporated to obtain pure FAME.
Fatty Acid Analysis
The GCMS instruments used were Variant-3900, GC/MS/MS Saturn 2000, CP-8400 autosampler and GC (Shimazu, G-5000A) was equipped with a Hydrogen generator (Whatman) with a Bonded OV-1 column. The GC instrument was firstly conditioned by setting the system according to the ideal conditions of the analysis, namely the injector temperature of 240 OC, detector temperature 280 OC, the separation system was set with an initial temperature of 50OC, held for 2 minutes, then the temperature was raised at a speed of 4 OC/minute until reach temperatures of 240 OC. The temperature of 240OC was then held for 2 minutes. So the total duration of analysis was 51.5 minutes for each sample. The standard FAME and the FAME sample crystallized by the three methods were then injected into the previously set GCMS system.
Statistical Analysis
Data tables and figures are presented in the format of mean +standard deviation (SD) and analyzed with one-way ANOVA using the Microsoft Excel program with the significance of the data difference was set at P <0.05 [17], [18]. All experiments were carried out as three-time.
Results and Disscussion
The fatty acid content in MAM
MAM waste hydrolysis was conducted to break the bonds between fatty acids and glycerol [1]. This process aims to facilitate the process of separation of fatty acids in the oil. Fatty acids naturally appear in the form of triacylglycerol, that glycerol which is esterified with three fatty acids [1], [22]. The crystallization method using acetone or n-hexane was known to be able to produce the best separation compared to other solvents [1].
GC analysis of MAM materials and fractions obtained from the three crystallization methods can be seen in table 1 and fig 1. GC data show that waste materials contain very diverse fatty acids. MUFA was the highest number of fatty acids, which was 53.48%, then SFA 28.05%, and PUFA 18.47%. EA was the most dominant fatty acid in MAM that was equal to 47.95% and contributes 89.65% of the total MUFA. Palmitic acid was the most dominant fatty acid in SFA group which was 13.58% while the most dominant in the PUFA group was EPA which was 8.08%. The presence of n-3 fatty acids in MAM beside a large amount of EA makes it as very potential material to be recycled into n-3 rich oils that beneficial to health. EA is a geometrical isomer of oleic acid and has a trans configuration. Therefore, EA is known as trans-FA [1].
The content of EA in oil poses a threat to the health of the Indonesian population. Although the oil is specialized as animal feed, trans-FA will diffuse to animal products so that their eggs and meat will be contaminated with trans-FA. trans-FA have been proven to correlate with the emergence of cancer risk factors [23], precipitating atherosclerosis [24], breast cancer [25], and increasing hepatic lipogenesis as a hepatotoxic risk factor [26]. trans-FA can also have side effects on fetal growth and development [27]. trans-FA can also diffuse into the mammary glands of nursing mothers so that it affects the quality of breast milk [28].