Effect of frozen storage on the biochemical composition of five commercial freshwater fish species from River Nile, Sudan

Abstract Postharvest processing and preservation of fish have great influence on fish quality and consumption. Freshwater fish in Sudan are facing problems related to bad handling and improper storage which reduce their quality. This study investigated the changes in the chemical composition, mineral contents, pH and acid value during storage (−18°C) of five commercial fish species (Bagras bayad, Lates niloticus L., Mormyrus casahive L., Oreochromis nilotica L., and Synodrontis schall) from the River Nile coast of Sudan. The fish species are rich in protein (17.22%–23.60%) but have low fat and ash contents. Frozen storage of the fishes for 45 days reduces their protein contents while the fat and ash contents were increased (p ≤ .05). Potassium and iron are the predominant major and trace minerals and their values were increased with storage period. The pH range from 5.74 (O. niloticus) to 6.24 (B. bayad) while acid value range from 0.02 (M. casahive) to 0.12 (L. niloticus). Both pH and acid values increased with storage period. In conclusion, storage of these fish species for up to 45 days did not adversely affect their nutritional value.

Generally, fish has been an important source of foods for human since it is among the cheapest source of protein, highly digestible and rich in unsaturated fatty acids. Moreover, it contains high amount of other nutrients such as minerals and essential amino acids for the development of functional and structural proteins (Mahboob et al., 2015). Increase in human population has led to increase in demand for alternative source of proteins and the gap in fish supplies and as human food are expected to be filled by aquaculture industries (Naylor et al., 2000).
The River Nile in Sudan is rich in fish biodiversity with approximately of 128 species belonging to 27 families (Witte et al., 2009).
In spite of high fish potentialities in Sudan, yet consumption of fish meat is still quite low compared to that of red meat and this may probably be due to poor postharvest processing and preservation.
Freshwater fish products are known to easily deteriorate during post mortem storage and processing as a result of autolytic degradation, microbiological spoilage, and lipid oxidation Subramanian, 2007). Also, freshwater fish and its products in Sudan are facing problems related to bad handling and improper storage which reduce their quality. Inadequate storage techniques would imply a substantial shortfall in fish availability thereby affecting the animal protein intake of the people and has a large economic impact (Adejonwo et al., 2010).
Freezing storage is an important postmortem method for preservation of fish and it is considered as the best way of preserving the quality of fish if proper care at each step of freezing is done (Foucat et al., 2001). Fish quality also deteriorate to some extend during freezing storage and the extent of deterioration is usually determined by measuring the sensory, chemical, and physical changes (Liu et al., 2009;Obemeata et al., 2011). In order to increase the utilization of commonly consumed fresh water fishes obtained from River Nile in Sudan, changes in quality of the fishes during freezing storage need to be examined. Therefore, the present study evaluated the changes in the chemical composition, pH, acid value, and mineral composition of five commercial fishes; Bulti (Oreochromis

| Collection of samples
Five species of freshwater fish from River Nile commonly consumed in Sudan were used in this study. Families, genera, species as well as local and English names of these fishes are presented in Table 1. Fresh fish samples (4 kg each) of the same freshness, catch period, weight (480-600 g/fish), and approximate length (25-30 cm/fish) were collected randomly from fishermen. The samples were then transported within 2-3 hr in ice boxes (4°C) to the laboratories of Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum. All reagents used were of analytical grade and procured from Fisher Scientific Co., Rochester, New York.

| Sample preparation
Fish samples were thoroughly washed with clean water to remove all contaminants or unwanted particles and dirt, disemboweled and beheaded. The nonedible portions (offal, head, viscera, and scales) were removed and weighed to determine percentage of edible portion in each fish. Fish muscles were separated from the bones and the leans muscles remaining were chopped into pieces (0.25 cm), homogenate thoroughly and divided into four equal portions.
Each portion was assigned randomly to one of four storage period (0, 15, 30, and 45 days), packed in polythene bags and frozen in Gyrofreezer set at −18°C. Samples were taken at each storage period, thawed at room temperature (25°C), and then minced before each analysis.

| Chemical composition
The chemical compositions (moisture, crude protein, fat, and ash) of the fresh fishes were determined according to AOAC (2005) method. The moisture content was determined by oven drying method (Method No. 934.01) at 105°C (AOAC, 2005). The crude protein content was determined using Kjeldahl method (Method No. 978.04) and 6.25 was used as conversion factor (AOAC, 2005). The crude fat content was determined using Soxhlet method (Method No. 930.09) and petroleum ether as extracting solvent (AOAC, 2005).
The ash content was measured using incineration method (Method No. 930.05) at 550°C (AOAC, 2005). The results were expressed as percentage of wet weight basis.

| pH determination
Exactly, 50 ml of distilled water was added to 10 g of fresh minced fish samples. The mixture was stirred well for about 5 min, and contents were then allowed to settle. The pH of the mixture was recorded using HI 255 combined pH meter.

| Determination of acid value
Acid value was analyzed according to official method Cd 3a-63 (AOCS, 2017). One ml of oil or fat extracted from fish samples was transferred into a glass vial and dissolved in mixture of 100 ml of ethanol and diethyl ether (1:1; v/v), heated gently and titrated with

| Statistical analysis
Data were analyzed using the Software of the Statistical Analysis System (SAS). Experiments were carried out in triplicates. The data were assigned in Completely Randomized Design (CDR) and subjected to one-way analysis of variance (ANOVA). Mean separation was done using DMRT tests and significance level was accepted at the probability level of (p ≤ .05).

| Effect of species and storage on chemical composition of fish fillets
The chemical composition of fishes could greatly influence the postharvest processes and storage and also assist in determining the suitability of different species to specific processing and storage techniques (Optstvedt, 2000). As shown in Table 2 (Kirschnik et al., 2006). The decline in moisture content during the earlier stages of storage might be due to sublimation of surface water of fish in the freezer . However, moisture losses in the later stage of freezing may be due to myofibrillar distortion resulting in poor water denaturation ability in the muscles of fish fillets . In addition, water loss by evaporation is greater during the precooling period, since the mass transfer rate is reduced when the product freeze. The high moisture content of the fish fillet would increase the deterioration level of fish when kept for long time due to the increased activity of the microorganisms associated with the high moisture content (Love, 1980;Oluwaniyi & Dosumu, 2009). Generally, storage at −18°C for 45 days reduced the moisture content of the investigated samples with a reduction rate of 0.77% (Bulti) to 1.95% (Gargour). Storage time and temperature are the major factors implicated in the loss of quality and shelf life of fish (Rahman, 2007) which result in hard, dry and fibrous fish products, hence reduced eating quality. Moreover, liquid losses may also directly result in economic losses (Obuz and Dikeman, 2003 (Aberoumand, 2013;Arekemase et al., 2010;Ekpenyong & Ibok, 2012;Saliu, 2008) and to changes in the proportion of chemical composition and protein breakdown (Kjaersgård et al., 2006). Also, there may be leaching of nitrogen during thawing which in turn causes a reduction in the protein content Arannilewa et al., 2005). Furthermore, fish undergoes rapid protein degradation due to the action of endogenous and bacterial enzymes after death of fishes (Saeed & Howell, 2002). Deterioration of fish quality due to protein denaturation can therefore be checked by using as low temperature as possible, preferably at −18°C. Storage temperature and time have great impact on the degree of protein denaturation (Herrero et al., 2005). The shelf life of fish products, therefore, is markedly extended when products are stored at low temperatures (Herrero et al., 2005). Other possible reasons for decrease in protein content might be the loss of ammonia (NH 3 ), volatile amines, conversion of nitrogen to other nonprotein nitrogen molecules, amino acid destruction, and formation of protein-fat complexes that results in decrease in protein content (Saeed & Howell, 2002). Present study shows that storage can cause decrease in the protein content of freshwater fish from River Nile (Sudan). Thus, it is better to consume the fishes before 45 day of storage or to be involved in a technological process after freezing at −18°C for 45 days.
The fat contents of fillet samples of all examined species, excluding Bulti, were significantly (p ≤ .05) increased throughout the storage period compared to fresh samples (Table 2). Although, fat content of Bulti was not changed at 15th day of storage, however, it increased (p ≤ .05) significantly as the storage period progressed.
Highest fat content was found in all the species at the end of the storage period. Similar increases in fat content during storage have been reported in Tilapia fish fillets (Emire & Gebremariam, 2010) and TA B L E 3 Effect of specie and storage on the mineral contents (mg/100 g) of fresh water fishes from River Nile, Sudan  (Özyurt et al., 2005). Although the fat content was increased in all fishes as the storage period elevated, but the trend of increasing differed significantly (p ≤ .05) between fish species.
The resultant increased fat content can be ascribed to the fact that there is an inverse relationship (r = −0.99) between the moisture and lipid contents of fish flesh Inhamuns & Bueno Franco, 2001;Oliveira et al., 2003).
The highest ash contents for all investigated fish species were perceived in the 45th day of the storage, while the lowest ash values were recorded in the initial day of storage (Table 2). However, the differences were not significant except in Bulti and Ijeel at the end of storage period as compared to day 0. Similarly, higher ash percentage was found in Tilapia fish after 240 days of storage at −18°C as compared to fresh sample. Conversely, Okeyo et al. (2009) who studied L. niloticus fish found that ash content was not significantly (p ≥ .05) related to storage time. Loss of water in any food substances produce an uneven increase in the percentage of other nutrients (Castrillon et al., 2008), therefore, the consequential increase in ash content during freezing might be due to the decrease in moisture and protein contents of the studied fish samples.

| Effect of species and storage on mineral composition of fish fillets
The five fish species showed different mineral concentrations throughout the different storage intervals (Table 3). Fish absorb minerals not only from their diets but also from the surrounding water via their gills and skin (Nurnadia et al., 2013). As shown in Table 3 Generally, the impact of storage on the mineral contents of freshwater fishes was differed depending on the types of species and studied minerals as well as storage period.

| Effect of species and storage on pH and acid values of fish fillets
The pH and acid value follows the same trend throughout the storage period for all samples ( Storage average 0.05 D ± 0.14 0.17 C ± 0.13 0.24 B ± 0.14 0.33 A ± 0.14 Note: Means (n = 3) not sharing a common letter in columns and rows are significantly different at p ≤ .05.

F I G U R E 1
The HJ-biplot based on principal component analysis for biochemical composition of fleshes of five freshwater fish species from River Nile as affected by frozen storage at −18°C for 0, 15, 30, and 45 days the pH between these studies perhaps due to the differences in the catching season, geographical location, fish size, and water composition. The increase in pH values during frozen storage may be due to the accumulation of alkaline compounds, such as ammonia, mainly derived from microbial actions (Liu et al., 2013).
The acid values obtained from extracted oil of stored fillet samples of Bulti, Gargour, Bayad and Ijeel were significantly (p ≤ .05) increased following the initial 15 days of storage to between 0.08 and 0.27 mg KOH/g (Table 4) However, results of the present study were less than the limit, which mean that (the oil of) fish stored at −18°C are suitable for consumption up to 45 days.

| Multivariate analysis
To profoundly estimate the combined effect of storage period and fish species on the biochemical composition of five freshwater fishes from River Nile, principal component analysis using HJ-Biplot was conducted. The results clearly showed an excellent contribution of the principle components axes (PC1, 53.11% and PC2, 31.55%) to the total variability (84.66%) of the blotted data ( Figure 1). Clustering analysis indicated four groups of fish species and storage times based on their combined impact on the biochemical composition of the fish fleshes. The first group (triangle symbol) composed of Gargour (

| CON CLUS IONS
In conclusion, the five fish species are good source of protein, potassium, sodium, and iron. Storage of the fishes for up to 45 days greatly reduces their protein contents while their fats, ash, and mineral contents were increased. The increase in pH of the fish species during storage could promote microbial growth, thus, further study should be conducted to investigate the microbiological characteristics of these fishes at these storage conditions.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

E TH I C A L A PPROVA L
The study did not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data that support the findings of this study will be available from corresponding author upon request.