Analysis and Evaluation of Nutritional Composition of Farmed Male Pufferfish (Takifugu obscurus)

Liya WANG 1, Ningping TAO 2 College of Food Science & Technology Shanghai Ocean University Shanghai, China 1.E-mail：[email protected] 2.E-mail: [email protected]

Abstract: The nutritional composition of the main tissues(meat, testes and liver) of farmed Takifugu obscures were investigated. All tissues reported in this study showed higher contents of crude protein in meat (18.44±0.11 g/100g) and testes(17.15±0.15 g/100g), lower contents in liver (3.89±0.16g/100g ). At the same time, liver (63.86±0.93 g/100g) contain higher contents of crude fat, testes (1.82±0.13 g/100g) and meat(1.31 ±0.21 g/100g) contain lower crude fat contents. Concerning the minerals, amounts of the macroelement K were the highest elements in meat, testes and liver. Zn were the most abundant microelement in meat and testes; Fe were the most abundant microelement in liver. In addition, amino acids (AA) were rich in testes and meat. The contents of total AA (TAA) in meat and testes were 80.08 ±0.75g/100g and 63.33±0.10 g/100g, respectively. The ratios of total essential AA (TEAA) to TAA in meat and testes were 40.60% and 39.33%, respectively. The TEAA/TAA were comparable to the reference values recommended by FAO/WHO. The AA contents of meat were dominated by glutamic acid, testes were dominated by arginine. According to amino acid score (AAS), the first limiting acid of meat was tryptophan; the first limiting acid of testes were (phenylalanine+ tyrosine). Key words: farmed pufferfish; meat; testes; liver; compenent;nutritional evaluation Compared with the general fish, puffer fish meat is 1. Introduction high in protein, low fat, and rich in essential amino Puffer fish, also name bubble fish, warm water acids, minerals. In addition to meat, sexual gland also marine benthic fish. Generalized puffer fish include rich in nutritional value, testis are rich in protamine, all species of the dolphin-shaped mesh, but in fact DNA, Zn and Se, and had good name of “beauties puffer means a class of highly toxic but delicious milk” in the history . Currently, the study reported on economic fish which closely related to our food, the nutritional value of the puffer fish’s testes and culture and history. China is the production and liver is not yet complete. This article analyze and marketing power of puffer fish in the world. The evaluate farmed Takifugu obscurus’ nutritional and annual allowable catch is more than 100,000 tons, quality of testes and liver tissue, in order to provide a accounting for about 70% of the world's total output, theoretical basis of effectively development and and its 70% to 80% for export, mainly to Japan, South utilization the resources of farmed Takifugu obscurus. [1] Korea . In recent years, China has made significant 2. Material and Methods progress in puffer fish farming industry. Farmed fugu fish is mainly based on the produce poison mechanism, 2.1 Material the growth of ecological and physiological Takifugu obscurus collected from the breeding requirements, by using a series of core technology base of Jiangsu Ocean Group Co., Ltd. in April 2010, such as artificial breeding, control toxic and healthy [2] taken 8 male samples which are all 2 ages old, farming, to control its poison in food range . In gonadal maturation, uniform size, weight (328±36)g which farming Takifugu obscurus fish is unique and body length(20±1)g. After conventional biological Chinese migration of fish. Its individual large, measurements, quickly slaughtered, try removing fast-growing, fresh meat, nutrient-rich, are popular [3] other debris attached to the organization, such as with consumers. Yin Ning confirmed that under the glands, fat, connective tissue, blood clots and dirt, condition of freshwater aquaculture with bait gives, then washed clean, the liver weight (43±14)g, testis supplemented by certain technical measures, even if weight for (20±7)g. After homogenate, the fish Takifugu obscurus reach sexual maturity, their ovarian packing in zip lock bag, the liver and the testis are sub and liver are non-toxic. Professor Hua Yuanyu, puffer package in thread mouth bottle, quick freeze under- fish farming expert in Nanjing Normal University who think Fugu obscurus after generations of nurturing by 80 ℃stand-by. fully freshwater environments, can produce toxic 2.2 Determination of Nutrients generation low than the generation, and safe to eat "[4].

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Nutrients are determined with reference to the (FAO/World Health Organization (WHO)) in 1991 [9]. relevant standard. The water is referencing to AOAC, The amino acid score (AAS), essential amino acid 2007, methods 950.46; crude protein reference to index (EAAI), the branched chain amino acids/ the AOAC, 2007, method 981.10; crude fat reference to Aromatic value amino acids (TBCAA,/TArAA) AOAC, 2007, method 948.22; ash with reference to Calculated as follows: AOAC, 2007, method 938.08[5]. Amino acid is AAS（%）= Test protein amino acid content (g/100g referencing Adeyeye[6] etc, hydrolysis by 6mol/L protein)/FAO scoring model amino acid content (g/100g hydrochloric acid, assay by Japan's Hitachi L-8800 protein)×100 amino acid automatic analyzer. Because tryptophan is n 100ab 100 100h destroyed in hydrochloric acid hydrolysis, so EAAI     referencing to the determination of Wu Fang etc. ae b e h e [7] .Using 5mol/L NaOH which containing 0.5% Wherein: the n is comparing the number of soluble starch solution hydrolysis, plus essential amino acids; the a, b, c, ..., h are essential KH2PO4-NaOH buffer, measured by Shimadzu amino acids content of test protein (g/100g protein); ae, RF-5301pc fluorescence spectrophotometer. b , c , ..., h are essential amino acids content of egg [8] e e e Referencing the determination of to Borges et al , proteins, (g/100g protein). measured constants and trace elements K, Na, Ca, Mg, Zn, Fe, Cu by TAS-990 flame atomic absorption 2.4 Statistical Approach spectrophotometer of Beijing Purkinje General Using SPSS13.0 software on variance analysis Instrument Co., Ltd. Se being determinate by PE what measured data of general nutrients and elemental ELAN60000 model ICP-MS of Beijing Bode analysis, ±S.D is statistical results, P <0.05 is HengYue Electronics Co., Ltd.. P measured by significant difference. UV-1100 spectrophotometer that produced by Beauty Spectrum of Instrument Co., Ltd., Shanghai, China. 3. Results and Discussion 2.3 Evaluation Method of Nutritional Quality 3.1 General Nutrition Composition Analyses Nutritional quality evaluation based on the amino acid score mode proposed by the FAO/WHO

Table.1 Proximate composition in the tissues of farmed Takifugu obscures (g/100g, wet, mean±S.D., n=3)

Tissues Moisture Crude Protein Crude Fat Ash Content Meat 79.73±0.52a 18.44±0.11a 1.31±0.21b 1.42±0.04b Testes 79.53±0.90a 17.15±0.15b 1.82±0.13b 2.22±0.08a Liver 29.81±0.23b 3.89±0.16c 63.86±0.93a 0.64±0.02c The different letters in same column mean statistically significant difference (P < 0.05) Table. 2 Amino acids composition and content in the tissues of farmed Takifugu obscurus（%,dry,mean±SD.,n = 3）

Amino Acid Meat Testes Liver Amino Acid Meat Testes Liver Asp** 5.92±0.05 a 4.39±0.01b 0.42±0.02c Lys* 7.79±0.06 a 7.19±0.02 b 0.42±0.03c Thr* 3.78±0.05 a 3.01±0.03 b 0.27±0.02c His 1.99±0.03 a 1.17±0.00 b 0.15±0.01c Ser 3.61±0.05 3.14±0.02 0.30±0.02a Arg** 5.64±0.06 b 8.63±0.01 a 0.39±0.03c Glu** 11.97±0.18 a 5.99±0.03 b 0.69±0.04c Pro 3.69±0.19 a 2.92±0.09 b 0.29±0.02c Gly** 5.38±0.04 5.92±0.08 0.52±0.06a Trp* 0.54±0.02 b 1.06±0.02 a 0.14±0.01c Ala** 4.86±0.05 a 4.27±0.01 b 0.35±0.02c TAA 80.08±0.75 a 63.33±0.10 b 5.66±0.66c Cys 1.82±0.01 a 1.19±0.01 b 0.15±0.00c EAA 32.51±0.30 a 24.91±0.10 b 2.21±0.12c Val*c 4.12±0.03 a 3.05±0.01 b 0.30±0.01c UAA 47.57±0.52 a 38.42±0.11 b 3.45±0.21c Met* 3.24±0.04 a 2.07±0.01 b 0.21±0.01c NEAA 33.72±0.06 a 29.21±0.07 b 2.36±0.15c Ile*c 3.48±0.11 a 2.20±0.01 b 0.18±0.01c TEAA/TAA 40.60 39.33 39.05 Leu*c 6.80±0.05 a 4.35±0.01 b 0.45±0.03c TEAA/TNEAA 68.34 62.21 64.06 Tyrd 2.74±0.04 a 1.75±0.05 b 0.20±0.01c TUAA/TAA 42.11 46.12 41.76 Phe*d 2.76±0.05 a 1.99±0.01b 0.23±0.02c The different letters in same column mean statistically significant difference (P < 0.05) TAA: total amino acids; * TEAA: total essential amino acids; TNEAA: total nonessential amino acids;** TUAA: total umami amino acids;

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The nutritional composition of the meat, testes and about 40%, TEAA / TNEAA is 60%. Shows that liver of farmed Takifugu obscures were investigated, protein amino acid nutritional value of farmed and the results are shown in Table 1. Liver moisture Takifugu obscurus’ fish meat, testis and liver is match content (29.81%), protein content (3.89%) and ash the standards-compliant mode. Asp, Glu, Ala, Gly and content (0.64%) are significantly lower compared with Arg in Amino acids are the main factor influent the the fish and testis, but high fat content (63.86%). food flavor, are collectively referred to as the Tetrodotoxin links protein together to exist as a delicious amino acids. Asp, Glu has the flavor complex in puffer body [10], so the protein content in characteristics. Ala, Gly have the sugariness the liver is lower. characteristics. Arg has raises fresh effect. The flavor Testis nutritional characteristics are consistent amino acid content in meat, testes and liver of farmed with the meat nutrition that is high-protein, low-fat. Obscure Puffer, respectively is for 47.57g/100g, 38.42 The crude protein content (18.44%) in fish is higher g/100g and 3.45g/100g, the ratio of the total amino than the testis (17.15%), fat and ash content of testes (TUAA/TAA) is for 42.11%, 46.12 % and 41.76%, (1.82%, 2.22%) are higher than the fish meat(1.31%, which are abundant. So, the meats, testes and liver of 1.42%). The result is agreement with the measured the puffer fish taste very delicious, and testis taste results of blue crab by Su Xiurong[11]. During better. That may be related to the different maturation, the fish has been basically not feeding, organizations accumulate flavor amino acid in which have the higher accumulation nutrients of different level[14]. Testes delicious was one of the gonad fat and ash content in order to achieve the level reasons of puffer fish testis reputation as "beauties of breeding. Protein content high in fish meat, which milk may be associated with its own genetic characteristics, Table.3 Comparison of AAS and EAAI of farmed Takifugu also with the maternal compensation of fish gonadal development, which mean under certain conditions, FAO/WHO AAS the parent transport essential nutrients from the fish to EAA (1991) (g/100g protein). Meat Testes Liver meet the needs of gonadal development [12]. The nutritional composition of the fish affected and Ile 2.8 137 94 116 regulated by body weight, food (feeding level, diet Leu 6.6 113 79 123 composition and hunger status) temperature, the Lys 5.8 148 148 131 special stages (breeding) of the life cycle, annual Met+Cysa 2.5 222 156 260 changes in the aquatic environment, and the b differences between species [13]. Phe +Tyr 6.3 96 71 123 Thr 3.4 122 106 143 3.2 Amino Composition and Evaluation Trp 1.1 54 115 230 The amino acid composition and content of testes Val 3.5 129 104 155 and liver in the farmed Fugu puffer fish were measured, and the results are shown in Table 2. Three EAAI - 71.72 61.32 86.02 Phenylalanine + Tyrosine; b Methionine + Cysteine. contain common 18 amino acids, including 8 essential The contents of TEAA of FAO/WHO (1991) standard were 32.0 amino acids and 10 non-essential amino acids. Total (g/100g protein) amino acids is highest (80.08g/100g) in fish, testis The contents of TEAA of meat, testes and liver were 36.22, 29.61, followed by (63.33g/100g), liver (5.66g/minimum 40.60, respectively (g/100g protein)

100g)is the lowest. The highest content is Glu (11.97 Essential amino acid content (g/100g Protein)of g/100g) in fish meat, as part of Glu transformed from [10] fish meat, testes, and live, the ideal model of amino Gln , the lowest levels is Trp (0.54g/100g). The acid recommended by the FAO/WHO and the highest concentrations is Arg (8.63g/100g) in testis, is essential amino acids in egg protein is shown in table 1.5 times in fish content (5.64g/100g). The highest 3. Fish meat, testes and liver of essential amino acids concentrations are Glu (0.69g/100g) in liver, the (g/100g Protein) are 36.22, 29.61 and 40.60 lowest levels is Trp (0.14 g/100g). respectively, all closed to or even higher than that Essential amino acids (TEAA) of fish meat, testis essential amino acids total (g/100g Protein) 32.0 and liver were 32.51g/100g, 24.91g/100g and which recommended by the FAO/WHO. Separately 2.21g/100g, accounting for the ratio of the total calculated the amino acid score (AAS) in fish meat, content of amino acids (TEAA/TAA) were 40.60%, testis, and liver, and essential amino acid index (EAAI) 39.33% and 39.05%. The ratio of essential amino according to the FAO/WHO. The results are in Table acids and non-essential amino acids (TEAA/TNEAA) 3. First limiting amino acid in fish and testis as the were 68.34%, 62.21% and 64.06%, respectively. AAS, were Trp and (Phe+Tyr), in the liver without According to The ideal mode of FAO/WHO, good limiting amino acid. The AAS essential amino acids quality protein constitute amino acids TEAA / TAA is in the fish meat and testes is higher than or close to

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100 (food protein amino acid pattern is closer to The different letters in same column mean human proteins constitute mode, the higher its statistically significant difference. The fish meat is nutritional value), liver AAS scores were higher than high with Zn and low with Ca. The Zn content in 100. Rated highest amino acid in the three tissues is testes is 3 to 4 times of the content in the meat. Hill Met + Cys, which indicate all rich in sulfur-containing and Matron put forward that nature similar elements’ amino acids [15]. EAAI is one of the commonly biological function is antagonistic to each other. In indicator used to evaluate the nutritional value of terms of fish, its body contains constant ratio of K/Na protein. EAAI in Fish meat, testis and the liver were and Ca/P. K/Na in fish meat, testis and liver are>1.0. 71.72, 61.32 and 86.02, close to the reported EAAI in This shows the Puffer fish, is high-K and low Na food, farmed Takifugu obscurus (56.02) [16] ,mandarin fish and more favorable to hypertension patients for eating. (62.30), grass carp (60.59) [17] and such as a variety of Ca/P are ratio of 0.04 to 0.23, so liver, testes, and fish freshwater fish. meat are low Ca high P food. Usually Ca/P should Analysis based on amino acid score, the essential be<0.5, otherwise require additional supplemental amino acid content during the three is very rich, and intake of Ca in [8]. form a more balanced, respectively close to 100. So, There have varied different ability to accumulate amino acid composition in fish meat, testes and liver mineral elements in different parts of the puffer fish, is in line with human needs. View of the total amino which may be relevant to the mineral needs of the acid content in the liver significantly lower. Overall puffer fish and its detoxification mechanisms. Puffer consideration, it is recommended to choose to eat fish fish contain tetrodotoxin, strongest toxicity in liver meat and testes to supply amino acid content. (toxicity of the edible range), tetrodotoxin can block Na+influx. In addition, minerals involved in 3.3 Mineral Substances detoxification mechanisms, so the lower mineral Determination of mineral elements in the content in the liver [10]. During the puffer fish sexual organization, from Table 4 is known that mineral maturity, gonadal and fish meat need to store energy. element is rich in fish meat, testes and liver. Overall, So, mineral content is higher in the fish meat and mineral content of the fish meat and testes was testis. significantly higher than the liver. The highest concentrations in the constant element are K (content 4. Conclusion of fish meat, testes and liver are 102.10mg/100g, Comprehensive analysis of the study results, fish 1534.16mg/100g, 1462.39mg/100g), the highest meat and testis of farmed Takifugu obscurus are rich content of trace elements in fish meat and testes is Zn in protein and amino acids, which total content (content respectively. 11.88 mg/100g, 7.06mg/100g respectively up to 80.08±0.75g/100g and 63.33 ± respectively), and in the liver is Fe (4.94mg/100g). 0.10g/100g that have balanced composition proportion. Table.4 Contents of of minerals in the tissues of farmed Essential amino acids composition mode meets the Takifugu obscures (mg/100g,dry, mean±S.D.,n=3) needs of the human body, flavor amino acids account for the high ratio of the total amino acids, are Mineral Meat Testes Liver high-quality source of protein. Fat content is high

a a b (63.86±0.93g/100g) in liver, which abundant with a K 1462.39±45.07 1534.16±49.76 102.10±11.96 more reasonable proportion mineral elements. K, Na 720.81±28.22b 862.04±23.76a 89.87±8.74c Zn-rich in fish meat and testis, and liver is rich in Fe. P 302.26±16.34b 466.50±4.70a 32.03±1.35c First limiting amino acids are Trp according to AAS, in fish meat and testes respectively are Phe+Tyr and a b c Mg 134.79±7.26 75.44±4.37 8.76±0.50 Ile. Therefore, farmed Takifugu obscurus is a higher Ca 33.00±5.76a 16.63±0.87b 6.37±1.15c nutritional value delicious fish that very worthy of development and utilization freshwater fish resources. Fe* 2.88±0.53b 6.60±1.29a 4.94±0.54a We will further research on its nutritional value Zn* 7.06±0.60b 11.88±0.96a 3.68±0.06c divided into age/gender/geographical/season.

b b a Cu* 0.22±0.04 0.24±0.07 0.51±0.03 Thanks a a b Se* 0.12±0.00 0.12±0.00 0.001±0.00 Thank the Shanghai Education Commission of "Food K/Na 1.61 1.78 1.14 Quality and Safety" Key Discipline Project (J507040), Ca/P 0.11 0.04 0.20 Shanghai Science and Technology Engineering Center building (11DZ2280300) and Zhenjiang CAPE The different letters in same column mean statistically significant difference (P < 0.05)。 commerce Yangtze River ecological farming center" Study of nutritional evaluation and flavor comparative of farmed fugu" (D-8006-12-0052) for support the project .

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