H. PUGILINUS MEAT. fatty acids, vitamins and minerals accompanied by low fat and calories. In the

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42 BIOCHEMICAL COMPOSITION OF C. RAMOSUS AND H. PUGILINUS MEAT INTRODUCTION Sea foods have been widely regarded as healthy component of our diets for hundreds of years, providing an excellent source of high quality protein, omega - 3 fatty acids, vitamins and minerals accompanied by low fat and calories. In the present study, the biochemical composition such as protein, carbohydrate, lipid, cholesterol and total free sugars were analysed in the raw edible meat of gastropods. The other parameters such as vitamins, heavy metals, glucose, and protein bound sugar, minerals and fatty acid profile were also assessed. MATERIALS AND METHODS Biochemical analysis Fresh raw meat of C. ramosus and H. pugilinus was procured from the local shell meat dealer. The edible portions such as foot and adductor muscles were separated out and washed thoroughly. The meat samples were dried in a hot air oven at 50-55 C. The dried samples were powdered and used for the biochemical study. Moisture Fifty grams of the meat was taken and it was allowed to dry separately in a mechanical drier at 60 C until a constant weight was reached. The percentage of moisture content was calculated by using the formula

EN Net weight (g) - Dry weight (g) Moisture content (%) = ----------------------------------------X 100 Net weight (g) Carbohydrates The carbohydrate content of the C. ramosus and H. pugilinus meat powder was estimated by following the method of Dubois et al. (1956). To 20 mg of dried powdered tissue, 1 ml of phenol solution, 5 ml of con H2SO4 were added and made up to 10 ml using distilled water. This mixture was incubated for 15 minutes at 29 C to develop the colour. The colour intensity was measured in a spectrometer at 490nm. The sugar content was determined from calibrated standard curve of D-glucose and expressed as mg of sugar per gram dry weight or in percentage. Std Value x OD of sample Carbohydrate % = ----------------------- X 100 Weight of the sample Proteins The protein content was estimated by following the method of Raymont et al. (1964). The dried and powdered meat (0.2g) was taken and ground with 5 ml of distilled water and the extract was centrifuged at 4000 rpm for 10 minutes and the supernatant solution was used for the estimation of protein. To 1 ml of supernatant 4 ml of Biuret reagent was added and incubated for 30 minutes and the colour was read at 540 nm using spectrophotometer. The protein content was calculated by referring to the standard graph of BSA.

El OD of sample x Std. value x Total volume Protein(%) = ------------------------------------------------------- X 100 Wet weight of sample x Volume of extract Total lipids Total lipids of the tissue samples were analysed by following the gravimetric method of Foich et al. (1957). One gram of tissue was homogenized in 20 ml of chloroform - methanol mixture at 2.1 ratios. It was then left undisturbed for 2 hrs in the dark and then filtered through a Whatman No.1 filter paper and preserved. The residue was re extracted in the same solvent (half of the original volume) and filtered. Both the filtrates were cooled and the total volume was noted. To the filtrate 1/ 5 the volume of 0.6 % saline was added and the mixture was transferred to a separating funnel and left undistributed overnight in the dark. The lower layer was carefully taken out from the separating funnel in a pre-weighted beaker and 4 ml of benzene and 8 ml of ethanol was added. This mixture was evaporated to dry at room temperature. The beaker (after total evaporation) was weighted again and from the difference, the weight of the lipid in the tissue was calculated. Total free sugars Total free sugars of the tissue samples were analysed by following the method of Roe (1955). 100 mg of tissue was homogenized in 1 ml of 10% Trichloro Acetic Acid (TCA) and centrifuged at 2500 rpm for 10 minutes and the supernatant was taken for analysis. 200 microliter of supernatant was mixed with 300 micro liter of double distilled water and 5 ml of anthrone reagent was added and boiled for 15 minutes in a water bath and cooled in dark at room temperature for 30 minutes. The OD value was read at 620 nm and the free sugars were calculated by using the formula

OD of unknown x concentration of standard x volume of the homogenate x 100 45 OD of standard x 1000 x Volume of the homogenate taken for analysis x 100 The protein bound sugars The protein bound sugars were analysed using Anthrone method after precipitating the protein with hydrochloric acid (Caroll et al., 1956). 100 mg of tissue was homogenized in 1 ml of 10% of TCA and centrifuged at 2500 rpm for 10 minutes. To the precipitate, 1 ml of IN HCI was added and the tube was closed with marble and placed in an oven at 80 C for 18 hrs for hydrolyses and the hydrolysate was used for analysis. To 0.05 ml of the hydrolysate, 0.45 ml of double distilled water was added. To this 5 ml of anthrone reagent was added and the mixture was heated in a water bath for 15 minutes. Then it was cooled in dark at room temperature for 30 minutes and the OD value was read at 620 nm in a spectrophotometer. The protein bound sugars were calculated by referring the OD using the standard graph and the following formula OD of unknown x concentration of standard x volume of the homogenate x 100 OD of standard x 1000 x Volume of the homogenate taken for analysis x 100 Glucose Glucose of the samples was analysed by following the method of Miller (1972). 100 mg of tissue was homogenized in 1 ml of 10% TCA and this was centrifuged at 2500 rpm for 10 minutes and the supernatant was taken for analysis. To 0.2 ml of supernatant, 0.8 ml of double distilled water was added and to this 4 ml of 0-toludine boric acid reagent was added. This mixture was heated in a boiling

water bath for 15 minutes and then cooled under running tap water. The blue colour developed was read spectrophotometrically at 640 rim. The concentration of glucose in the sample was calculated by referring of OD using the standard graph and the following formula and the results are expressed as mg/g. OD of unknown x concentration of standard x volume of the homogenate x 100 OD of standard x 1000 x Volume of the homogenate taken for analysis x 100 Free cholesterol Free cholesterol was estimated by following Liebermann - Burchard test (Liebermann, 1885; Burchard, 1889). Thirty grams of the total lipid content was dissolved in 5 ml of chloroform. One ml of this and 1 ml of standard solution were taken in separated test tubes and evaporated to dry in a water bath at 80 C. Then 3 ml of Liebermann Burchard reagent was added and the mixture was kept for 30 minutes in the dark and the OD was read at 620 nm in a spectrophotometer. A standard graph was drawn using the OD observed for different concentration and the concentration of cholesterol was calculated using the standard graph. Vitamins and minerals The vitamins and minerals were analysed at A to Z pharmaceuticals (p) Ltd., Chennai following the method of Sethi (1997). The dried and finely powdered meat samples were used for the analysis Fatty acid profile The analysis of the following fatty acid profile of C. ramosus and H pugilinus were done.

Lipid Extraction bog of meat samples of C. ramosus and H. pugilinus were extracted for total lipid content by following the method of Bligh and Dyer (1959). The lipid was extracted using the solvents such as methanol and chloroform. 5g of dried powdered meat was weighed accurately and 12m1 of distilled was added to this and mixed well. To this 30ml of methanol and 15 ml of chloroform was added and mixed well for 2 minutes. Additional 15ml chloroform was added and mixed for 30 seconds. Finally 15m1 of water was added and mixed again for 30 seconds. The contents were transferred to a centrifuge tube, and centrifuged for 1 Sminutes at 3000rpm. After it was centrifuged, the water/ methanol phase at the top of the tube was removed, and the plug of solids was loosened. Then the chloroform phase containing the oil was filtered and poured into a 50m1 volumetric flask. The solid was transferred to the filter paper and rinsed with 20ml chloroform, and filled to the mark with chloroform. lomi of the chloroform extract was evaporated to dryness in an oven and then weighed, and the lipid content was calculated by the formula Dry weight of oil x 50 % lipid - X 100 ml x weight of sample Methylation The fatty acids present in C. ramosus and H. pugilinus meat lipids were converted to Fatty Acid Methyl Ester (FAME) using the BF 3 - Methanol method described in AOAC (AOAC, 1990). 250 mg of lipid was taken in a 50 ml round bottom flask to which 4 ml of 0.5M alcoholic NaOH was added and refluxed for 5-10 minutes. 5 ml of BF3 methanol was added to the mixture and refluxed for 1 minute. The contents were transferred to a stoppered test tube and 15 ml of

48 saturated NaCl solution was added. The FAME was extracted twice with 5 ml of hexane. The upper hexane layer was collected into a screw cap test tube and dried under the stream of nitrogen to get a final concentration of 100 mg / ml hexane. G C analysis Fatty acid composition was determined by gas chromatography. The quantification was done by the method described by Candela et al. (1996) with slight modification. The Fatty Acid Methyl Esters (FAME) prepared by BF3 methanol was quantitatively separated on a Perkin Elmer Autosystem XL gas chromatograph fitted with a Flame ionization Detector (FID). A fused silica capillary column (PE-225) (0.25mm ID x 30 m length) was used. The operating conditions were injector temperature 2500 C, and detector temperature 300 C. A temperature gradient programme was followed with initial oven temperature set at 70 C for 1 mm, which was then increased to 80 C at the rate of 3 C / min and then to 220 C at the rate of 10 C / mm. The carrier gas used was nitrogen at 20-psi pressure. Peaks were identified by comparison of their retention time with those of standard mixtures (Sigma Chemical Co., St. Louis, USA, 99% purity specified for GC). RESULTS The biochemical compositions of C. ramosus and H. pugilinus meat are given in Table 1.

we Table 1. Biochemical composition of C. ramosus and H. pugilinus Parameters C. ramosus H. pugiinus Protein (%) 14.35 6.09 Carbohydrates (%) 4.82 4.12 Total lipids (mg/g) 2.7 3.0 Moisture (%) 75.88 69.5 Cholesterol (mg/g) 0.597 0.717 Total free sugars (mg/g) 0.004 0.001 Glucose (mg/g) 0.168 0.215 Protein bound sugars (mg/g) 0.757 0.906 The protein content 14.35% and 6.09%; carbohydrate, 4.82% and 4.12%; and lipid, 2.7mg and 3.0mg/bOg were observed in C. ramosus and H. pugilinus meat respectively. The level of glucose, protein bound sugars and total free sugars were less than lmg/g in both C. ramosus and H. Pugilinus. The vitamin content (per 100g) in C. ramosus and H. pugilinus meat is shown in Table 2.

50 Table 2: Vitamin content in C. ramosus and H. pugiinus meat Parameters C. ramosus meat H. pugiinus meat Vitamin B 1 (mg) 0.12 0.12 Vitamin B 2 (mg) 0.34 0.34 Vitamin B 6 (mg) 0.42 0.23 Vitamin B 12 (Meg) 0.34 1.034 Niacinamide (mg) 1.02 0.014 Folic acid (mg) 0.01 0.39 Vitamin C (mg) 56.6 4.45 Vitamin A (mg) 1.034 2.07 Vitamin D (IU) 48.5 24.7 Vitamin E (mg) 31.3 26.8 Vitamin K (mg) 23.5 32.5 Biotin (mg) Below detectable level Below detectable level The Vitamins such as Vitamin B 1 (0.12 and 0.12 mg/100g), B 2 (0.32 and 0.34mg/10g), B 6 (0.42 and 0.23mg/10g) B12 (0.34 and 0.34 mcg/loog), Niacinamide (1.023 and 0.014 mg/100g), Folic acid (0.01 and 0.39mg/10g), C (56.6 and 4.45 mg/100g), A (1.034 and 2.07mg/10g), D (48.5 and 24.711J) E (31.3 and 26.8mg! bog), K (23.5 and 32.5 mg/1 OOg) were present, whereas Biotin in both the samples were below the detectable levels.

The minerals and trace metals (per 100g) in C. ramosus and H. pugilinus meat are shown in Table 3. 51 Table 3: Minerals and Trace metals in C. ramosus and H. pugiinus meat Parameters C. ramosus H. pugilinus Sodium (mg) 101.54 101.56 Potassium (mg) 112.4 112.67 Calcium (mg) 486.5 435.7 Iron (mg) 0.97 0.56 Copper (mg) 0.12 0.12 Phosphorus (mg) 1.13 1.12 Mercury Nil Nil Cadmium Nil Nil Arsenic Nil Nil Nickel Nil Nil Zinc (JDpm) 5 2 The minerals and trace metals such as sodium (101.54 and 101.56 mg/100g), potassium (112.4 and 112.67 mg/100g), calcium (486.5 and 435.7 mg/100g), iron (0.97 and 0.56 mg/100g), copper (0.12 and 0.12mg/10g) and Phosphorus (1.13 and 1.12 mg/i OOg) were present, whereas in trace metals zinc (5 and 2ppm) was present, but mercury, cadmium, arsenic and nickel were absent in both the meat samples. Fatty Acid Profile Analysis The fatty acid profile of C. ramosus and H. pugilinus are given in Table 4 and Figs I and 2.

52 Table 4. Weight percent of some fatty acids of C. ramosus and H. pugiinus meat Fatty Acids Weight (%) Saturated Fatty Acid C12:OLauricacid 1.00 0.41 C14:OMyristicacid 1.65 3.30 C16:OPalmiticacid 9.03 14.23 C17:0 Methyl hepta deconate 1.37 1.21 C18:0 Stearic acid 8.00 10.09 C20:0 Arachidic acid - 2.67 C23:0 Tricosanoic acid 5.91 1.85 Monounsaturated Fatty Acid (MUFA) C14:1 Myristoleic acid - 1.28 C16:1 Palmitoleic acid - 1.03 C18:lElaidicacid 1.59 2.74 C22:1 Erucic acid - 1.32 Polyunsaturated Fatty Acid (PUFA) C18:2 Linoleic acid 2.62 2.13 C18:2 Linoeladic acid 4.00 3.38 C18:3 Linolenic acid 1.91 1.08 C20:2Methyl11,14, eicosadienote 3.47 1.56 C20:4 Arachidonic acid 10.36 4.48 C20:5 Eicosapentaenoic acid 3.27 1.91 C20:3 8, 11,14 icosatrienoic acid - 2.20 C20:3 11, 14, 17 icosatrienoic acid - 12.90 C20:5 5,8,1 1,14,l7icosa pentaenoic acid - 4.74 C22:2 13, 16 docosadienoic acid - 1.51 C22:6 Docosahexaenoic acid 4.54 0.69 * - Not Determined

53 The presence of major saturated Fatty acids (weight %) such as Lauric acid (C12: 0), Myristic acid (C14: 0), Palmitic acid Methyl heptadeconate, Stearic acid (C18:0), Tricosanoic acid were present in C. ramosus and H. pugilinus meat. The saturated fatty acid, Arachidic acid (C20:0) was found only in H. pugilinus sample. In C. ramosus raw meat the monounsaturated fatty acid, Oleic acid was present. The Monounsaturated Fatty Acid (MUFA) present in H. pugilinus meat samples were Myristoleic acid (C14:1), palmitoleic acid (C16:1), elaidic acid (C18:1) and erucic acid (C22:1) respectively. The major Polyunsaturated Fatty Acid (PUFA) present in both meat samples were Linoleic acid (C18:2) Linoeladic acid (C18:2), Linolenic acid (C18:3), Methyl 11, 14 eicosadienote, Arachidonic acid and Docosa Hexaenoic acid (C22:6). In H. pugilinus meat samples other PUFA such as 11, 14 eicosadienoic acid (C20:2), 8, 11, 14 eicosatrienoic acid (C20:3), 11, 14, 17 icosatrienoic acid (C20:3 n-3) and 13, 16 docosadienoic acid (C22:2). I Retention time (mins) Fig. 1. Peaks of fatty acid profile in C. ramosus meat

54 Retention time (mins) Fig. 2. Peaks of fatty acid profile in H. pugilinus meat DISCUSSION The gastropods, C. ramosus and H. pugilinus meat possess 75.88 and 69.5% moisture content respectively. Ramesh and Ayyakkannu (1995) have reported the moisture content in fresh foot (67.4%) and columella muscle (70%) of C. ramosus. The moisture content of foot and adductor muscle of Pleuroploca trapezium ranged from 68 to 78% during Jan. 2001 to Dec.2001 (Shanthini, 2003). The level of protein was estimated as 14.35% in C. ramosus whereas in H. pugilinus meat, it was 6.09%. Mukundan (1968) has reported that molluscs have approximately 8-10% of proteins (by weight) 4-5% of carbohydrates 2-3% of minerals, but 1-2% of fat. In clam meat, Dore (1941) observed high protein content of 12.77g1100g, carbohydrate 0.97g1100g and low lipid content of 0.97g I 100g. Suryanarayanan and Nair (1976) recorded a protein range of 60.42 86.08% in males and 61.98 85.78% in females of the gastropod Cellana radiata. Giese (1969) reported that protein to be the dominant organic constituent in molluscs.

55 The carbohydrates in seafood are low when compared to protein. In the present study the carbohydrate level was estimated as 4.82% in C. ramosus and 4.12% in H. pugilinus. In molluscs, generally the carbohydrate reserves may be utilized under unfavourable conditions and the great variations in tissues indicate the level of mobilizable carbohydrate reserves, which may fluctuate widely and rapidly in response to fluctuations in conditions affecting the position of the animal (Ansell et al., 1973). Low lipid level was observed in both meat samples. The fatty acid content in shellfish is comparatively low because the lipid content in them is low (Ackman, 2000). He also reported that molluscs such as oysters, clams etc., are lean and lipid content ranging from 1-3% and in crustaceans the fat content ranges from 0.5-1.5%. Sarvaiya (1977) has reported that the fat contents may very between 1-14.5% in molluscs (lowest being in Murex virgineus and the highest in Crassostrea cucullata). Venkatraman and Chari (1951) correlated low levels of fat in Crassostrea madrasensis with the growth and maturation of the gonads. The cholesterol level in C. ramosus and H. pugilinus meat was 0.597 and 0.717 mg/100g respectively. Ackman (2000) has reported 35-56 mg of cholesterol estimated in mollusc body. The other parameters such as total free sugars, glucose and protein bound sugars were below lmglg level in both the meat samples. Samuel (2003) has reported similar levels of total free sugars, glucose and protein bound sugars in mantle and nidamental glands of Sepia pharaonis. The fat-soluble vitamins such as vitamin A, D, E and K and water-soluble vitamins (B 1, B 2, B6, B 12, niacinamide, folic acid and biotin) were also present in C. ramosus and H. pugilinus meat. Lall and Parazo (1995) have reported that the snail flesh contains 831U 100g -1 of vitamin A and 3.50 (mg 1 00g' ) of vitamin E. In the present study, low level of vitamin A and higher amount of Vitamin B content

56 was observed. The other fat-soluble vitamins like vitamin D and K were also present. The water soluble vitamin C content in C. ramosus and H. pugilinus meat were 56.6 and 4.45 mg/100g whereas in Pleuroploca trapezium, low level of vitamin C (0.189 mg/bog) was estimated (Shanthini, 2003). Fish and shellfish, like other living organisms, contain most of the 90 naturally occurring elements. The macro elements such as calcium, magnesium, phosphorus, sodium, potassium and chlorine are present in gram per kilogram quantities. The remaining elements occur in the body in much lower concentrations (mg or p.g per kg). In the present study, sodium, potassium, calcium and phosphorus were observed in appreciable amount in both the meat samples. Higher sodium content was estimated in both the meat and it was little bit lower in P. trapezium (Shanthini, 2003). The calcium content is higher in both the meat samples. Dore (1941) has reported that crustacean and molluscs have two to three times the calcium content of the finfish. Oysters are an exceptionally good source of iron. Other shellfish considered to be good sources of iron are clams (3ounces/85g raw has 5.2mg of iron) and shrimp, which contain about 2.6mg of iron in 3 ounces/85g of canned product. Molluscs contain more copper in muscle and in their organs than marine plants, crustaceans and fish (Eisler, 1979). Molluscs use copper in a unique way, by producing haemocyanins to carry oxygen to their tissues (Lall, 1995). The Cu level in molluscs are 0.1-0.3 (40) p./g. In the present study, the iron and copper content of the C. ramosus and H. pugilinus meat were 0.97, 0.56, and 0.12 and 0.12 mg/100g respectively. The trace elements such as mercury, cadmium, lead, arsenic and nickel were not detected in both the meat samples. These metals are particularly toxic in high

57 concentration and usually inhibit enzymatic activities (Vallee and Wacker, 1970). Zinc is an important element performing a variety of functions in the body, as it is a factor for a number of enzymes. Molluscs contain the greatest concentration of zinc. In general, fish have low zinc contents when compared to molluscs and crustaceans. The level of Zn observed in squid and cuttle fish were 24.25 and 24.02 ppm respectively (Khasim, 1994), where as in fishes 5-10 ppm zinc was detect ed (Kureishy et al., 1981). In the present study, low levels of zinc were observed in C. ramosus and H. pugilinus meat samples. Fish and shellfish are the main sources of omega - 3 fatty acids (Ackman, 1989). Consumption of seafood, the primary dietary source of the long chain n - 3 fatty acids eicosapentaenoic acid (EPA, (20:5n - 3) and docosahexaenoic acid (DHA, (22:6 n - 3), increases the levels of these fatty acids in cell membranes and results in shifts in eicosanoid production that might reduce platelet aggregation and coronary spasm. Addison et al. (1972) and Kryznowek (1985) reported crab as good source of EPA. Rehbung et al. (1977) reported that palmitic acid, stearic acid, Oleic acid, linolenic acid, arachidonic acid, docosatrienoic acid, docosa pentaenoic acid, docosahexaenoic acid and eicosapentaenoic acid as major fatty acids in the gastropod, Trochus niloticus. In the present study the major polyunsaturated fatty (health beneficial) such as Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were present. The other fatty acids such as Lauric acid, Myristic acid, Palmitic acid, Stearic acid, Oleic acid, Linoleic acid, Linolenic acid, Arachidonic acid, heptadeconate, icosadienoic acid and docosadienoic acid were also present. Shanthini (2003) has reported some similar fatty acids such as Lauric acid, Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Myristoleic acid Oleic acid, Linoleic acid, Linolenic acid, EPA and Arachidonic acid in Pleuroploca trapezium meat.

58 The nutritive value of C. ramosus and H. pugilinus meat such as protein, carbohydrate, lipid, vitamins, minerals and fatty acid profile were observed through the present study. This information would be very useful in the development and popularization of value added products.