Storage stability and quality of polyunsaturated fatty acid rich oil fraction from Longtail tuna (Thunnus tonggol) head using supercritical extraction

Abstract

The polyunsaturated fatty acids (PUFA)-rich fish oil fractionated from tuna head using supercritical carbon dioxide (SC-CO₂) were stored at 4°C and –18°C for 60 days to study the storage stability. The changes in fatty acids, iodine value (IV), peroxide value (PV), acid value (AV), and saponification value (SV) up to 30 days were found to be negligible, then started to decrease from 45 and the significant changes were observed at 60 days. At 4°C, the PV, AV, and SV were increased from 1.74 to 6.72 meq O₂/kg oil, 1.36 to 5.56 mg KOH/g oil, and 182.73 to 190.74 mg KOH/g oil, respectively, while IV and PUFA were decreased from 185.43 to 175.35 g I/100 g oil and 43.07 to 33.94% at 60 days. It is concluded that SC-CO₂ extracted PUFA-rich fraction could be maintained in good quality for consumption at both temperature up to 60 days.

Durante 60 días, se almacenó a 4°C y a –18°C aceite de pescado rico en ácidos grasos poliinsaturados (AGPI), extraído de la cabeza de atún usando dióxido de carbono supercrítico (CO₂-SC), con el fin de estudiar su estabilidad durante el almacenamiento. Los resultados obtenidos demuestran que, hasta los 30 días, los cambios registrados en los ácidos grasos en lo que respecta al valor de yodo (VY), al valor de peróxido (VP), al valor ácido (VA) y al valor de saponificación (VS) fueron insignificantes, empezando a disminuir a partir de los 45 días para mostrar cambios significativos a los 60 días. En este sentido, se constató que a una temperatura de 4°C, el VP, el VA y el VS aumentaron de 1,74 a 6,72 meq O₂/kg aceite, de 1,36 a 5,56 mg KOH/g aceite y de 182,73 a 190,74 mg KOH/g aceite, en cada uno de los períodos señalados respectivamente, mientras que el VY y los AGPI disminuyeron de 185,43 a 175,35 g I/100 g aceite y de 43,07 a 33,94% a los 60 días. Se concluye que la fracción rica en AGPI, extraída por medio de SC-CO₂, puede mantener su buena calidad para el consumo, almacenándose a ambas temperaturas hasta por 60 días.

Palabras claves:

• fraccionamiento SC-CO₂
• aceite de pescado rico en AGPI
• subproductos de pescado
• oxidación de lípidos; condición de almacenamiento

Keywords:

• SC-CO₂ fractionation
• PUFA rich fish oil
• fish by-products
• lipid oxidation
• storage condition

Introduction

Tuna fish is a very important and significant source of economy in many countries. The longtail tuna (Thunnus tonggol) is the largest neritic tuna fishery in Indian Ocean. Neritic tuna has become the main targeted species in Southeast Asian region since 1982 because of the high demand and attractive prices offered by tuna canneries (Nootmorn, Jaiyen, & Rodpradit, 2011). During canning process of tuna fish, a large amount (~60% of total weight) of solid wastes (mainly head, skin, viscera) are generated as by-products which are usually discarded as processing leftover or sometimes used as animal feeds (Chantachum, Benjakul, & Sriwirat, 2000). In our previous study, a new process was introduced for changing fish wastes/by-products into omega-3 fish oil for the utilization of 100% fish wastes that can be achieved for human consumption. Longtail tuna head was rich in omega-3 fish oil, especially high amount of docosahexaenoic acid (DHA) (19.7%) (Sahena et al., 2013). Head of little tuna Euthynnus alletteratus represents an important source of polyunsaturated fatty acids (PUFA) (41.43–47.71%), especially omega-3 DHA (29.34–34.02%) (Selmi & Sadok, 2010). There are great differences in lipid storage between different fish species and various parts of the fish organ due to their size and age, location and season of catch, gender and food habit, reproductive cycle, salinity, and temperature (Boran, Karaçam, & Boran, 2006; Huss, 1988; Sahena et al., 2010).

The importance of omega-3 fish oil for human nutrition as well as disease prevention is well recognized. Many clinical and epidemiologic studies have shown significant roles of omega-3 PUFA in the prevention and treatment for cardiovascular diseases, hypertension, cancer, arthritis, and various mental illnesses such as, depression, dementia, and attention-deficit hyperactivity disorder (Uauy & Valenzuela, 2000). Moreover, DHA is considered as a promising pharmaceutical because it is important for the development of brain and central nervous system for the infant (Uauy & Valenzuela, 2000). Since PUFAs are regarded as essential fatty acids (FAs), it could be used as additives in several food products such as eggs, milks, pasta, bread, yogurt, and certain cereals such as rice and oats (Martin et al., 2006). Therefore, the unutilized sources of omega-3 PUFA need to be explored due to their significant role in human health.

Extraction and fractionation of fish oil by supercritical carbon dioxide (SC-CO₂₎ is a promising alternative to conventional separation method. Degradation or decomposition of thermolabile compounds cannot be avoided in conventional method, since relatively high temperature are required (Corrêa, Peixoto, Gonçalves, & Cabral, 2008), in addition with hazards associated with the use of flammable and toxic organic solvents. A SC-CO₂ fractionation process offers the advantages of high purity, selective fractionation, single step processing, and environmental friendly. In addition, it can be efficiently managed at mild operating temperatures without exposing the extract to oxygen, thereby preserving the integrity of the PUFA of interest and avoiding solvent residues in the final product (Jachmanián, Margenat, Torres, & Grompone, 2007; Staby & Mollerup, 1993). Fractionation and purification of fish oil omega-3 PUFA have been studied by many researchers during the past 10 years (Catchpole, Grey, & Noermark, 2000; Corrêa et al., 2008; Jachmanián et al., 2007; Létisse & Comeau, 2008; Perretti et al., 2007).

Due to wide ranges of application in pharmaceuticals, nutraceuticals and food industries, further study is needed on the quality and stability of omega-3 PUFA. Despite their health benefit, omega-3 PUFA of fish oil are highly susceptible to oxidative deterioration. The rate of oxidation of omega-3 fish oil could always be high due to the presence of high unsaturation. Therefore, the omega-3 base products are highly sensitive to oxidative spoilage (Huss, 1988). The important factors that influence oxidative spoilage of the omega-3 base products are oxygen, temperature, metal, water, and light. Quality of fish oil will be decreased with increase in temperature and storage time. The unpleasant flavor and odorous of the product could be the result of the increment of oxidative spoilage of omega-3 PUFA at very low peroxide value (PV) (Boran et al., 2006). Apart of unpleasant taste and odour, oxidation of omega-3 PUFA could also affect to the safety and quality of the products (Huss, 1988). Bimbo (1998) reported the quality guidelines for crude fish oil, where the ranges of PV were 3–20 meq/Kg, free FAs ((% oleic acid) 1–7% (usually 2–5%), and p-Anisidine number 4–20.

Compositions of lipid and its stability is required to know about proper utilization of newly explored underutilized resource (Navarro-García, Ramírez-Suárez, Cota-Quiñones, Márquez-Farías, & Bringas-Alvarado, 2010). However, available report on head oil PUFA extracted by SC-CO₂ from Thunnus tonggol in Malaysia and in the world is scarce. The storage stability of the omega-3 PUFA could be the major concern to maintain the product’s quality, especially for scale-up production. Hence, the aim of this study was to evaluate the quality and stability of the fractionated PUFA rich fish oil from Thunnus tonggol head stored up to 60 days at two different temperatures (4°C and –18°C) by determining the changes of FA constituents, acid value (AV), iodine value (IV), PV, and saponification value (SV).

Materials and methods

Sample preparation

Thunnus tonggol were obtained from the fish landing center of Batu Maung, Pulau Penang, Malaysia. The fresh fish samples were collected in an insulated icebox and transported to the laboratory of School of Industrial Technology, Universiti Sains Malaysia, Malaysia. The fish samples were immediately de-headed manually. The heads were then freeze dried (Model: LABCONCO, USA) at a drying temperature of –47°C and vacuumed at 0.133 bar for 3 days. The samples were then kept in desiccators until further use. The dried heads were grounded with a dry mixer (Waring, Laboratory, USA) into particle sizes ranging from 0.2 to 0.5 mm by sieving.

Fractionation of oil by SC-CO₂

The experimental set-up for the SC-CO₂ extraction process was assembled according to Norulaini et al. (2009). The ISCO SFE System (ISCO Inc., Lincoln, NE, USA) consisted of a supercritical fluid extractor (ISCO, SFX 220), a controller (ISCO, SFX 200), a carbon dioxide cylinder, a chiller (Yih Der BL-730), two syringe pumps (ISCO, Model 100DX): a CO₂ pump and a co-solvent pump, and restrictor temperature controller associated with two coaxially heated capillary restrictors (ISCO). The CO₂ pump was fitted with a cooling jacket to deliver CO₂, and the co-solvent pump was fitted to deliver co-solvents as modifiers/entrainers. To cool up the pump’s head, an ethylene glycol-deionized water mixture (50:50, v/v) was circulated through the cooling jacket using a refrigerated bath circulator (model 631D, Tech-Lab Manufacturing Sdn. Bhd., Selangor, Malaysia), which can chill the coolant down to 0°C. In each experiment, 2 g of grounded sample (dry weight) was loaded into a 2 ml sample cartridge and placed in the ISCO extraction chamber and allowed to equilibrate at desired temperature. The restrictor was maintained at 65°C to avoid problems of restrictor plugging. Each extracts with ethanol were collected separately into a preweighed blue cap bottle yield trap through a restrictor. The trap was cooled with an ice water mixture. The oil trap containing the extracted oil with ethanol, as a residue of co-solvent, were evaporated under vacuum at 40°C using a rotary evaporator (Büchi-Rotavapor, Switzerland), and then placed in the oven at 45°C for 30 min before being transferred into the desiccators.

The extractions were performed at optimized condition with CO₂ and ethanol (as a co-solvent) at 3 ml min-1 (2.4 ml CO₂ and 0.6 ml ethanol/min, v/v), for 120 minutes at 65°C temperature and 40 MPa pressure (Sahena et al., 2013). Simultaneous extraction and fractionation of yield were performed into six fractions (each fraction 20 mins). The yield of these six continuous extraction processes of every 20 minutes was collected in different yield trap successively which is denoted as fractional yields (F1, F2, F3, F4, F5, and F6 labeled as 1st, 2nd, 3rd, 4th, 5th, and 6th, respectively). In this study, the last three fractions (F4, F5, F6) were dominant in PUFA and monounsaturated fatty acid (MUFA). These predominant PUFA- and MUFA-rich fractions 4, 5, and 6 were mixed vigorously which is regarded as mixed fraction of PUFA were then stored up to 60 days in two different temperatures at 4°C and –18°C to investigate quality and stability of PUFA. In storage period of 60 days, aliquots were analyzed from each bottle of sample at various time intervals to measure the extent of lipid oxidation. The FAs composition, PV, IV, SV, and AV of mixed oil fraction were analysed at both 4°C and –18°C in every 15 days interval up to 60 days.

Analysis of FA constituent by gas chromatography

The FA constituents of mixed oil fractions obtained from fraction type A, B, and C were analyzed to determine the FA profile using gas chromatography with flame ionization detector (GC-2010 Plus AOC-5000, Shimadzu, Osaka, Japan). Fatty acid methyl ester (FAME) was prepared by dissolving 50 mg of sample into 0.95 ml n-hexane, and 0.05 ml 1 M sodium methoxide (30% methanol in sodium methoxide). The mixture was then shaken vigorously using an auto-vortexer (Janke and Kunkel, VF2, Germany) for 30 s and stored for another 5 min so it formed a bilayer. The clear upper layer containing the FAMEs (1 μl) was pipetted off and injected into a GC using an external standard method (AOCS, 2003). Supelco 37 component FAME mixtures and standard Menhaden oil (PUFA-3) (Sigma-Aldrich, Supelco, Bellefonte, Pa., USA) was used as reference standard fish oil (purity 99%). The BPX70 [70% Cyanopropyl polysilphenylene-siloxane (30 m × 0.32 mm × 0.25 µm film thickness), SGE France] was purchased from Sigma-Aldrich Co., USA. The oven temperature was set at 140°C, held for 2 min, increased with a heating rate of 5°C/min up to the final temperature of 250°C, and then held for 10 min at 250°C. Identification of chromatographic peaks was performed using the retention time of FAME standard. Results of FAs analysis were reported as an average of three analyses for each sample investigated.

Determination of AV

The AV is the number of milligrams of potassium hydroxide necessary to neutralize the free acids in 1 g of sample. A known mass of the fat is dissolved in neutralized isopropanol and the free FAs are neutralized with standard alkali. The acidity is the content of free FA conventionally expressed as a percentage (m/m) of oleic acid or any relevant acid. Determination of free FA was carried out according to AOCS official method no. Cd 3d-63 (AOCS, 2003).

The sample was weighed into an Erlenmeyer flask. Then the sample was liquefied with 50 ml of 2-propanol (neutralized solvent). The flask was placed on hot plate and temperature regulated to about 40°C. Phenolphthalein indicator was then added to the sample. The sample was gently shaken while titrated with standard sodium hydroxide (0.1N) to the first permanent pink color.

Determination of PVs

The PV is expressed in terms of milliequivalents of active oxygen per kilogram of oil which oxidize potassium iodide under the condition specified. The best test for autoxidation (oxidative rancidity) is determination of the PV. Peroxides are intermediates in the autoxidation reaction. The determination of PV was carried out according to AOCS official method no. Cd 8–53 (AOCS, 2003).

About 5 g sample was weighed and dissolved with 30 ml acetic acid chloroform solution. Then, 0.5 ml saturated potassium iodide was added, then the solution was swirled for 1 min and then 30 ml distilled water was added. The sample was vigorously shaken while titrated with standard sodium thiosulphate (0.005 mol/L) until the blue color disappears.

Determination of SV

The SV is the number of milligrams of potassium hydroxide required to saponify 1 g of fat under the condition specified. It is a measure of the average molecular weight of all the FAs present. The glycerides present are split by alcoholic alkali and any free FAs are neutralized. Excess alkali is back titrated with hydrochloride acid in the presence of an indicator. The determination of SV was carried out according to AOCS official method no. Cd 3–25 (AOCS, 2003).

The sample was weighed and saponified by ethanolic potassium hydroxide (KOH) with some boiling aids and it was refluxed for 30 minutes. Excess KOH was then titrated with 0.5 Mol/L hydrochloric acid using phenolphthalein solution as an indicator until the pink color of the indicator disappeared. A blank determination was conducted simultaneously for the control.

Determination of IV

The IV is a measure of the unsaturation of fats and oils. IV of fish oil were determined according to AOCS official method no. Cd 1–25 (AOCS, 2003).

Statistical analyses

Experiments were performed in triplicate and each set of yields were averaged. The weighted means were derived from an analysis of variance by Statistica version 11.0 (StatSoft Inc., Tulsa, Okla., USA).

Results and discussion

The changes in FA constituents, PV, IV, SV, and AV of mixed oil fractions extracted from Thunnus tonggol head using SC-CO₂ were investigated for the storage stability at different time and temperature. Tables 1 and 2 shows the changes in FA constituents of PUFA-rich fractions (mixed fractions of 4 to 6) during storage time and temperature. This mixed fraction is predominant of PUFA and MUFA with little amount of saturated fatty acids (SFA). Thus, the mixed fractions were analysed to investigate the storage stability of the FA at 4°C and –18°C. However, negligible changes were found in SFA and MUFA during storage at both 4°C and –18°C for up to 60 days storage. The changes in PUFA’s storage at 4°C were negligible up to 30 days and then started to decrease from 45 days and the changes were not significant. The significant changes were observed at 60 days; thus, the study was not continued for more than 60 days. However, similar trends were observed in both omega-3,6 and other PUFAs; therefore, the amount of unidentified FAs were increased significantly with storage time and assumed that some part of the PUFA might be converted to either SFA or MUFA. Whereas, at storage –18°C (Table 2), the quality of the oil was much better than at storage 4°C in retaining the quality and quantity of the FA compositions similar up to 60 days.

Table 1. Changes in fatty acid composition of PUFA rich fraction of oil extracted from Thunnus tonggol head during storage at 4°C (values are mean ± SD).

Cambios en la composición de ácidos grasos de la fracción de aceite rica en AGPI extraída de cabeza de Thunnus tonggol ocurridos durante el almacenamiento a 4°C (valores son medias ± DE).

		Days of storage
Fatty acids (%)	Control	15 days	30 days	45 days	60 days
C14:0	0.75 ± 0.08^b	0.87 ± 0.05^b	1.01 ± 0.07^b	1.56 ± 0.06^ab	2.14 ± 0.06^a
C15:0	0.54 ± 0.05^b	0.63 ± 0.09^b	0.94 ± 0.09^b	1.25 ± 0.09^ab	1.99 ± 0.04^a
C16:0	7.78 ± 0.10^b	7.93 ± 0.08^b	8.51 ± 0.18^b	8.87 ± 0.12^ab	10.01 ± 0.25^a
C17:0	2.67 ± 0.06^b	2.75 ± 0.07^b	3.25 ± 0.15^b	3.71 ± 0.08^ab	4.26 ± 0.18^a
C18:0	6.11 ± 0.12^b	6.34 ± 0.14^b	6.87 ± 0.25^b	7.21 ± 0.17^ab	8.17 ± 0.15^a
C22:0	1.46 ± 0.04^b	1.51 ± 0.11^b	1.93 ± 0.13^b	2.17 ± 0.09^ab	2.58 ± 0.08^a
16:1n7	4.68 ± 0.18^b	4.55 ± 0.11^b	4.03 ± 0.12^b	3.68 ± 0.10^ab	3.02 ± 0.09^a
18:1n9	19.73 ± 0.34^a	19.05 ± 0.72^a	18.74 ± 0.44^a	18.17 ± 0.36^ab	17.13 ± 0.36^b
17:1	3.44 ± 0.12^a	3.28 ± 0.14^a	3.17 ± 0.18^a	2.94 ± 0.07^a	2.24 ± 0.08^b
20:1n9	2.53 ± 0.09^a	2.38 ± 0.06^a	2.04 ± 0.06^a	1.73 ± 0.08^a	1.22 ± 0.07^a
22:1n9	2.35 ± 0.12^a	2.21 ± 0.05^a	1.86 ± 0.09^a	1.53 ± 0.07^a	1.18 ± 0.05^a
18:2n6	3.19 ± 0.10^a	3.07 ± 0.08^a	2.86 ± 0.17^a	2.65 ± 0.10^a	2.01 ± 0.10^b
18:3n3	2.55 ± 0.08^a	2.37 ± 0.14^a	2.01 ± 0.12^a	1.69 ± 0.08^a	1.19 ± 0.05^b
20:4n6	3.27 ± 0.13^a	3.19 ± 0.19^a	2.96 ± 0.07^a	2.71 ± 0.15^a	2.12 ± 0.06^b
20:5n3	5.14 ± 0.17^a	4.95 ± 0.13^a	4.47 ± 0.14^a	4.02 ± 0.12^a	3.24 ± 0.11^b
22:5n3	5.76 ± 0.08^a	5.66 ± 0.16^a	5.21 ± 0.18^a	4.97 ± 0.11^a	4.32 ± 0.14^b
22:6n3	23.17 ± 0.57^a	23.01 ± 0.96^a	22.87 ± 0.79^a	22.52 ± 0.87^ab	21.06 ± 0.57^b
Total SFA	19.31 ± 0.46^a	20.03 ± 0.78^a	22.51 ± 0.56^ab	24.77 ± 0.79^b	29.15 ± 0.72^c
Total MUFA	32.72 ± 0.73^a	31.47 ± 0.85^a	29.84 ± 0.74^ab	28.05 ± 0.82^b	24.79 ± 0.79^c
Total PUFA	43.07 ± 0.88^a	42.25 ± 0.91^a	40.38 ± 0.83^a	38.56 ± 0.76^ab	33.94 ± 0.68^b
Non identified	4.91 ± 0.15^cd	6.25 ± 0.14^c	7.27 ± 0.27^b	8.62 ± 0.23^b	12.12 ± 0.19^a

Note: Different letters in the same row indicating the significant differences (P < 0.05).Nota: letras distintas en la misma fila que indica las diferencias significativas (P < 0,05).

Table 2. Changes in fatty acid composition of PUFA rich fraction of fish oil extracted from Thunnus tonggol head during storage at –18°C (values are mean ± SD).

Cambios en la composición de ácidos grasos de la fracción de aceite rica en AGPI extraída de cabeza de Thunnus tonggol ocurridos durante el almacenamiento a −18°C (valores son medias ± DE).

		Days of storage
Fatty acids (%)	Control	15 days	30 days	45 days	60 days
C14:0	0.75 ± 0.03^a	0.79 ± 0.08^a	1.01 ± 0.08^a	1.24 ± 0.09^a	1.35 ± 0.12^a
C15:0	0.54 ± 0.06^a	0.51 ± 0.05^a	0.77 ± 0.06^a	0.93 ± 0.05^a	1.07 ± 0.07^a
C16:0	7.78 ± 0.10^a	7.81 ± 0.16^a	7.95 ± 0.24^a	8.21 ± 0.25^a	8.46 ± 0.24^a
C17:0	2.67 ± 0.05^a	2.74 ± 0.08^a	2.93 ± 0.15^a	3.09 ± 0.10^a	3.28 ± 0.09^a
C18:0	6.11 ± 0.13^a	6.21 ± 0.11^a	6.43 ± 0.28^a	6.55 ± 0.08^a	6.69 ± 0.10^a
C22:0	1.46 ± 0.06^b	1.55 ± 0.11^a	1.69 ± 0.08^a	1.75 ± 0.05^a	1.88 ± 0.13^a
16:1n7	4.68 ± 0.08^a	4.62 ± 0.09^a	4.31 ± 0.07^a	4.15 ± 0.06^a	4.08 ± 0.14^a
18:1n9	19.73 ± 0.73^a	19.71 ± 0.42^a	19.51 ± 0.46^a	19.23 ± 0.51^a	18.98 ± 0.47^a
17:1	3.44 ± 0.14^a	3.38 ± 0.10^a	3.17 ± 0.19^a	3.05 ± 0.07^a	2.83 ± 0.06^a
20:1n9	2.53 ± 0.07^a	2.47 ± 0.06^a	2.21 ± 0.07^a	2.06 ± 0.08^a	1.89 ± 0.04^a
22:1n9	2.35 ± 0.05^a	2.31 ± 0.12^a	2.24 ± 0.08^a	2.18 ± 0.03^a	2.11 ± 0.11^a
18:2n6	3.19 ± 0.16^a	3.16 ± 0.09^a	3.09 ± 0.14^a	2.97 ± 0.10^a	2.86 ± 0.08^a
18:3n3	2.55 ± 0.09^a	2.51 ± 0.10^a	2.47 ± 0.11^a	2.29 ± 0.11^a	2.03 ± 0.05^a
20:4n6	3.27 ± 0.12^a	3.23 ± 0.17^a	3.19 ± 0.15^a	3.11 ± 0.07^a	3.02 ± 0.15^a
20:5n3	5.14 ± 0.10^a	5.07 ± 0.21^a	4.93 ± 0.09^a	4.88 ± 0.21^a	4.81 ± 0.19^a
22:5n3	5.76 ± 0.11^a	5.71 ± 0.17^a	5.62 ± 0.07^a	5.48 ± 0.16^a	5.37 ± 0.21^a
22:6n3	23.17 ± 0.72^a	23.11 ± 0.84^a	23.09 ± 0.62^a	22.96 ± 0.94^a	22.74 ± 0.79^a
Total SFA	19.31 ± 0.65^a	19.61 ± 0.79^a	20.78 ± 0.58^a	21.77 ± 0.73^a	22.73 ± 0.58^a
Total MUFA	32.72 ± 0.89^a	32.49 ± 0.68^a	31.44 ± 0.93^a	30.67 ± 0.68^a	29.89 ± 0.72^a
Total PUFA	43.07 ± 0.93^a	42.79 ± 0.87^a	42.39 ± 0.75^a	41.69 ± 0.85^a	40.83 ± 0.89^a
Non identified	4.91 ± 0.26^b	5.11 ± 0.27^ab	5.39 ± 0.34^a	5.87 ± 0.11^a	6.55 ± 0.21^a

Note: Different letters in the same row indicating the significant differences (P < 0.05).Nota: letras distintas en la misma fila que indica las diferencias significativas (P < 0,05).

The SFA were increased from 19.31% to 29.15% and 19.31% to 22.73% when stored at 4°C and –18°C (Tables 1 and 2), respectively, for 60 days, while MUFA and PUFA content were decreased from 32.72% to 24.79% and 43.07% to 33.94% at 4°C and 32.72% to 29.89% and 43.07% to 40.83% at –18°C (Tables 1 and 2), respectively. DHA, the major PUFA, which cover more than 50% of total PUFA, also decreased from 23.17% to 21.06% at 4°C and 22.74% at –18°C for 60 days storage (Tables 1 and 2). The changes in most of the SFA were significant at 60 days whereas PUFAs and MUFA started to decrease negligibly until 45 days and the changes were found to be significant at 60 days when stored at 4°C. The unidentified FAs increased slightly (non-significant) with storage time up to 15 days and then significantly increased from 30 to 60 days, and are also assumed that some part of these unidentified FAs could either be SFA or MUFA.

Jaswir, Osman, Khatib, and Chowdhury (2009) studied storage stability of fish oil from different species including Indian mackerel. Authors stored fish oil at 4°C and –27°C for 3 weeks and analysed FAs to compare with the fresh sample. They found slight decrease in MUFA and PUFA at both conditions while –27°C was found to be better than 4°C in retaining the FA compositions of the fish oil. Their findings are in line with our observation of this study. Authors also reported slight increase in SFA simultaneously at both conditions; thus, the more possibility of some part of our unidentified FAs could either be SFA or MUFA. These results are in agreement with the findings of our study. Moreover, conjugated FAs could be produced when the omega-3 PUFA are oxidized and these compounds can absorb the UV light at 232–234 nm (Kulås & Ackman, 2001). Navarro-García et al. (2010) studied the effect of storage time for 87 days at 25°C on the quality of liver oil from two commercial rajiform species. Authors reported a decrease in DHA during the storage time for both species as oxidation proceeded after a certain period of time. The significant decrease in DHA started from 31 days and the changes were identical up to 45 days. Then, another decrease was found at 52 days up to 87 days for both the species. Our observation is in similar trend with the report of Navarro-García et al. (2010) for the PUFA exactly for DHA at 4°C, whereas at –18°C found to be better in retaining the quality of the FAs up to 60 days.

The mixed fractions were also analysed to evaluate the chemical quality with various parameters, including IV, PV, AV, and SV at 4°C and –18°C for 0 (fresh), 15, 30, 45, and 60 days of storage (Table 3). In IV, a gradual and significant decrease was found from 185.43 to 177.92 g I/100 g oil with storage time up to 45 days and was drastically decreased to 175.35 g I/100 g oil at 60 days when the sample was stored at 4°C (Table 3). Similar trends were also observed for the storage temperature at –18°C, where IV was slightly decreased from 185.43 to 180.91 g I/100 g at 60 days storage time (Table 3). This was due to the degree of unsaturation of the samples that were decreased with storage time at both temperatures. Although the unsaturated FAs did not decrease significantly for the sample stored at –18°C up to 60 days, the slight decrease in the degree of unsaturation may affect the IV to significantly decrease.

On the other hand, the PV, AV, and SV for most of the sample stored at both 4°C and –18°C were increased significantly with storage time up to 60 days. PV, AV, and SV were increased from 1.74 to 6.72 meq O₂/kg oil, 1.36 to 5.56 mg KOH/g oil, and 182.73 to 190.74 mg KOH/g oil, respectively, when the samples were stored at 4°C (Table 3). Similar trends were also found for the storage temperature at –18°C, where PV, AV, and SV were increased from 1.74 to 2.81 meq O₂/kg oil, 1.36 to 2.54 mg KOH/g oil, and 182.73 to 186.19 mg KOH/g oil, respectively (Table 3). The oxidative deterioration of the sample occurred with storage time is obvious for unsaturated oil. This peroxidation might affect all the chemical properties of IV, PV, AV, and SV of the sample during storage at both temperatures. However, the level of this oxidative deterioration and the changes in the chemical properties in this study were in acceptable limit, and did not affect to the oil quality. The unsaturated oil sample preserved at –18°C provided better quality than the sample preserved at 4°C, in all respect. Boran et al. (2006) studied chemical quality of fish oils extracted from horse mackerel, shad, garfish, and golden mullet during storage at 4°C and –18°C for 150 days. Authors reported that PV, AV, and SV gradually increased, while IV decreased during storage at both temperatures, indicating that the degree of unsaturation of the oil decreased. The IV is a measure of the total number of unsaturated double bonds present in any oil. The differences in IV during storage of oil sample might have caused the increase of oxidation rate of the sample. According to Augustin and Berry (1983), excessive deterioration of the oil during storage caused a significant change in IV. On the other hand, SV increased, meaning that molecular weight of the samples increased with storage time at both temperatures. This is due to oxidative peroxidation that produces aldehydes or ketones contributed to the increase in SV. However, authors observed the rate of increases in PV, AV, and SV contrarily decreased in IV, were lower at –18°C than at 4°C. This observation indicates that storage temperature at –18°C could be better than 4°C, although a little deterioration occurred at both temperatures and that were in the acceptable level up to 60 days. The acceptability limit for PV of crude fish oil is 7–8 meq O₂/kg oil as reported by Huss (1988). In our study, the PV reached to 6.72 meq O₂/kg at 4°C and 2.81 at –18°C for 60 days which did not exceed the acceptability limit and remains quality good for the consumption. On the other hand, Verma, Srikar, Sudhakara, and Sarma (1995) reported that PV of sardine oil stored at –20°C for 150 days increased from 4.12 to 18.6 that exceeded the acceptability limit. Therefore, the study was not further continued to be experimented after 60 days. Moreover, the mixed fractions of MUFA- and PUFA-rich fish oil with little amount of SFA could attribute to the less oxidation than the purified PUFA.

Table 3. Quality changes in fish oil fraction from Thunnus tonggol head during storage at 4°C and –18°C temperature (Values are mean ± SD).

Cambios de calidad en la fracción de aceite de pescado de cabeza de Thunnus tonggol ocurridos durante el almacenamiento a 4°C y −18°C (valores son medias ± DE).

		Days of storage
Chemical properties	Control	15 days	30 days	45 days	60 days
At 4°C
Peroxide value (meq O2/kg oil)	1.74 ± 0.15^c	2.08 ± 0.17^bc	3.10 ± 0.14^b	3.98 ± 0.16^ab	6.72 ± 0.17^a
Acid value (mg KOH/g oil)	1.36 ± 0.11^c	2.16 ± 0.15^bc	2.99 ± 0.10^b	3.47 ± 0.13^b	5.56 ± 0.21^a
Iodine value(g I/100 g oil)	185.43 ± 1.2^a	184.72 ± 0.87^ab	180.37 ± 0.84^b	177.92 ± 0.69^c	175.35 ± 0.82^d
Saponification value (mg KOH/g oil)	182.73 ± 0.95^d	183.73 ± 0.98^cd	185.02 ± 0.77^b	188.26 ± 0.95^b	190.74 ± 0.76^a
At –18°C
Peroxide value (meq O2/kg oil)	1.74 ± 0.13^a	1.96 ± 0.09^a	2.28 ± 0.15^a	2.55 ± 0.11^a	2.81 ± 0.20^a
Acid value (mg KOH/g oil)	1.36 ± 0.08^a	1.52 ± 0.17^a	1.83 ± 0.10^a	2.11 ± 0.08^a	2.54 ± 0.16^a
Iodine value(g I/100 g oil)	185.43 ± 0.92^a	185.07 ± 0.86^a	183.76 ± 0.82^a	182.32 ± 0.75^a	180.91 ± 0.69^a
Saponification value (mg KOH/g oil)	182.73 ± 0.78^a	182.89 ± 0.94^a	183.56 ± 0.97^a	184.85 ± 0.86^a	186.19 ± 0.81^a

Note: Different letters in the same row indicating the significant differences (P < 0.05).Nota: letras distintas en la misma fila que indica las diferencias significativas (P < 0,05).

Jaswir et al. (2009) studied the PV and AV for Indian mackerel oil stored up to 3 weeks at 4°C and –27°C. The PV increased from trace value to 2.11 and 0.70 at 4°C and –27°C. Similarly, the AV increased from 1.00 to 4.86 and 2.45 at 4°C and –27°C, respectively. On the other hand, the IV decreased from 178.2 to 169.0 and 173.5 at 4°C and –27°C, respectively. Authors concluded that –27°C could be the better storage condition for the PUFA-rich fish oil compared to 4°C. However, the rate of increment of PV and AV and decrease of IV are similar to our observation for 30 days at both 4°C and –18°C. Bimbo (1998) stated that the acceptability limit for AV is 7 to 8 mg KOH/g and increase in AV is generally associated with lipase activity. In our observation, the AV is within the acceptability limit to be stored at 4°C and –18°C for 60 days (Table 3).

Conclusion

The result of this study on the effect of storage temperature and time on the quality and stability of PUFA fraction of Thunnus tonggol showed better quality at –18°C than the sample stored at 4°C. The oxidative deterioration of the sample occurred with storage time is obvious for any polyunsaturated fish oil. The level of this oxidative deterioration and the changes in the chemical properties of the sample in this study were in acceptable limit, and did not affect to the oil quality up to 60 days at both –18°C and 4°C. However, the purified PUFA, especially DHA, preserved at –18°C found to be better than 4°C in retaining the quality and stability for up to 60 days.

Acknowledgments

The authors gratefully acknowledge the financial support under Research Universiti Grant no. 1001/PTEKIND/845032 of Universiti Sains Malaysia, Malaysia to carry out this study.