Nutrient composition analysis to investigate the richness Rita Rita in essential nutritional components.

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Rita rita, an important member of Bagridae family, contribute largely to riverine fisheries in the Indian subcontinent [1]. It enjoys high consumer preference as a valuable food fish due to its muscle texture, good taste and lesser intramuscular spines. The meat quality and texture is mainly determined by biological factors such as muscle protein content, organization, and composition [2]. Earlier we have generated information on muscleproteome profileandfunctionalgenomics aspects of this catfish and we could identify proteins in muscle proteome associated with muscle texture, flesh quality such as elasticity, firmness, water holding capacity etc. which could possibly be linked to its taste and high consumer preference [3].

Besides quality animal proteins, fish is also considered to be an important source of essential and functional amino acids, fatty acids and minerals that are essential in human health and nutrition [4]. As nutritional information on of this species is meagre, we have carried out the nutrient composition analysis to investigate the richness of the species in essential nutritional component and how it can contribute to nutritional security. As the taste, nutrition, muscle composition and quality of fish meat are known to vary with age, growth, season etc., a comparative nutrient profiling of three different size groups of the fish has been carried out.

Materials and Methods

Sample collection

The fishes were collected from river Ganga at Allahabad, Farakka and Kolkata. After collection, the individual length (cm) and weight (gm) of the fishes were recorded. The fishes were divided into three groups based on body weight as small (200-400 g), medium (500-800 g) and large (1200-1800 g). The authors confirm that all the research done meet the ethical guidelines, including adherence to the legal requirements of the study country.

Proximate composition

Fishes were cleaned, degutted, filleted and were stored at -40°C until further use. The proximate compositions (moisture, crude fat, crude protein and ash) were determined as per standard protocol [5]. Fish fillets of small (n=12), medium (n=10), large (n=6) samples were homogenized with the help of a mixer grinder. The minced samples were kept in an oven at 105±2°C overnight until constant weight was obtained. The crude protein and crude fat contents were estimated by Kjeldahl and Soxhlet methods, respectively [6]. Ash was obtained after incineration of moisture free dry sample in a muffle furnace at 600°C for 6 h until weight became constant. The ash content was determined gravimetrically and expressed as percentage.

Amino acids

Amino acid composition was determined following standard protocol [7] and has been described earlier [8]. Briefly, 50 mg muscle samples of individual fishes from small, medium and large size category were hydrolyzed with 6N hydrochloric acid at 110°C under anaerobic condition for 12 h. The hydrolyzed samples were neutralized with 6N NaOH and were derivatized using a kit (AccQ-Fluor Reagent, WAT052880, Waters). The derivatized sample were injected in HPLC (1525, Waters) equipped with a C18 RP column and a fluorescence detector (2475, Waters) with excitation and emission wavelengths 250 nm and 395 nm, respectively. The amino acids were identified and quantified by comparing the retention times and peak areas of standards (WAT088122, Waters). For the tryptophan analysis, minced meat was digested with 5% (w/v) NaOH for 12 h and neutralized to pH 7.0 with 6N HCl. Tryptophan content was measured spectrophotometrically at 530 nm [9].

Fatty Acids

Total lipid of the fish was extracted following the standard protocol [10]. Briefly, 30 gm wet fish muscle was homogenised with 2:1 mixture of chloroform and methanol to extract the total lipid content of the tissue. Fatty acid methyl esters (FAMEs) were prepared by transesterification with boron trifluoride (BF3) in methanol from lipid fraction according to Metcalfe et al [11]. The FAMEs were quantified by injecting into gas chromatography–mass spectrometry (GC–MS) (ITQ 900, Thermo Scientific). The FAME 0.5 µL (30:1 split ratio) was injected into GC-MS column and identified, quantified using a GC (Trace GC Ultra, Thermo Scientific) equipped with a capillary column (TR FAME 30 m × 0.25 mm, 0.25 µm film thickness) and an MS attached to it. The individual constituents showed by GC–MS were identified and quantified by comparing the retention times and peak areas to those of standards (ME-14-KT and ME-19-KT, SUPELCO Analytical).

Minerals

Fish samples of different size group were analyzed in ICP Spectrometer (iCAP 6300 Radial, Thermo Scientific) after digesting the samples using nitric acid and hydrogen peroxide in a microwave oven. Quantification was done by comparing with multielement standard IV, MERCK (NIST) for sodium, potassium, calcium, magnesium, iron, copper, zinc and manganese and Trace Cert (NIST) for phosphorous.

Statistical analysis

Results are given as mean ± standrad deviation of the mean and subjected to analysis of variance (ANOVA) using SPSS 16.0 software. The occurrence of significance was established at p ≤ 0.05.

Results and discussion

Proximate composition

The proximate composition of different size groups of Rita rita is expressed in percentage of wet tissue (Table 1). The average moisture, crude protein, crude fat and ash content of different size groups of fishes were estimated to be 79.09%, 18.12%, 1.78% and 1.00%, respectively (Fig. 1). Protein content of Rita rita ranged between 17.2-19.55%. Protein content of medium size (19.55%) is comparatively higher than other size groups and similar to Indian major carps (IMCs) Catla catla (19.60%), Labeo rohita (19.10%) and Cirrihinus mrigala (19.10%) [12]. Average protein content of Rita rita (18.12%) is higher than that of many other catfishes like Mystus vittatus (15.62%), Clarius batrachus (14.78%), Wallago attu (17.00%) and is similar to bagridae catfish Sperata aor (19.05%) [13, 14] and Sperata seenghala (20.06%) [8]. Crude fat content is significantly higher in large size. Average crude fat content of Rita rita was found to be 1.78%, which is very low as compared to other catfishes such as Heteropneustes fossilis (3.45%), Clarias batrachus (7.90%), M. vittatus (7.53%) and IMC L. rohita (4.33%) [13]. Average crude fat content of Rita rita is similar to that of its close relative S. aor (1.78%) [13, 14] and S. seenghala (1.40%) [8]. The high protein and low fat content of Rita rita indicates that this catfish can be exploited as a good source of lean meat, which has a good demand.

Amino acid composition

Amino acids are the building blocks of body proteins that are responsible for growth, repair and maintenance of cell [15]. Based on the growth or nitrogen balance of animals, amino acids have been traditionally classified as nutritionally ‘essential’ or ‘nonessential’ as the dietary requirements of amino acid depend on species, developmental stage, physiological status, the microbiota in the lumen of the small intestine, environmental factors, and pathological states etc. [16]. Thus, some of the amino acid that are synthesized by animals have been classified as conditionally essential (CEAA) because rates of their utilization are greater than rates of their synthesis under certain conditions which include glutamine, arginine, proline, glycine and taurine [17]. However, recent concept of functional amino acids mention about amino acids that participate in and regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of the organisms and the amino acids of significance in human nutrition are arginine, cystein, leucine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, glycine, proline and taurine [17].

Seventeen different amino acid were detected in muscle extract of Rita rita. The amino acid profiles of different size groups of Rita rita are expressed in percentage of total amino acid (Table 2). Total essential amino acid was 72.15%, 86.32%, and 55.12% in small, medium and large size groups, respectively. Essential amino acid content was found to be higher in medium size groups than small and large. The non-essential amino acid contents were 7.65%, 12.20% and 10.97% in small, medium and large, respectively.

Among essential amino acids (EAAs), leucine (17.06%) is the predominant one. Leucine is the only dietary amino acid that can stimulate muscle protein synthesis [18] and has important therapeutic role in stress conditions like burn, trauma and sepsis [19]. As a dietary supplement, leucine has been found to slow the degradation of muscle tissue by increasing the synthesis of muscle proteins. Leucine content is significantly high in medium size fishes (18.67%). Average leucine content of Rita rita was higher than other freshwater catfishes like S. seenghala (0.7%), H. fossilis (8.2%) and C. batrachus (8.1%) and Indian major carps (IMCs) C. catla (7.7%), L. rohita (9.0%) and C. mrigala (8.4%) [20]. Phenylalanine is an essential amino acid and a precursor for biosynthesis of amino acid tyrosine and the biogenic amines dopamine, norepinephrine and epinephrine. Average phenylalanine content of Rita rita (12.46%) was also higher than S. seenghala (0.4%), H. fossilis (3.7%), C. batrachus (3.7%). Glutamic acid plays an important role in amino acid metabolism because of its role in transamination reactions and is necessary for the synthesis of key molecules, such as glutathione which are required for removal of highly toxic peroxides and the polyglutamatedfolate cofactors. Glutamic acid content of medium size fishes (10.55%) is significantly higher in medium size than both small and large size. Glutamic acid content of Rita rita (11.66%) lower than IMCs C. catla (13.8%), L. rohita (14.6%), C. mrigala (14.8%) and freshwater catfishes H. fossilis (16.0%), C. batrachus (14.5%) but higher than giant catfish S. seenghala (1.6%) [19]. Comparative amino acid (leucine, phenylalanine and glutamic acid) content in different catfishes was presented in Fig. 2. Among non-essential amino acids, alanine (5.83%) and aspartic acid (2.94%) were found to be the dominant ones.

Fatty acid composition

The profiles and percentage composition of 27 different fatty acids in Rita rita are presented in Table 3. GC- MS fingerprint of fatty acid profile shows that total saturated fatty acid content in small, medium and large size fishes are 20.82%, 26.59% and 24.05%. Saturated fatty acid (SFA) content is lower in small fishes than medium and large fishes. Monounsaturated fatty acid (MUFA) content was found to be increasing with age and size of fish (small size (41.91%), medium (47.52%) and large (49.37%) size). Total polyunsaturated fatty acid (PUFA) distribution in different size groups of fishes: small (12.1%)> large (9.88%)> medium (8.78%).

In SFA, palmitic acid (C16:0) was found to be dominant one (11.02%). It was reported that palmitic acid was the predominant in SFA group as found in other catfishes like the channel catfish Ictalurus punctatus (19.2%), Pangasius hypophthalmus (42.63%) [21], riverine catfish S. seenghala (21.10%) [8] and in rainbow trout (Oncorhynchus mykiss) (21.3%) [21]. High amount of monounsaturated fatty acids (MUFAs) (46.27%) were found in freshwater catfish Rita rita. Oleic acid (C18:1) was found to be the major MUFAs. The level of oleic acid was 26.56%. Oleic acid content was lower in Rita rita than other catfishes like S. seenghala (28.36%) [8], Pangasius hypophthalmus (34.69%) [20]. The level docosahexaenoic acid (DHA) (C22:6) found to be (3.97%), which was the highest among all PUFAs, followed by eicosapentaenoic acid (EPA) (C20:5), arachidonic acid (C20:4). The amount of EPA and DHA was found to be 7.90%. EPA and DHA content were lesser than other catfish S. seenghala (10.56%) [8] and small indigenous fish Puntius sophore (9.47%) [22].

Mineral Content

Minerals are required in very trace amount; but, they are vital for maintaining proper homeostasis inside the body. The minerals that were detected in Rita rita are sodium (Na), potassium (K), calcium (Ca), magnesium (Mn), phosphorous (P) (macro minerals) and iron (Fe), zinc (Zn) and manganese (Mg) (micro minerals).The mineral profiles of Rita rita are presented (expressed in mg/kg) in Table 4.

Among the macrominerals, potassium is the predominant one followed by phosphorous, sodium, calcium, and magnesium. Potassium works in conjunction with sodium to control the water balance of the body. Potassium content is significantly high in medium size fishes than small and large size. The FDA-recommended daily allowance for potassium is 3,500 mg, needed for a healthy nervous system and a regular heart rhythm. Average potassium content is higher in Rita rita (15633.33 mg/kg) than other riverine catfishes like S. seenghala (13780.01 mg/kg), fresh water catfish H. fossilis (1320.67 mg/kg), walking catfish C. batrachus (3110.64 mg/kg) [23] and almost two times higher than that of nutrient dense small indigenous fish P. sophore (2283.7 mg/kg) [22]. Phosphorus is involved in many functions besides forming bones and teeth. Like calcium, fish meat is commonly regarded as valuable source phosphorus. Average phosphorus content is almost ten times higher in Rita rita (13333.00 mg/kg) than other catfishes like C. batrachus (1300.59 mg/kg) and H. fossilis (1070.09mg/kg) [23]. Sodium content was also significantly high in medium size fishes like potassium. Average, sodium content is higher in Rita rita (2700 mg/kg) than other riverine catfishes like S. seenghala (1983.11mg/kg), fresh water catfish H. fossilis (2040.38 mg/kg) and similar as walking catfish Clarias batrachus (2080.00 mg/kg) [23]. It is well known that the calcium is very much necessary for normal functioning of muscle, nervous system and strong bones (formation and mineralization). Calcium content is significantly higher in large size than small and medium .Average calcium content of this fish (1400 mg/kg) is found to be lower in comparison to other catfishes like S. seenghala (4581.15mg/kg), H. fossilis (1950.35 mg/kg), C. batrachus (2250.75 mg/kg) but higher than Indian major carps L. rohita (862.8 mg/kg) [12, 23].

The microminerals, average iron, zinc and manganese content is 64.26, 37.52, 6.66 mg/kg, respectively. Iron is essential for synthesis of haemoglobin in red blood cells which is important for transporting oxygen to all parts of the body. Iron deficiency is associated with anemia, impaired brain function and in infants is associated with poor learning ability and poor behavior. Thus, the high iron content makes this species nutritionally superior as such consumers have a strong perception that this fish is highly nutritious and thus is market demand for this fish. Iron content is significantly higher in small size groups than medium and large size. Iron content of Rita rita is higher than other catfishes like S. seenghala (45.1 mg/kg), C batrachus (18.9 mg/kg) and H. fossilis (27.1 mg/kg) and Indian major carps (IMCs) Catla catla (16 mg/kg), Labeo rohita (22 mg/kg), Cirrihinus mrigala (3 mg/kg) [22]. Zinc is an important micromineral required for growth and development as well as for the proper functioning of immune system, cell growth and healthy skin. Zinc acts as a co-factor for many enzymes required in metabolism. It also plays a vital role in cell division, cell growth and wound healing. Zinc content is lower in small size than medium and large. Same as iron, zinc content also high in Rita rita (37.52 mg/kg) than S.seenghala (29.4 mg/kg), C.batrachus (12.9 mg/kg) and H.fossilis (13.00 mg/kg) and Indian major carps (IMCs) Catla catla (13.0 mg/kg), Labeo rohita (19.0 mg/kg), Cirrihinus mrigala (3 mg/kg) [23] but lower than small indigenous fish P. sophore. Manganese content is higher than riverine catfish S. seenghala (2.36 mg/kg). Comparative micro mineral (Fe, Zn) content in different catfishes was presented in Fig. 3.

Based on nutritional composition of Rita rita, it can be concluded that high protein and low fat makes this species nutritious as a good source of lean meat. Along with protein it is also a good source of important amino acids like leucine, phenylalanine and glutamic acid. This fish is also rich in minerals potassium, phosphorous, iron and zinc. Moreover, comparative analysis of muscle composition of different size group of Rita rita showed that the medium size (500-800g) fish is nutritionally superior to both small and large size groups. The nutritional information on Rita rita indicates that this fish species can play important role in fighting protein-calorie malnutrition prevalent in socio-economically underprivileged population, including the fishermen communities and thus merits to be domesticated and brought under aquaculture. Moreover, as reports [24] indicate, this species has become critically endangered in neighbouring countries like Bangladesh, special attention is necessary to revive and stabilize the population [25].

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