Fatty Acid-Related Health Lipid Index of Raw and Fried Nile Tilapia ( Oreochromis niloticus ) Fish Muscle

Fried food consumption is popular in most parts of the world including Ethiopia. Among many fried products available in Ethiopia, fried ﬁsh is most commonly consumed in Hawassa Town due to the easy access to the ﬁsh from the lake. Recently, there is growing concern among fryers to recycle the oil while frying ﬁsh. However, there is limited evidence about the frying eﬀect on the fatty-acid-related health lipid index of fried ﬁsh. Thus, the study was aimed to determine the fatty acid proﬁle and the fatty-acid-related health lipid of raw and fried ﬁsh. Raw and fried ﬁsh were taken from the Hawassa open ﬁsh market. Fatty acid proﬁles were analyzed using a gas chromatography-mass spectrophotometer (GCMS), and the health lipid index was determined by calculation using the recommended formula. JMP pro 13 version software was used for data analysis. Our result showed that raw ﬁsh had a high amount of essential fatty acid, nutritive value index, hypocholesterolemic ratio, and peroxidizability index. In contrary to this, the fried ﬁsh had a high amount of trans -fatty acids, nonessential fatty acids, atherogenic index, and thrombogenic index. In conclusion, the fried ﬁsh loses its fatty-acid-related nutritional quality in uncontrolled frying conditions. Therefore, frying needs to be controlled, as it risks


Introduction
Fish is a source of essential nutrients and is highly promoted by nutritionists and health-care workers because it has a high amount of double-bonded fatty acids and plays an important role in chronic disease prevention [1]. According to Leung et al. [2], complimentary food including fish for infants is highly recommended. However, according to Gadiraju et al. [3], fish is highly susceptible to safety and quality deterioration, and this leads the fish to lose its nutritional quality due to processing.
Currently, fried food consumption is highly accustomed as it has a good sensory appeal, such as crispy texture, color, and taste [4], and consumption of fried fish has become more common in the world including Ethiopia. Oke et al. [5] stated that changes such as fat absorption, moisture loss, and oxidation reactions occur during frying, and this generates free radicals that affect the safety of fish and considered a risk factor for chronic diseases which can limit the life span of the consumer. us, according to FAO [6], a risk inspection and control system should be established for minimizing such problems.
People living around the lake Hawassa (Ethiopia) are highly vulnerable to the health problem associated with fried fish consumption. Around the lake, in the open fish market, three main products are served (fish soup, fried fish, and fish fillet), and Nile tilapia fish is one of the important commercial fish at lake Hawassa [7]. e main fish processing method across Hawassa open fish markets is frying, and no studies have been conducted about identifying the impact of frying on the nutritional quality of fish muscle from the Hawassa open fish market; thus, it is necessary to study the fatty-acid-related nutritional quality of fish muscle, and this would in turn help in identifying the key risks and intervene across the fish value chain, mainly frying in the open fish market. erefore, the current study aims to characterize the fatty acid profile of raw and fried fish from the Hawassa open fish market and identify the nutritional quality index to show the risk and direction of the management system.

Description of the Sample Collection Area.
Hawassa is the capital city of the southern region and Sidamo regional state of Ethiopia. Hawassa city is 275 km far from the capital city of Ethiopia (Addis Ababa), and the city is known by its lake called Lake Hawassa, which is about 95 sq km wide and stretches 16 km from northeast to southwest and 8 km from east to west [8]. e lake depth is 21.6 m. Geographically, Lake Hawassa is found in 6 033′-7 033′ N and 38 022′-38 029′ E. e lake has no outflow and receives inflow from the factory via the Tikur Wuha River [9]. e lake is named yefiker Hayek in Amharic meaning adorable lake. e lake is very useful economically for the entrepreneurs. e lake has more than three fish species, and Nile tilapia (Koroso) is the dominant fish species found in the lake [8]. e lake serves three types of fish products, fillet, fried fish, and fish soup [10].

Sample Collection and Preparation.
Sampling operation was carried out during the dry season. Raw fish was collected from Hawassa Lake (Gudumale Park), and the fishes were immediately dissected in the field and cleaned, descaled, and eviscerated manually using a sterile plastic knife [11]. e edible part (flesh and skin) was transferred into plastic bags and kept in an icebox at a temperature of 4°C with a fish/ice ratio of 1 : 2 and then transported to the laboratory. e fried fish was randomly collected from the Hawassa fried fish selling market (Fikir Hayik). Raw and fried fishes were grounded to 0.3 mm size, and the powder was stored in desiccators for analysis. Samples were homogenized by passing them twice through a mincer with 4 mm holes and mixed thoroughly. Uniformity of the homogenate was ensured by further mixing. e resultant homogenate was packed into several small, convenient sterile containers and stored at 0°C until analysis. e experiment was conducted in duplicate for all analyses.

Determination of Fatty Acid Profiles.
e fatty acid profile was determined by using a gas chromatography-mass spectrophotometer (GCMS). Lipid was extracted following the standard procedure in [12]. A homogenized fried fish sample of 5 g was weighed into a conical flask and dried for 1 hr at 105°C. e flask was cooled to room temperature, and 50 ml of 4 M hydrochloric acid was added; then, the solution was boiled for 1 hr. Water (150 ml) was added, then the solution was filtered by fluted filter paper, and washed until a neutral reaction to litmus paper. e filter paper was dried for 1 hr at 105°C and inserted into an extraction thimble of the Soxhlet apparatus. Fat extraction was continued with hexane using a Soxhlet for 6 hr. Hexane was evaporated using a rotary evaporator after extraction, and it was kept for derivatization.
Exactly 200 mg of oil was weighed into the bottom of the screw-capped tube (Teflon-lined). 2 ml of 2N KOH was added in methanol. e tube was closed tightly and heated by shaking on a water bath at 80oC for 1 hr and cooled, and then, 5 ml of 5% HCl was added in methanol. e tube was closed tightly again and heated by shaking on a water bath at 80°C for 1 hr. After cooling, 5 ml of hexane and 5 ml of water were added to the tube and mixed, and then, hexane was collected from the layer after short centrifugation. e solvent layer was washed by diluting potassium bicarbonate to remove excess acid and dried over anhydrous sodium or magnesium sulfate. Potassium bicarbonate was recovered after removal of the solvent by evaporation under reduced pressure on a rotary film evaporator. en, the extract was transferred into a GC vial and injected into it. All the analyses were carried out in triplicates.

Indexes of Lipid Quality.
From fatty-acid composition data, the atherogenicity index and thrombogenicity index were calculated as follows: Atherogenicity index (AI): it is the relationship between the sum of the main saturated fatty acids and main classes of unsaturated, the former being considered proatherogenic (favoring the adhesion of lipids to cells of the immunological and circulatory system) and the latter being antiatherogenic (inhibiting the aggregation of plaque and diminishing the levels of esterified fatty acid, cholesterol, and phospholipids, thereby preventing the appearance of micro-and macrocoronary diseases [13]. e following equation was applied: rombogenicity index (TI): is the tendency to form clots in blood vessels. It is defined as the relationship between prothrombogenic (saturated) and the antithrombogenic fatty acids (MUFAs, PUFAs-n6, and PUFAs-n3) [13]. 2

Journal of Food Quality
Hypocholesterolaemic index (h/HI): it is the cholesterol/saturated fat index. Low CSI indicates low saturated fat and cholesterol and low atheroginicity [14].

Data
Analyses. e analyses were performed twice for all samples, and closely agreeing replicates were obtained. e data were analyzed using JMP pro 13, one-way ANOVA. e means were separated using Tukey's HSD test at p < 0.05.

Assessment of Consumption, Frying, and Marketing of Fish at the Hawassa Lakeside.
A preliminary study about the consumption, frying, and marketing of fish at the Hawassa lakeside (Gudumale Park, Tikur wuha, and Fikir Hayik) was conducted. ere are more than 18 unions on the lakeside. e preliminary assessments showed more than 60 fishes are fried at the lakeside by each union. us, more than 1080 fried fishes are marketable per day. At a time, a maximum of 5 fishes are fried using a metal pan after 5 min of catching, and the frying time is 10 min. e most commonly fried, consumed fish in the lakeside is Nile tilapia (Koroso), and palm oil is the commonly used oil for frying at the lakeside. After frying, all unions put fried fishes in the open air by hanging on wood to collect the oil leaked from fried fish, and the maximum duration of storing fried fishes before serving to customers is 30 min. e unions use the oil several times as they are not well informed about food safety. us, the fish can be exposed to many quality and safety degradation.

Identified Fatty Acids in Raw and Commercial Fried Fish.
A total of 22 fatty acids were identified in raw and fried Nile tilapia fish muscles (Table 1) Among the identified fatty acids, cis-monounsaturated and -polyunsaturated, omega-3, and omega-6 are the most important contributors to the enrichment of aromatic components [17] and are considered as highly nutritional because of their protective role against cardiovascular diseases and the ability to reduce susceptibility to mental illness, decrease brain and eye function in infants, and alleviate rheumatoid arthritis symptoms [18]. On the contrary, saturated fatty acids cause a rise in the blood cholesterol level as they are easily deposited on the walls of the arteries [19].
Saturated fatty acids and trans-fatty acids of commercially fried tilapia fish were higher as compared to raw fish. e increase in saturated fatty acids might be because the frying oil is palm oil, and palm oil is high in saturated fat. Similarly, the uncontrolled frying method might result in a high amount of saturated fatty acids in commercially fried fish. Wang et al. [20] stated that a higher temperature can accelerate the reaction rates of oxidation, and more trans-fat could be generated in the frying oil and penetrate the frying fish. A similar finding was reported in [21]. Conversely, cis-and essential fatty acids were lower in the fried fish compared to raw tilapia fish. e decrease in cis-and essential fatty acids are associated with the increase in trans-and saturated fatty acids. According to Saguy [22], the increase in trans-fatty acids might be due to the decrease in cis-fatty acids by thermal oxidation, and cis-fatty acids can be converted or isomerized into trans-fatty acids. e increment of saturated fatty acids and trans-fatty acids was supported by a strong significant negative correlation with the decrement of essential fatty acids (omega-3 and omega-6) and cis-fatty acids (%) at both α � 0.05 and 0.01 with rcalc � −1.

Nutritional Quality Index (NQI) of Raw and Commercial
Fried Fish. Fried fish consumption is popular worldwide including in Ethiopia. Recently, a few studies have been conducted and stated that Nile tilapia fish is the major fish being fried and consumed in the Hawassa open market   [7]. e frying oil used in an open fish market is palm oil because the oil is cheap and provided through cooperatives easily; this oil has been reused for a long time, and this might affect the fatty-acid-related nutritional quality index. Our study showed, in commercially fried fish, the saturated fatty acid, atherogenic index, thrombogenic index, and hypercholesterolemic index have been increased. On contrary to this, the unsaturated fatty acid, hypocholesterolemic index, and nutritive value index have been decreased. erefore, frying noticeably decreases the nutritional quality of fish muscle. e summation of saturated fatty acids, unsaturated fatty acids including essential, trans-, and cis-fatty acids, and the nutritional quality index is detailed in Table 3. e total saturated fatty acids, cis-fatty acids, trans-fatty acids, omega-3 fatty acids, and omega-6 fatty acids of raw and commercial fried fish were ranging from 38. 27 e PUFA/SFA and ω-3/ω−6 are commonly used parameters to judge the nutritional quality and healthiness of intramuscular fat for human consumption. Kang et al. [23] reported that a balanced intake of dietary PUFA/SFA is very important in regulating serum cholesterol.
Indeed, the ratio of UFA/SFA and PUFA/SFA greater than 0.45 is recommended in human diets for cardiovascular (CVD) and chronic disease prevention [24]. In the meantime, a diet with a ratio of UFA/SFA and PUFA/SFA below 0.45 is considered undesirable for consumption due to its potential to induce cholesterol increase in the blood [25]. e result of this study showed that the UFA/SFA and PUFA/SFA of raw fish are about 1.16% and 0.44%, which is beneficial for human health. However, the ratio of PUFA/SFA for commercially fried fish is 0.20 which is below the recommended level, and the commercially fried fish is not considered as a balanced human diet and it cannot improve the balance of fatty acids in the tissue. According to Wood et al., 2003, the recent focus of nutritionists is omega-3 and omega-6 fatty acids, and the high proportion of PUFA is not necessarily healthy unless it is not balanced with the ω−3/ω−6 ratio.
Omega-3, omega-6, and the ratio of ω−6/ω−3 are principal fatty acids to control the hypocholesterolemic index. Omega-3 and omega-6 fatty acids regulate the thrombogenic and atherogenic index, respectively. Wolosyzyn et al. [24] reported that healthy meat and meat kind products are characterized by a low atherogenic index and high h/H index. Attia et al. [26] also reported that meat and meat kind products with a low thrombogenicity decrease threat of atrial fibrillation. According to Fernandes et al. [14], if the ratio of ω−6/ω−3 is less than 4, then the diet has a desirable quantity of omega-3 and omega-6 fatty acids and reduces cardiovascular diseases. In our study, the ratio of omega-6 and omega-3 fatty acids of raw and commercial fried Nile tilapia fish was 4.76% and 19.98%, respectively, and this showed that raw fish is good and beneficial for human health since the ratio is not as such deviated from the standard. However, the ratio ω−6/ω−3 for commercially fried fish is significantly higher than the standard value, and consumption of the commercially fried fish couldnot be beneficial for human health and can expose to noncommunicable diseases.
In addition to the ratio of UFA/SFA, PUFA/SFA, and ω−6/ω−3, it is important to evaluate DHA, EPA, atherogenicity, thrombogenicity, nutritive value, and hypocholesterolemic/hypercholesterolemic index (HH) as they indicate the contribution of fish fatty acids to human health. Maki et al. [27] reported that DHA decreases the concentration of low-density lipoprotein cholesterol in plasma and EPA is recognized as the most important essential fatty acid of the ω−3 series in the human diet because it is the precursor to the formation of ω−3 series eicosanoids [28]. In the study, DHA and EPA of raw and commercially fried Nile tilapia fish vary from 1.02-0.25% and 0.82-0.1%, respectively. e study revealed that raw fish had higher DHA and EPA compared to commercially fried fish. e lower DHA and EPA in commercially fried fish might be because double bonds are highly labile for oxidation during heating, owing to the presence of (pi) bonds, which consequently produces degradable products, though the essential fatty acid would be degraded [29].

Nutritive Value of Raw and Commercial Fried Fish.
e ratio of the nutritive value index of raw and commercial fried fish is shown in (Figure 1). Atherogenicity indicates the adhesion of lipids to cells of the immunological and circulatory system, inhibiting the aggregation of plaque and diminishing the levels of esterified fatty acid, cholesterol, and phospholipids, thereby preventing the appearance of microand macrocoronary diseases, and myristic and palmitic acids are among the most atherogenic agents [30]. rombogenicity showed the tendency to form clots in the blood vessels [31]. Stearic is thought to be neutral to atherogenicity but considered to be thrombogenic [32]. According to Estuary et al. [33] and Karimian et al. [34], the higher index of atherogenicity and thrombogenicity values is responsible for the formation of atheroma and stimulates the aggregation of platelets in the cardiovascular system. Hence, the lower values are desirable in the prevention of cardiovascular disorders. e current study showed IA and IT of raw and commercial fried Nile tilapia fish ranged from 0.60-1.01% and 0.85-1.58%, respectively. is indicates frying leads to increase IA and IT which is considered to be low in nutritional quality. e index of the ratio between hypocholesterolemic and hypercholesterolemic of fatty acid (h/H index) is another indicator of the effect of specific fatty acids on cholesterol metabolism. e higher value of this index is considered to be desirable.
us, nutritionally higher h/H values are considered more beneficial for human health. In the current study, the h/H index obtained in raw fish is about 1.81 and the h/H index for commercially fried fish is 1.15. is indicates raw fish is more healthy and beneficial as compared to fried ones. e highest NVI value was found in raw fish Journal of Food Quality 5 which about 1.23 as compared to commercially fried Nile tilapia fish whose NVI value is 0.91. e highest NVI value in raw fish is because raw fish had the highest proportion of C 18 : 0, C 18 : 1n−9, and the lowest percentage of C 16 : 0.

Comparison of the Hypocholesterolemic and Peroxidizability Index of Raw and Commercial Fried
Fish. e hypocholesterolemic, hepercholestrolemic, and peroxidizability index of raw and commercial fried fish are shown in Figure 2. Peroxidizability index (PI) is another parameter to indicate the relationship between the fatty acid composition of a tissue and its susceptibility to oxidation. e PI index assesses the stability of PUFA included in food products and protects from possible oxidation. e higher PI value is equivalent to the greater protective potential for coronary artery disease. According to Kang et al. [23], Sinanoglou et al. [35], and Ska1ecki et al. [36], excessive intake of PUFA leads to oxidative stress because lipid is susceptible to lipid peroxidation. Oxidative stress is associated with the formation of lipid peroxides, and has been suggested as contributing to pathological processes in aging and many diseases such as atherosclerosis.
In the current study, the PI value of raw and commercial fried fish ranged from 38.27%-15.45%.
us, the study revealed that commercially fried Nile tilapia fish had a lower PI value compared to raw fish, and the lower PI value indicates low level of PUFA for auto-oxidation. However, raw  fish had high PUFA and susceptible to auto-oxidation. e higher tendency to lipid oxidation was found in different kinds of marine fish fillet by Ghaeni et al. [31] and in crab edible tissue by Fernandes et al. [14].

Conclusions and Recommendations
e dominant saturated fatty acids found in Nile tilapia fish were palmitic, myristic acid, and methyl stearate. Oleic acid, linoleic acid, and palmitoleic acid were the highest cis-fatty acids. Saturated and trans-fatty acids were correlated positively. Similarly, unsaturated and cis-fatty acids were found to have a positive correlation. Regarding the nutritional quality index, raw fish was found with a high amount of essential fatty acids, h/H index, nutritive value index, and peroxidizability index which is considered beneficial for human health. Conversely, in the commercially fried fish, a high amount of IA and IT was found, which could have a negative influence on human health. us, it could be recommended to set the frying time and temperature during fish frying, and further analysis should be conducted about the frying oil's effect on the nutritional composition of fried fish.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding authorship and/or publication of this article.

Authors' Contributions
Aemiro Tadesse is the principal investigator, designed the study, took part in the sample collection process and laboratory experiment, entered and analyzed the data, and wrote the manuscript of the current study. Derese Tamiru oversaw the whole sample collection process, giving comments and suggestion during the manuscript write up. e authors read and approved the final manuscript to be submitted.
Acknowledgments e current study was funded by Hawassa University as a part, and the authors are grateful for the fund and help. e donors had no direct involvement in data collection, analysis, interpretations, and experimental work. e authors also acknowledge the School of Nutrition, Food Science, and Technology of Hawassa University for support given during the sample collection processes.