Sky Journal of Biochemistry Research Vol. 2(3), pp. 9 - 21, June, 2013 Available online http://www.skyjournals.org/SJBR ©2013 Sky Journals

Full Length Research Paper

Effect of processing on the vitamins (A and E) and fatty acid content of trachurush trachurus

O.T. Adeyemi1, O.O. Osilesi1, F. Onajobi1, O. Adebawo1 and A. J. Afolayan2*

1Department of Biochemistry, Babcock University, Ilishan Remo, Ogun state, Nigeria.

2Botany Department, University of Fort Hare, Alice 5700, South Africa.

Accepted 3 April, 2013

The effects of different cooking methods (poaching, wood and coal smoking) on vitamins A, E and fatty acid composition in the fillet and SHB (skin, head and bone) of Trachurush trachurus were determined. Vitamins and fatty acid analysis were done by standard methods. All processing methods, except for wood smoked fillet significantly (p<0.05) reduced level of vitamin A in both Fillet and SHB, but increased (p<0.05) levels of vitamin E in the SHB; whereas the same significantly increased fatty acid contents in the fillet and SHB. Highest total polyunsaturated fatty acid (PUFA) of 251.78% was recorded by the coal smoke method in the fillet and wood smoked method (230.97%) in the SHB. Highest ω-3 fatty acid concentration was obtained for coal and wood smoked fillet and SHB which was lowest in the poached fish. The poached fillet was the highest in total saturated fatty acid (456.40%) while the least value was obtained in charcoal smoked fillet (370.19%). Wood smoking of both fish varieties was the highest in eicosapentaenoic acid (EPA) (30.51 ± 1.82

– 32.17 ± 1.09).

Comparing both raw and cooked fish samples, data obtained demonstrated that processed SHB and fillet could be a veritable source of valuable ingredients for human consumption and animal feeds. Key words: Effect of processing, Trachurus trachurus, vitamins (A and E), fatty acid.

INTRODUCTION Horse mackerel (or scads), T. trachurus (Linnaeus, 1758), also known as Kote in Nigeria West Africa belongs to the Order: Perciformes and fish family Carangidae. It is silver in colour, with bits of olive green and a white belly. It is abundant and widespread in the tropical and temperate East Atlantic and Mediterranean Sea, ranging from Norway to South Africa (where it is considered a distinct sub-specie T. trachurus capensis) (Smith-Vaniz, 1986; ICES 2006). The horse mackerel is edible and can be smoked, fried, salted and baked etc. It is an important commercial fish (Food and Agriculture Organization, 1992), whose fat content may vary greatly between individual fish caught in the same haul, and this could cause problems for processors requiring fish with a uniform fat level.

Fish provides between 30% and 80% of the total animal protein intake of the coastal people of West Africa (Teutscher, 1990; Saisithi, 1994). It contains unsaturated *Corresponding author: E-mail: aafolayan@ufh.ac.za. Fax: +27866282295.

fatty acids, vitamins and minerals (Andrew, 2001; McCullough et al., 2002; Gokoglu et al., 2004; Ahmed, 2010). Marine fishes like tuna are good source of unsaturated fatty acids of the ω-3 family. All the vitamins necessary for good health in humans and domestic animals (that is, A, D, E, K, B complex and C) are present to some extent in fish, which varies widely from even different parts of the same fish, species to species and throughout the year. There is little Vitamin C found in seafood, but it is considered an excellent source of the B complex vitamins, particularly niacin, B12 and B6. Thiamine is also found in seafood but in fair amounts (Venturi and Venturi, 2007).

Often, the parts of a fish not normally eaten, such as the liver and the gut, are known to contain greater quantities of oil-soluble vitamins than the flesh; the livers of cod and halibut for example contain almost all of the vitamins A and D present in those species (Smith and Guentzel, 2010). In contrast, the same two vitamins in eels, for example, are present mainly in the flesh. While the water-soluble vitamins in fish, although present in the skin, the liver and gut, are more uniformly distributed, and the flesh usually contains more than half the total amount

10 Sky J. Biochem. Res. present in the fish. The roe, when present, is also a good source of these vitamins (Gesch et al., 2002; Amado, 2009). In general the vitamin content of white fish muscle is similar to that of lean meat and, with the exception of vitamin C, can usually make a significant contribution to the total vitamin intake of man and domestic animals (Amado, 2009). Also the vitamin content of fish is not markedly affected by careful processing or by preservation, provided storage is not very prolonged (Calder, 2010). In addition, fishes are one of the most important sources of fatty acids. The type and amount of fatty acids in fish tissues vary mainly with what the fish eats, but other factors may also influence their fatty acid composition. Specie, size or age, reproductive status, geographic location and season, all influence fat ncontent and composition of fish muscle (Nettleton, 1985; Ackman, 1989; Lavniegos and Lopez-Cortes, 1997; Satio et al., 1999). The fatty acid composition of fish oil is very important for its functional properties (Von Schacky et al., 1999; Nestel, 2000), particularly in the correct ratio between omega-3 and omega-6 fatty acids (Perretti et al., 2007). The best nutritional ratio- of omega-3 in our diet not only boost the human immunity but reduces the inflammatory stimulus in the liver, so that the appropriate inflammation caused when the immune system defends against an invader does not age us as much (Mehmet et al., 2007). Fish species do not synthesize omega-3 fatty acids but they obtain them from sea life such as krill and algae which they feed on. These is evident in the lipids of cold-water and marine fish (such as salmon, mackerel, halibut, sardines, tuna, and herring) (Lovell, 1988), which are characterized by their high proportion of polyunsaturated fatty acids, such as the nutritionally important Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but these polyunsaturated fatty acids are highly susceptible to autoxidation (oxidation at normal temperatures due to contact with air) because of their high degree of unsaturation (Gunstone and Norris, 1983). Not all fish products contain appreciable amounts of omega-3 fatty acids (Lovell, 1988; Kris-Etherton et al., 2002). Generally, fish have relatively low saturated fatty acid (SFA) (<30%) content, except for certain species (Ackman, 1989; Nettleton and Exler, 1992); however, the SFA percentage of fish lipid increases with rising water temperature (Reiser et al., 1963; Tawfik, 2009). Essential fatty acids (EFAs) are similar to vitamins in their importance to overall health in humans (Kelley et al., 2009). However, vitamins are required in small dietary quantities (μg/day), while EFAs are a macronutrient (i.e., necessary in g/day) (Kruger and Horrobin, 1997). A joint study released by the Food and Agriculture Organization and the World Health Organization recommends that at least 3% of our daily calorie intake be in the form of EFA (Kelley et al., 2009). Lack of these omega acids causes significant metabolism disorders. Resent studies have linked omega-3 deficiencies to chronic health problems of

diabetes, cancer, arthritis, inflammatory diseases, depression, heart disease, hypertension, memory problems, weight gain and some allergies and skin conditions (Andrew and Stoll, 2001; Pam Rotella, 2006). Also, evidence has shown that omega-3 fatty acid deficiencies cause a number of moderate to extreme illnesses in both the mother and child (Pam Rotella, 2006). In the mother, an omega-3 fatty acid deficiency is connected with postpartum depression, which is “moderate to severe depression in a woman after she has given birth, which may occur soon after delivery or up to a year later” (Rogge, 2008). It also impacts pre-term birth, breast milk production, development of gestational diabetes, preeclampsia (a type of high blood pressure), and overall ongoing health. In the child, the mother’s deficiency in omega-3 fatty acids impairs brain, visual, cardiovascular, immune and nervous system development (Rogge, 2008). Fish, plant, and nut oils are the primary dietary source of omega-3 fatty acids. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are found in cold-water fish such as salmon, mackerel, halibut, sardines, tuna, and herring (Kruger and Horrobin, 1997). ALA is found in flaxseeds, flaxseed oil, canola (rapeseed) oil, soybeans, soybean oil, pumpkin seeds, pumpkin seed oil, purslane, perilla seed oil, walnuts, and walnut oil. Other sources of omega-3 fatty acids include sea life such as krill and algae (Kelley et al., 2009).

In this connection, the consumption of T. trachurus species has recently increased. It is generally marketed as fresh, chilled, frozen or smoked. Although this fish is commonly consumed as pan-fried by the Nigerians people, preferences of the consumers for cooking methods are increasingly changing. The Nigerian consumer, however, has minimal knowledge about nutritive values of raw and cooked fish. Therefore the objective of this work was to carry out a comparative study on the effect of poaching, wood (Acacia seyal and Citrus lemon) and charcoal smoking on the vitamins (A and E) and fatty acid contents of T. trachurus. Also noting that there remains paucity of scientific information on the raw and processed skin, head and bones (SHB) of this fish, which may contain vital nutritive values that are considered insignificant by majority of the people but are most often thrown away during processing. PROCEDURE Determination of vitamins A and E Carotenoids (Vitamin A) 0.20 g of each finely ground sample was weighed into a flat bottom reflux flask. The above mixture was heated in a boiling water bath for 1 h with constant shaking. The mixture was cooled rapidly and 30 ml of water was

added. The hydrolysate obtained was transferred into a separating funnel. The solution was re-extracted three times with 25 ml of chloroform. 2 g anhydrous Na2SO4 was added to the extract to remove any traces of water, the mixture was then filtered into a 100 ml volumetric flask and made up to mark with chloroform. Standard solution of β-carotene Vitamin A of range 0-50 mg/ml with chloroform by dissolving 0.003 g of standard β -carotene in 100 ml of chloroform. Gradient of different standard prepared were determined with references then absorbance from which average gradient was taken to calculate Vitamin A (β-carotene in mg/100 g). Absorbance of sample and standards were read at a wavelength of 328 nm using a spectronic 21D Spectrophotometer (Otunola et al., 2010). Carotenoids (µg/100g) = Absorbance of sample x Average gradient x 10 Wt. of sample x 1000

Vitamin A (I/U) = Absorbance of sample 328nm x 1020

0.6

Where; 1020 = Gradient factor (Neeld & Pearson, 1962) 0.6= Factor converting β-carotene to IU vitamin A α-Tocopherol (Vitamin E) The method as described by Yilmaz et al. (2004) was used.

Solutions Stock solution of α-Tocopherol was prepared at a concentration 0f 300 µg/ml in absolute ethanol. Working standard was prepared by diluting the stock solution at thee concentrations of 0, 10, 20, 30, 40, and 50 µg/ml with absolute ethanol in volumetric flasks (10 ml). Absolute ethanol was used has blank solution. Procedure 0.2 g extracted fish oil was accurately transferred into 100 ml calibrated flask and 75 ml of absolute ethanol was added. The flask was placed in an ultrasonicator for 50 min at 54°C and diluted with absolute ethanol to mark. The mixture was filtered through nylon membrane of 0.45 µm pore size (47 mm filter membrane, Phenommenex, USA). 1 mm of this solution was transferred into the 10 ml calibrated flask and diluted with absolute ethanol to mark. UV-3000 PC Spectrophotometer Optima Scientific was used to measure the absorbance at 281 nm. Fatty acid determination Oil extraction Crude fat was extracted from the finely ground fish

Adeyemi et al. 11 samples using the Soxhlet extraction method described by the AOAC, (2002). 2.0 g of moisture free sample was weighed into a fat free thimble, plugged with cotton wool and then introduced into the extraction tube. A clean dry boiling flask was weighed (W1) and 250 ml of 40- 60

°C

petroleum ether was introduced into the flask and sample was extracted for 6 h continuously adopted according to the EPA 3540 Method (Reinik et al., 2007). The extract was concentrated in a rotary evaporator (RE-100, Stone Staffordshire, and England) at 60°C to 2 ml. This was repeated for other samples. Then the remaining solvent was removed from the extracted oil by placing the flask in the fume hood at 25°C for 45 min. and weighed (W2). The percent crude fat was calculated by the following formula: % Fat = W2- W1 × 100 Wt of sample

Where W1 = weight of empty flask; W2 = weight of flask and fat deposit. FAME analysis Methyl esters were prepared by trans methylation using 2M KOH in methanol and n-heptane according to the method described by Ichihara et al. (1996) modified by Suseno et al. (2011). The extracted fish oil (0.1 g) was dissolved in 2 ml hexane. After the addition of 4 ml of 2M methanolic KOH, the tube was votexed for 2 min at room temperature, and then centrifuged at 4000 rpm for 10 min. The hexane layer was then taken for analysis by gas chromatography (GC). The fatty acid composition was analysed by GC Shimadzu 2010 with an auto sampler (Shimadzu, Japan) equipped with a flame ionization detector and a fused silica capillary SGE column (30 m x 0.32 mm, ID x 0.25 lm, BP20 0.25 UM, USA). The oven temperature was 140

0C, held for 5 min, raised to 200°C

at a rate of 4°C/min and to 220°C at a rate of 1°C/min, while the injector and the detector temperatures were set at 220°C and 280°C, respectively. The sample size was 4 μl and the carrier gas was controlled at 16 psi. The split used was 1:100. Fatty acids were identified by comparing the retention times of FAME with a standard 37 component FAME mixture (Supelco). Three replicate GC analyses were performed per sample and the results were expressed in GC area % as a mean value and ± standard deviation. RESULTS

Vitamin content The changes in content of fish samples after cooking processes are as shown in Figures 1 and 2

12 Sky J. Biochem. Res.

Figure 1. Vitamin A and E Composition (µg/100g) of Processed Trachurus trachurus Fillet* Values with different superscripts along a row are

significantly different (p < 0.05). CSKF: charcoal smoked kote fillet; WSKF: wood smoked kote fillet; SKF: poached kote fillet; RKF: Raw kote fillet.

Figure 2. Vitamin Composition (µg/100g) of Processed T. trachurus SHB.*Values with different superscripts along a row are significantly

different (p < 0.05). WSSHB: wood smoked kote SHB; CSSHB: charcoal smoked kote SHB; PSHB: poached kote SHB; RSHB: Raw kote SHB.

respectively. T. trachurus was observed to have higher (P < 0.05) contents of Vitamin E than A, in both fillet and SHB. The highest (P < 0.05) vitamin A and E values for cooked horse mackerel was seen in the fillets of raw and wood smoked horse mackerel (215.87μg / 100 g and

1002.10 mg / 100g). Vitamins A and E have important roles in human metabolism, immune and digestive system. It is reported that the human body needs 1.50mg vitamin A and 17 mg vitamin E daily (Belitz et al., 2001). Result from this study indicates that 100g of raw Atlantic

Horse mackerel was higher in Vitamin E (446.82 mg) than vitamin A (215.87 mg) in the fillet, sufficient for the bodies’ daily requirement. Thus, values of RKF were significantly (p < 0.05) higher than the 0.04mg (vitamin A) and 1.5mg (vitamin E) reported by Belitz et al. (2001) which, may be, has a result of variability observed majorly in lipo-soluble vitamins (Dias et al., 2003). Moreover, Vitamins A and E were observed to be higher (p < 0.05) in the fish fillet than in the SHB, but reduced significantly (p < 0.05) with all the processing methods, except for vitamin E in WSCF as compared with the raw fish sample.

Furthermore, T.hjyu76 trachurus SHB also contained high (p < 0.05) contents of Vitamin A (94.64 mg) and vitamin E (335.62 mg). Where poaching, coal and wood smoking methods significantly (P < 0.05) increased levels of Vitamins A and E in the SHB compared with th78tyuhj aggregation (Bouts and Gasthuys (2003). Also, vitamin E helps to prevent per oxidation of polyunsaturated fatty acids of phospholipids and cholesterol in cellular and sub-cellular membranes. In general, because of per oxidative damage to cellular membranes, nutritional muscular dystrophy, fatty liver degeneration, anaemia, exudative diathesis, erythrocyte haemolysis, haemorrhages, de-pigmentation and reduction of fertility are observed in fish in the deficiency of vitamin E (He and Lawrence, 1993; Mehrad, 2012). Therefore based on the amount of vitamins A and E in this study, both T. trachurus fillet and SHB are a good source of these vitamins.

Fatty acid composition The fatty acid (F.A) composition of T. trachurus fillet and SHB are shown in Tables 1 and 2 and Figures 3a - 4b respectively. A total of 34 F.A in T. trachurus (T.t) fillet and 32 F.A in T. trachurus SHB were identified in this study. All processing methods increased significantly the composition of saturated (SFA), polyunsaturated fatty acid (PUFA) of T. trachurus fillet and SHB. The highest values of SFA and PUFA were found to be 456.40% and 251.78% in the poached and charcoal smoked fillet, while those of the T. trachurus SHB were found to be 456.60%, and 230.42%, respectively in the charcoal and wood smoked SHB. Accordingly, the SFA level T. trachurus fillet and SHB were the highest among the other fatty acid groups, with T. trachurus SHB recording the highest values of SFA (Figure 3a). The PUFA / SFA ratios which were highest in CSKF fillet and WSHB are 0.68 and 0.55 respectively (Figure 3b). Among SFA, WSKF had the highest levels of F.A in the following order; stearic acid > magaric acid > nonadecanoic acid > lauric acid > pentadecanoic acid > palmitic acid in the fillet, while in the SHB charcoal smoking process recorded the most abundant levels of F.A in the following

Adeyemi et al. 13 order: caproic acid > lauric acid > stearic acid > nonadecanoic acid > behenic acid respectively. Significant differences (P < 0.05) were observed between fish fillet and SHB for all the processing methods, with the fish fillets having the highest values in all the processing methods (Table 2).

T. trachurus feeds mainly on crustaceans, zooplankton, gadoids and herring which are rich in saturated fatty acids (SFA) and unsaturated fatty acids (UFA) (Macer, 1977; Dahl and Kirkegaard, 1987; Dahl and Kirkegaard, 1986; Murta et al., 1993; Naess and Bergh, 1994). The SFA level of raw T. trachurus was found to be the highest among all fatty acids. This result may be attributed to the effect of fish digestion. Fish species consume diets that are high in levels of C16:0; levels of C16:0 in both fish fillet and SHB were found also to be high (35.93 ± 2.60

and 38.80 ± 0.10) (Saglik and Imre, 2001), and all the processing methods (charcoal smoke, wood smoke and poaching) increased the levels of C16:0 in the fillet (37.28 ± 1.86, 38.28 ± 0.95 and 36.68 ± 4.68) (Alasalvar et al., 2002). However, all processing methods reduced the amount of C16:0 in the SHB (33.69 ± 2.32, 36.40 ± 1.89 and 35.16 ± 2.53), compared with the raw T. Trachurus (Table 2).

The level of total MUFA in fish fillet increased (p < 0.05) with all the processing methods (Figure 3a) and was found to be higher (p < 0.05) in WSKF (235.08%) than in the CSKF (370.19%) and PKF (388.82%) (Figure 3a), whereas the MUFA levels reduced (p < 0.05) in CSKF (161.11%), WSKF (195.98%) and PKF (188.68%) of the SHB as compared with the raw fish (197.35%) sample (Figure 4a). In addition, the C18:1n9 ratio of T. trachurus was found to be significantly higher in the raw SHB (38.51 ± 0.49) than that in the raw fish fillet (37.12 ± 2.01). Furthermore, some other significant differences (P < 0.05) were noted in both fillets and SHB of T. trachurus, in terms of C16:1, C20:1n9, and C17:1. Narcisa et al. (2001) reported that the total MUFA level in T. Trachurus fillet was 29.13% in autumn, 28.49% in winter, 30.40% in spring and 28.76% in summer. The level of MUFA in raw T. Trachurus fillet of present study is quite higher (p < 0.05) than the (229.64%) level reported by Narcisa et al. (2001) for raw T. Trachurus fillet. Nonetheless, value recorded for the raw (197.35%) SHB samples in present investigation, although promising, were significantly (p < 0.05) lower compared to the fish fillet (Figure 4b).

The PUFA level in raw T. Trachurus fillet (159.60%) was found to be considerably (p < 0.05) higher than in SHB (54.1%) (Figures 3b and 4b). In addition, CSKF had the highest (p < 0.05) value in the fillet (251.78%), while WSKSHB had the highest (p < 0.05) PUFA level in the SHB (230.97%) samples. The level of EPA (C20:5ω3) in the raw T. trachurus flesh was higher (p < 0.05) than those previously reported in marine fish such as bluefish (Narcisa et al., 2001; Saglik and Imre, 2001) and cultured sea bass (Alasalvar et al., 2002). Although EPA in the RKF was (25%) the same was undetected in the RSHB,

14 Sky J. Biochem. Res.

Table 1. Fatty acid composition (%) of processed T. trachurus Fillet. Data= Mean ± SD, n=3.

*Values are expressed as percentages of total fatty acids. ** (a-b) means in a row with the different letters are significantly different (P < 0.05). CSKF: charcoal smoked kote fillet; WSKF: wood smoked kote fillet; PKF: poached kote fillet; RKF: Raw kote fillet. SFA= Saturated fatty acid; MUFA= Monounsaturated fatty acid; PUFA= Poly unsaturated fatty acid; UFA: Unsaturated fatty acid; Σn-3= Sum of ω3 fatty acid; Σn-6= Sum of ω6 fatty acid N.D= Not detected. The values of (SFA, MUFA and PUFA) have exceeded 100% because minor fatty acids have been reported.

Table 2. Fatty acid composition (%) of processed T. trachurus SHB. Data= Mean ± SD, n=3.

FATTY ACID (%) ABBREVIATION RKF CSKF WSKF PKF

CAPROIC ACID C10:0 37.20 ± 0.00 a N.D 28.00 ± 2.40

c 37.00 ± 1.00

a

UNDECANOIC ACID C11:0 N.D 38.00 ± 0.00 a 28.00 ± 6.80

b 21.73 ± 3.70

c

LAURIC ACID C12:0 38.40 ± 0.00 a 38.00 ± 0.00

a 38.80 ± 0.00

a 27.88 ± 4.08

b

TRIDECANOIC ACID C13:0 37.47 ± 0.93 a 26.80 ± 0.00

d 29.43 ± 3.47

c 36.64 ± 1.10

b

MYRISTIC ACID C14:0 38.40 ± 0.41 b 37.18 ± 1.66

c 39.03 ± 0.17

a 37.23 ± 1.25

c

PENTADECANOIC ACID C15:0 38.32 ± 0.29 a 34.44 ± 4.31

b 38.48 ± 0.45

a 38.57 ± 0.44

a

PALMITIC ACID C16:0 35.93 ± 2.60 d 37.28 ± 1.86

b 38.28 ± 0.95

a 36.68 ± 4.68

c

MARGARIC ACID C17:0 34.08 ± 5.12 c 26.00 ± 0.00

d 39.20 ± 0.00

a 38.20 ± 1.27

b

STEARIC ACID C18:0 34.86 ± 3.01 b 39.60 ± 0.00

a 39.36 ± 0.16

a 39.04 ± 0.20

a

NONADECANOIC ACID C19:0 N.D 26.89 ± 2.52 b 39.20 ± 0.00

a 39.33 ± 0.13

a

ARACHIDIC ACID C20:0 33.07 ± 6.13 c 38.00 ± 1.13

a 31.04 ± 5.33

d 35.30 ± 4.03

b

BEHENIC ACID C22:0 N.D N.D N.D 38.40 ± 0.40 a

LIGNOCERIC ACID C24:0 N.D 28.00 ± 0.00 b N.D 30.40 ± 0.00

a

SFA 327.73 370.19 388.82 456.40 10 UNDECENOIC ACID C11:1 N.D 26.00 ± 0.00

a 25.20 ± 4.40

b N.D

DODECENOICACID C13:1 29.60 ± 0.00 a N.D N.D N.D

PENTADECENOIC ACID C15:1 38.00 ± 0.00 a 24.00 ± 0.00

b N.D N.D

PALMITOLEIC ACID C16:1 ω7 35.72 ± 1.71 b 37.26 ± 1.40

a 35.27 ± 1.13

c 33.05 ± 1.90

d

OLEIC ACID C18:1 ω9 37.12 ± 2.01 b 39.20 ± 0.25

a 34.89 ± 1.37

c 34.32 ± 1.60

c

10 NONADECENOIC ACID C19:1 N.D 39.47 ± 0.13 a 38.08 ± 1.24

b N.D

EICOSENOIC ACID C20:1 ω9 33.90 ± 5.70 b 30.67 ± 4.4

c 35.35 ± 2.84

a 30.40 ± 0.00

c

DOCOSENOIC C22:1 ω7 N.D 35.16 ± 2.36 a N.D N.D

ERUCIC ACID C22:1 ω9 34.50 ± 0.31 b N.D 37.76 ± 1.84

a 30.40 ± 0.00

c

NERVONIC ACID C24:1 ω9 24.80 ± 3.03 c N.D 28.53 ± 1.87

a 28.00 ± 0.00

b

MUFA 233.64 231.76 235.08 156.17

HEXADECATRIENOIC ACID (HTA) C16:3 ω3 34.53 ± 2.43 a 32.61 ± 2.08

b 31.33 ± 2.10

c 28.60 ± 4.68

c

α-LINOLEIC ACID (ALA) C18:3 ω3 32.40 ± 0.00 b 38.40 ± 0.40

a 29.40 ± 2.51

c 22.60 ± 2.27

c

EICOSATRINOIC ACID (ETE) C20:3 ω3 N.D 25.00 ± 0.00 a 25.30 ± 1.58

a 24.26 ± 0.53

b

EICOSATETRAENOICACID C20:4 ω3 N.D 24.44 ± 3.59 a N.D N.D

EICOSAPENTAENOIC ACID (EPA) C20:5 ω3 25.00 ± 3.80 b N.D 30.51 ± 1.82

a 22.60 ± 3.17

c

CIS 6, 9, 12, 15, 18 HENEICOSAPENTAENOIC ACID (HPA)

C21:5 ω3 N.D 36.90 ± 0.00 a N.D N.D

CIS 7, 10, 13, 16, 19 DOCOSAPENTAENOIC ACID (DPA)

C22:5 ω3 N.D 38.00 ± 0.00 a N.D N.D

DIHOMOGAMMALINOLENIC ACID C20:3 ω6 N.D N.D N.D 29.60 ± 0.00 a

ARACHIDONIC ACID C20:4 ω6 30.00 ± 8.00 a 21.60 ± 1.60

d 22.80 ± 6.00

c 29.12 ± 2.85

b

LINOLEIC ACID C18:2 ω6 37.67 ± 1.01 a 34.83 ± 1.51

c 36.11 ± 2.11

b 31.07 ± 6.55

d

EICOSADIENOICACID C20:2 ω6 N.D N.D 31.00 ± 2.20 a N.D

PUFA 159.60 251.78 206.45 187.85 Σn-3 91.93 195.35 116.54 98.06 Σn-6 67.67 56.43 89.91 89.79 Σn3/n6 1.36 3.47 1.30 1.09 DHA/EPA 0.00 N.D 0.00 0.00 Σ UFA 389.24 483.54 441.53 344.02 UFA/SFA 1.19 1.31 1.14 0.75 PUFA/SFA 0.49 0.68 0.53 0.41

Fatty Acid (%) ABBREVIATION RSHB CSHB WSHB PSHB

ACETIC C2:0 ND ND ND 15.20 ± 0.00 a

CAPROIC ACID C10:0 ND 30.40 ± 0.00 b 26.60 ± 11.40

c 34.60 ± 2.20

a

Table 2. Cont. Adeyemi et al. 15

* Values are expressed as percentages of total fatty acids. ** (a-b) means in a row with the different letters are significantly different (P < 0.05). CSSHB: charcoal smoked kote SHB; WSSHB: wood smoked kote SHB; PSHB: poached kote SHB; RSHB: Raw kote SHB. SFA= Saturated fatty acid; MUFA= Monounsaturated fatty acid; PUFA= Poly unsaturated fatty acid; UFA: Unsaturated fatty acid; Σn-3= Sum of ω3 fatty acid; Σn-6= Sum of ω6 fatty acid; N.D= Not detected. The values of (SFA, MUFA and PUFA) have exceeded 100% because minor fatty acids have been reported.

but increased (p<0.05) with all the processing methods in both the fish fillet and SHB compared to the raw fish samples (Figure 2). i.e. RKF (25.00 ± 3.80), CKF (N.D), WKF (30.51 ± 1.82), PKF (22.60 ± 3.17) in the fillet and RSHB (N.D), CSHB (24.42), WSHB (38.17%) and PSHB (28.20%) in the SHB, with the wood smoked fish varieties having significantly (P < 0.05) the highest value in both

fillet and SHB. Regardless of difference in levels of EPA and absence of DHA, T. trachurus can be considered as good sources of ω3 fatty acids such as EPA because, it contained a significantly (P < 0.05) high amount of EFA in the fillet, more so the human body can convert α-linolenic acid (ALA) to EPA and subsequently DHA (Cunnane, 2003). EPA levels obtained in this work

UNDECANOIC ACID C11:0 ND 23.50 ± 13.5 a 26.13 ± 6.73

b 21.73 ± 3.70

c

LAURIC ACID C12:0 ND 38.53 ± 0.35 a 38.00 ± 0.00

b 30.27 ± 4.06

c

TRIDECANOICACID C13:0 32.53 ± 1.54 b 32.17 ± 0.30

b 31.16 ± 3.02

c 35.50 ± 3.30

a

MYRISTIC ACID C14:0 35.43 ± 3.00 c 38.89 ± 0.30

a 37.17 ± 1.80

b 38.80 ± 0.21

a

PENTADECANOIC ACID C15:0 38.80 ± 0.10 a 34.52 ± 2.51

c 33.55 ± 2.34

d 35.20 ± 2.39

b

PALMITIC ACID C16:0 38.97 ± 0.30 a 33.69 ± 2.32

d 36.40 ± 1.89

b 35.16 ± 2.53

c

MARGARIC ACID C17:0 39.20 ± 0.40 a 34.13 ± 3.0

c 38.00 ± 1.60

b ND

STEARIC ACID C18:0 38.67 ± 0.37 b 39.27 ± 0.07

a 39.27 ± 0.16

a 39.12 ± 0.15

a

NONADECANOIC ACID C19:0 39.00 ± 0.40 a 39.00 ± 0.00

a 39.33 ± 0.13

a 39.50 ± 0.00

a

ARACHIDIC ACID C20:0 39.20 ± 0.16 a 37.60 ± 1.87

c 38.00 ± 1.60

b 34.80 ± 2.62

d

HENEICOSANOIC ACID C21:0 ND 35.80 ± 0.20 a ND ND

BEHENIC ACID C22:0 ND 38.50 ± 0.50 a ND 38.27 ± 0.13

a

LIGNOCERIC ACID C24:0 ND ND 38.40 ± 0.00 a 30.40 ± 0.00

b

SFA 302 456.60 422.01 428.55 DODECENOICACID C13:1 ND ND ND 33.20 ± 0.00

a

PENTADECENOIC ACID C15:1 ND ND 36.00 ± 0.00 a ND

PALMITOLEIC ACID C16:1 ω7 36.40 ± 1.31 a 30.58 ± 2.20

c 35.32 ± 2.15

b 36.40 ± 0.98

a

OLEIC ACID C18:1 ω9 38.51 ± 0.49 a 36.29 ± 1.14

b 33.33 ± 1.92

c 33.08 ± 1.71

c

10 NONADECENOIC ACID C19:1 38.67 ± 0.37 a ND ND ND

EICOSENOIC ACID C20:1 ω9 28.80 ± 5.60 c 36.53 ± 3.07

a 32.93 ± 6.67

b 27.60 ± 1.85

d

ERUCIC ACID C22:1 ω9 26.70 ± 3.73 c 27.70 ± 1.66

b 30.40 ± 0.00

a 30.40 ± 0.00

a

NERVONIC ACID C24:1 ω9 28.27 ± 2.13 b 30.00 ± 0.00

a 28.00 ± 0.00

b 28.00 ± 0.00

b

MUFA 197.35 161.11 195.98 188.68 HEXADECATRIENOIC ACID (HTA)

C16:3 ω3 ND 21.50 ± 3.50 c 30.80 ± 3.04

b 36.00 ± 0.00

a

α-LINOLEIC ACID (ALA) C18:3 ω3 16.40 ± 0.00 d 27.33 ± 2.75

b 32.73 ± 1.72

a 25.20 ± 2.10

c

EICOSATRINOIC ACID (ETE) C20:3 ω3 ND ND 23.20 ± 0.00 a ND

EICOSAPENTAENOIC ACID (EPA)

C20:5 ω3 ND 24.42 ± 1.93 c 32.17 ± 1.09

a 28.20 ± 2.50

b

DIHOMOGAMMALINOLENIC ACID

C20:3 ω6 ND ND 25.60 ± 0.00 a ND

LINOLEIC ACID C18:2 ω6 37.70 ± 1.77 a 18.00 ± 0.00

d 30.07 ± 3.96

c 35.07 ± 3.11

b

EICOSADIENOIC ACID C20:2 ω6 ND ND 32.40 ± 3.72 a ND

ARACHIDONIC ACID C20:4 ω6 ND 24.07 ± 4.22 a 24.00 ± 0.00

a 24.80 ± 2.47

a

PUFA 54.1 115.32 230.97 149.27 Σn-3 16.40 73.25 118.90 89.40 Σn-6 37.70 42.07 112.07 59.87 Σn3/n6 0.44 1.74 1.06 1.49 DHA/EPA 0 0 0 0 Σ Unsat 251.45 276.43 426.95 337.95 UNSFA/SFA 0.83 0.61 1.01 0.79 PUFA/SFA 0.18 0.25 0.55 0.35

16 Sky J. Biochem. Res.

Figure 3a. Fatty acid composition (%) of processed Trachurus trachurus fillet* Values are expressed as percentages of total fatty acids. *Means with identical colour on different levels are significantly different (P < 0.05). CSKF: charcoal smoked kote fillet; WSKF: wood smoked kote fillet; PKF: poached kote fillet; RKF: Raw kote fillet.

Figure 3b. Fatty acid composition (%) of processed T. trachurus Fillet

*Values are expressed as percentages of total fatty acids. *Means with identical colour on different levels are significantly different (P < 0.05). CSKF: charcoal smoked kote fillet; WSKF: wood smoked kote fillet; PKF: poached kote fillet; RKF: Raw kote fillet.

Adeyemi et al. 17

Figure 4a. Fatty acid composition (%) of processed T. trachurus SHB

*Values are expressed as percentages of total fatty acids. *Means with identical color on different levels are significantly different (P < 0.05). CSSHB: charcoal smoked kote SHB; WSSHB: wood smoked kote SHB; PSHB: poached kote SHB; RSHB: Raw kote SHB.

Figure 4b. Fatty acid composition (%) of processed T. trachurus SHB

*Values are expressed as percentages of total fatty acids. *Means with identical colour on different levels are significantly different (P < 0.05). CSSHB: charcoal smoked kote SHB; WSSHB: wood smoked kote SHB; PSHB: poached kote SHB; RSHB: Raw kote SHB.

18 Sky J. Biochem. Res.

is similar to the reports of Grigorakis et al. (2002) for Gilthead sea bream (Sparus aurata), Cahu et al. (2004) for Halibut (Hippoglossus hippoglossus), Alasalvar et al. (2002) and Fuentes et al. (2010) for Sea bass (Dicentrarchus labrax); consequently confirming the reports of Ackman (1982 and 1989) and Hossain, (2011) that pelagic fishes living in cold water usually have the highest content of EPA and DHA.

Arachidonic acid (C20:4ω6) levels in raw fish fillet was high (p < 0.05) i.e. 30.00 ± 8.00% and reduced with all processing method. But in the RSHB Arachidonic acid (C20:4ω6) was not detected but the same increased (p < 0.05) in the CSHB (24.07 ± 4.22%), WSHB (24.00 ± 0.00%) and PSHB (24.80 ± 2.47%). Also, the levels of ω3 and ω6 in decreasing order of magnitude was; WSKF > CSKF > PKF and WSKF > PKF > CSKF in the fillet; WSHB > PSHB > CSHB in the SHB for both ω3 and ω6 PUFA. Levels of ω3 and ω6 F.A in both the fish fillet and SHB increased (P<0.05) with in all the processing methods, except for the WSKF (1.30%) and PKF (1.09%) which significantly (P < 0.05) reduced compared with the RKF (1.36%). A high level of ω-3 in both fish fillet and SHB, as found in this study, is very important for human health because it prevents many health-related problems such as cardiovascular disease risk factors (Mayneris-Perxachs et al., 2010), while the low level of ω-6 fatty acids recorded in the fillet and SHB of T. trachurus were within the range for healthy living in humans, because a lesser quantity of ω6 than ω3 fatty acid is required by the human body (Stuchlík and Žák ,2002).

Lastly, the levels of ω-3/ω-6 ratio have been suggested to be a better index for comparing the relative nutritional value of different species (Piggot and Tucker, 1990). However, this index is of limited value without consideration of which fatty acids are present. Generally, C20 and C22 fatty acids are more nutritionally valuable than C18 fatty acids (Arts et al., 2001). Owning to their predominating quantity, two fatty acids namely DHA and EPA are responsible to the greatest extent for changes in the n-3/n6 ratios, a reliable indicator that enables a comparison of relative nutritive value of lipids. The n-3/n-6 ratios observed in present investigation as shown in Tables 1 and 2 were found to vary between 0.44 (SHB) to 1.36 (fillet). According to Sargent (1997) the optimum ratio of ω-3/ ω-6 PUFA should be 1:5 (0.2). The higher the n-3/n-6 ratios, the more able the body is to use the n-3 fats. A lower ratio means the enzymes that convert these fats into the forms in which they are active in the body are more likely to be used up by the n-6 PUFAs. All the fish samples in Table 1 showed higher ratios of n-3/n-6 PUFAs. Findings in present study are similar to the report of Ismail and Unusan (2007) and Hossain (2011), while both the fillet and SHB samples were seen to be good sources of essential and non essential amino acids. DISCUSSION Vitamins are essential for growth and metabolic

processes in an organism (Gaby, 2005). Deficiency of any vitamin may lead to different disorders and increase hazard of occurrence of “civilization” diseases, while excessive consumption or overdose of some of them is also very harmful (Dietary Reference Intakes, 2001; Bender, 2003). Our current finding showed that raw T.t fillet and SHB had high (p < 0.05) contents of vitamins A and E, which reduced (p < 0.05) with all the processing methods (poaching, coal and wood smoking) in the fillet, but increased (p < 0.05) in the SHB. Analysis of vitamin E in tissue has become more important, because tocopherol (vitamin E) takes part in providing of nutrients to cells. It strengthens blood vessel walls and protects erythrocytes against premature decomposition. It is used in the treatment of male sterility, muscle disorder, arteriosclerosis and heart diseases. Also, there is a possibility that vitamins A and E may act as cancer-chemo preventive agents (Gratzfeld-Hüsgen et al., 1991; Pemberton, 2006). In mammalians, vitamin E is necessary for the maintenance and protection of neuronal, muscular and reproductive tissue (Gratzfeld-Hüsgen et al., 1991). Vitamin E lubricates cell membranes and contributes to a healthy heart and lungs. Vitamin E is a main antioxidant that boosts cell regeneration and blocks harmful free-radical damage from prematurely aging the body. It is well known for its skin benefits and can be taken to prevent age spots and wrinkles or the pure oil can be applied directly to the skin as an anti-aging treatment (Null, 1998). Furthermore, Sarah (2010) reported that vitamin E helps improve liver function by protecting vitamin A. Other reports also stated that ‘when the two vitamins (Vitamin A and E) are combined, a potent liver tonic was achieved and both vitamins work together to strengthen and rejuvenate a tired liver (Iribhogbe et al., 2011). Vitamin A is well known for its vision-preserving properties. This vitamin guards against age-related vision problems and diseases such as mascular degeneration and night blindness. It is also essential to regulate oil production in the skin, keeping it soft, clear and youthful. Vitamin A is one of the fat-soluble vitamins and is of importance for normal growth and development of the human body. Mal-absorption results in disease of the liver, while its insufficiency results in extreme dryness of the skin and night blindness (Null, 1998). Thus, the presence of vitamins A and E in the fish fillet and SHB varieties would prevent oxidation of oxidisable components of both the raw, processed fish and compounded feed samples; hence save the feed from going rancid, which is in agreement with the works reported by Atteh (2004) and Muhammad et al. (2007). Conversely, current findings disagrees with the report of Aminullah Bhuiyan et al. (1993), who said that during smoking vitamin A decreased slightly, but not significantly (p > 0.05), while vitamin E remained unchanged in smoked Atlantic mackerel (Scomber scomber). Furthermore, vitamins A and E values recorded in the RCF are higher (P < 0.05) than the 0.04mg (vitamin A) and 1.5 mg (vitamin E) reported by Belitz et al. (2001),

this may be has a result of variability observed majorly in liposoluble vitamins (Dias et al., 2003). As a result of the high contents of vitamins A and E observed in present investigation, T.t fillet and SHB can be said to be good sources of this vitamins, particularly vitamin E.

Nutritional benefits of fish stems for most part, from its exceptionally advantageous fatty acid profile (Hossain, 2011). In recent years, increasing attention has been focused on the significance of ω3 polyunsaturated fatty acids (PUFAs) in human nutrition, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Saito et al., 1999).

In present investigation therefore, identified were a total of 34 fatty acids (F.A) in both raw and processed T. trachurus (T.t) fillet and 32 F.A in the SHB variety (Table 1). Raw T.t recorded high (p < 0.05) concentrations of 14:0, 16:0, 16:3w3, 18:3w3, 20:3w3, 18:2w6 and 20:4w6 in the fillet; as well as 14:0, 16:0, 18:1w9, 18:2w6, 18:2w6 and 18:3w3 in the SHB variety. The totals of unsaturated fatty acids, containing ω3 and ω6 double bonds in the fillet, ranged from 22.60 - 38.40% and 21.60 – 37.67% in the raw and processed T.t fillet; as well as 16.40-36.00% and 18.00% - 37.70% in raw and processed T.t SHB . Studies have reported that these fatty acids may help to prevent coronary attacks by reducing blood clotting rates as well as reducing cholesterol levels in the blood (Rawls, 1981). Also, Erucic acid (22:1w9), which has adverse effects on certain laboratory animals (e.g., heart lesions in male weanling rats) when fed at high levels, was quite low (p < 0.05) in this specie. The total for 22:1 isomers was about 0.3% of the total fatty acids. Hence, results of this study indicated that T. trachurus (T.t), contained appreciable (p < 0.05) amount of the essential and non essential fatty acids in both the fillet and SHB, with the fatty acid having similar patterns for both varieties, (i.e. high concentrations of (ω-3) and low levels of ω-6 in both fillet and SHB). Although the composition of fish and fish derived product is recommended as a means of preventing cardiovascular and other diseases, and has considerably increased over recent decades (Sargent et al., 1995). Yet, the lipid content and fatty acid profile of fish vary between and with species even in dark and white muscle, are also affected by many factors such as the temperature, salinity, season, size, age, species habitat, life stage, and the type and abundance of food, especially whether a species is herbivorous, omnivorous or carnivorous (Ackman, 1989; Sargent et al., 1995; Saito et al., 1999). Therefore, PUFA composition varies among different fish species of both freshwater and marine origins. Still, it is important for human health to increase the consumption of fish and its products, which are rich in ω-3 PUFA and poor in ω-6 PUFA series (Burr, 1989; Sargent, 1997). As a result, a number of countries (Canada, Sweden, United Kingdom, Australia, and Japan) as well as the World Health Organization and North Atlantic Treaty Organization have made formal population - based dietary recommendations for ω-3

Adeyemi et al. 19 PUFAs. Typical recommendations are 0.3 to 0.5 g/day of EPA + DHA and 0.8 to 1.1g of α-linolenic acid (Kris-Etherton et al., 2002; Mozaffarian and Rimm, 2006). These recommendations can easily be met by following the AHA Guidelines to consume two servings per week, of fatty fish (i.e. salmon, herring, and mackerel). Commercially prepared fried fish (e.g. from restaurants and fast food establishments), as well as many frozen convenience-type fried fish products should be avoided because they are low in ω-3 PUFAs and high in trans-fatty acids (Kris-Etherton et al., 2002). Nonetheless, for individuals with coronary heart disease, 1g/day of EPA + DHA has been shown to reduce coronary heart disease mortality (Kris-Etherton et al., 2002; Van de Werf et al., 2003). This could be achieved by consuming approximately 6 ounce (~170 g) serving / week of fish richest in ω-3 PUFAs (e.g., farmed salmon, anchovies, herring) (Mozaffarian and Rimm, 2006). On the other hand, Gebauer et al. (2006) reported that consuming approximately two servings of fish per week (8 ounce ~ 227g) may reduce the risk of mortality from coronary heart disease and that consuming EPA and DHA may reduce the risk of mortality from cardiovascular disease in people who have already experienced a cardiac event. Nonetheless, increase in consumption of fish in general with the hope of increasing the ω-3 PUFA concentration may not be the answer as they may cause a concomitant increase in the ω-6 concentration as well-especially in fish where ω-3 to ω-6 ratios are very small. Therefore, fish like kote with higher ω-3/ω-6 ratios should be recommended for consumption, because findings of present investigation has shown T. trachurus to be a good source of both essential, non essential amino acids, in addition to higher ω-3/ω-6 ratio. ACKNOWLEDGEMENT The authors thank National Research Foundation (NRF) of South Africa for their financial support. REFERENCES Ackman RG (1982). Fatty Acid Composition in Fish Oil. In: Barlow,

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