Fatty Acid and amino Acid profile of local squid flour (Loligo sp. ), shellfish flour (Ostrea sp. ), sea worm flour (Nereis sp. ) as artificial feed for domesticated vanamei broodstock Текст научной статьи по специальности «Фундаментальная медицина»

DOI https://doi.org/10.18551/rjoas.2018-09.56

FATTY ACID AND AMINO ACID PROFILE OF LOCAL SQUID FLOUR (LOLIGO SP.), SHELLFISH FLOUR (OSTREA SP.), SEA WORM FLOUR (NEREIS SP.) AS ARTIFICIAL FEED FOR DOMESTICATED VANAMEI BROODSTOCK

Suyuti Rb. Mohammad, Suprayitno Eddy, Widodo Maheno Sri

Postgraduate Program, Faculty of Fisheries and Marine Sciences, University of Brawijaya,

Indonesia

Triastutik Gemi

Ministry of Marine and Fisheries, Indonesia

*E-mail: suyut mohammad@yahoo.co.id

ABSTRACT

Natural feed such as Sea Worms (Nereis sp), Shellfish, Squid, and Earthworms are often used for penaeid shrimps reproductive growth. These include vanamei shrimp because they have sufficient nutritional content. However, the use of natural food has many disadvantages, as its availability is very limited and depends on the season. Sea worms are a vector of disease carriers, especially viruses and bacteria. Nutrients needed for reproductive growth and development are protein, fat, carbohydrates, vitamins and minerals other than water. Squid flour, sea worm flour, and shellfish flour are used in artificial feed. It contains nutrients in accordance with shrimp brood stock reproductive growth. It is an alternative substitute for fresh feed which has limitations and disadvantages. Consequently, the protein content of squid flour (Loligo. Sp), shellfish flour (Ostrea. Sp) and sea worm flour (Nereis. Sp) was 73.87%, 59.97%, and 54.35%. The total amino acid content was 69.20%, 56.66%, 50.75%, respectively. The total fatty acid content was 35.22%, 44.65%, and 22.87% respectively.

KEY WORDS

Reproduction, Vannamei Shrimp, nutrition, amino acid, fatty acids.

Squid, shellfish, sea worms and earthworms are feed possessing good nutrition components such as protein and fat. These are needed for fish and shrimp reproductive growth. Feed containing organic compounds, such as proteins, amino acids, and fatty acids encourage shrimp to approach feed source. (Kordi, 2007).

Natural food eligible to be used as feed are those sustainably available, possess high nutritional content, and does not carry disease. Nevertheless, sea worms availability are dependent on nature. Sea worms usage could be suppressed by combining sea worms with other natural feeds to produce high quality larval production (Sabrina et al., 2014). The provision of high protein nutrition is more widely used in natural foods such as worms, squid, and shellfish. Nevertheless, there's difficulty in regulating protein levels, therefore this technique still has limitations in its application. The disadvantages of these fresh food organisms are expensive costs, fluctuations in availability, inconsistent nutritional value, require a freezer, water contamination, lack of potential improvements, and an increased risk of transmission of pathogenic bacteria and viruses.

Squid is the most widely used food for vannamei shrimp gonads development (Ogle, 1991a). In a study conducted by Ogle (1991b), the squid was not favored and was placed under two artificial feeds for gonad maturation.

Important nutrients needed for shrimp growth are carbohydrates, fats, proteins, minerals and vitamins other than water. The most crucial component is proteins because the growth rate directly depends on the quality and quantity of the protein given.

Protein is a nutrient required for animals growth and survival. Aside from being used in body cell formation, it is also energy source the body lacks in carbohydrates and fats (Suprayitno and Titik, 2017). Proteins are composed of simple amino acid acids that form

polypeptide bonds. During the process of gonadal maturation and reproduction, there is an increase in the biosynthesis process. Therefore the protein requirement is assumed to peak when compared to the non-reproductive stage. The maturation process is a period of strong protein synthesis and increases protein requirements (Harrison, 1997).

MATERIALS AND METHODS OF RESEARCH

The tools used in this research are closed weigh bottles, desiccators, ovens, analytical balance, filter paper, 100 ml fat flask, soxhlet tools, fat-free cotton, 500 mL Kjeldahl flask, distillation equipment, electric heaters or bunsen burners, test tubes, spectrophotometers visible light, vortex, and hot plate.

The materials used in this study are Squid flour (Loligo sp), Shellfish Flour (Ostrea. Sp), Sea Worm Flour (Nereis sp), Petroleum Ether: 30-60°C, Ethyl ether, peroxide free, 95% Ethanol, Ammonium hydroxide, phenolphthalein indicator: 0.5% (w / v) in alcohol, selenium mixture; A mixture of 2.5 g of SeO2 powder, 100 g of K2SO4, and 20 g of CuSO4.5H2O or Selenium Mixture are ready to use specifically for the determination of N (from Merck), a mixed indicator; 0.1% bromocresol green solution and 0.1% methyl red solution in 95% alcohol separately. 10 mL bromocresol green with 2 mL of methyl red. The boric acid solution, H3BO3 2% b / v; 10 g H3BO3 solution in 500 mL of distilled water. The hydrochloric acid solution, 0.1 N. HCl NaOH sodium hydroxide solution 30% w / v; A solution of 150 g NaOH in 350 mL of water, stored in a rubber-lined bottle. The squid was procured from the waters of the northern coast of Java. Shellfish was procured from northern coastal areas of East Java. the Sea Worms were procured the southern coast of Bali island. On the other hand, Earthworms were procured from a cultivation facility in the Malang area.

The research method in this study was conducted in several stages. The first step was to determine the water content using the Oven method referring to SNI 01-2891-1992, Item 5.1; AOAC (2005) 930.15 (Temperature 135°C); Water Content Based on AOAC (2005) 930.15 (Temperature 135°C). The following is a work procedure for determining water content:

• Weigh the 2-gram sample on solder container which was measured beforehand

• Dry in an oven at 135oC for 2 hours

• Cool in a desiccator

• Repeat the work until a fixed weight was obtained, then the calculation was carried out using the following formula:

W1

Water content =--------x 100%

W

Where:

W = sample right before drying, in grams;

W1 = weight loss after drying, in grams.

The next step was to calculate fat content using direct extraction method (Soxhlet) with reference to SNI 01-2891-1992, Item 5.1; AOAC (2012) 930.15 (temperature 135 ° C); SNI 01-2891-1992. The procedure is described as follows:

• Weigh 7-10 g dry sample then put it into a paper sleeve covered with cotton.

• Insert into the Soxhlet device which has been connected with a fat flask containing dried boiling stones. Its weight was measured beforehand.

• Extract using hexane or other fat solvents for approximately 6 hours.

• Refine hexane and dry the fat extract in a drying oven at 105oC.

• Cool the sample and weigh it.

• Repeat drying process until a fixed weight is obtained.

• Calculate the fat content using the following formula:

Fat content (%) = W2-W1 x 100% W

Where:

W = sample weight, in grams;

W1 = weight before extraction, in grams;

W2 = weight after extraction, in grams.

The third stage measured crude protein level using the Kjeldahl method with reference to AOAC (2005) 960.52; SNI 01-2891-1992. The procedure is described as follows:

• Weighing a 0.1 g sample, then put it in a 500 mL Kjeldahl flask.

• Add 2 g of selen mixture and 10 mL of concentrated H2SO4.

• Boil it and the solution's color become clear and greenish (about 2 hours). Left the solution to cool.

• Insert the solution into the distiller and add 150 ml of distilled water, add 50 mL 40% NaOH, immediately close the distillation flask.

• Distill for 10 minutes, as a container use 10 mL of 2% boric acid solution (during the distillation process, the tip of the condenser pipe must always be immersed in boric solution).

• Rinse the ends of the pipe using distilled water.

• Titer with 0.01 N HCl solution

• Conduct blank determination. The protein content was calculated using the following formula:

Protein Content (%) = (V1-V2) x N x 0.014 x fk x fp x 100%

W

Where:

WExample = weight of sample (g); V1 = Volume HCl 0.1 N example (mL); V2 = Volume of HCl 0.1 N blank; NHCl = normality of HCl; Fk = conversion factor; fp = dilution factor.

The fourth step was to calculate the content of carbohydrates using the phenol-sulfuric acid method referring to Anal. Chem. 28 (1956) 350-356 (In-House Method). The following was a work procedure in calculating carbohydrate content: the first step was to produce a standard curve by dissolving standard starch 1000 mg / L pipetted to get the concentration of the standard series 10; 20; 30; 40; 50 and 60 ppm to a 50 mL flask. Take a 1 mL pipette of each standard solution to the test tube and add 1 mL of distilled water. Prepare a 2 mL blank solution of distilled water. Each solution was added with 1 mL of phenol solution 55% and did vortex. Add 5 mL of concentrated sulfuric acid quickly (dispenser). Left it for 10 minutes then measure absorbance at a wavelength of 490 nm2. The following procedure described sample analysis after creating a standard curve:

• Weigh 0.05-0.1 g of the sample into the test tube.

• Add 10 ml of concentrated H2SO4 and leave it.

• Dissolve it into 100 ml flask then filter it.

• Take as much as 1 mL of the sample solution and add 1 mL of distilled water

• Add 1 mL of 5% phenol solution and conduct vortex

• Add 5 mL of concentrated sulfuric acid quickly (dispenser)

• Leave the solution for 10 minutes

• Measure absorbance at a wavelength of 490 nm.

• Carbohydrate content was measured using the following formula:

Carbohydrate (%) = c/w x Vol Lar x W x 100

Where:

c = carbohydrate concentration as glucose from the standard curve (^g / g);

Vol. lar. — sample dissolution volume (mL); w — sample weight (g).

Use of the amino acid profile of each ingredient using HPLC (a reference to the In-house method / ICI Instrument Method, 1988) and the fatty acid profile of ingredients using GC.

RESULTS AND DISCUSSION

Based on the results of the proximate analysis, the amino acid and fatty acid profiles of each ingredient are exhibited in Table 1.

Table 1 - Results of proximate analysis, amino acids and fatty acids of each ingredient

Parameter Result Unit

Squid flour Shellfish flour Nereis Flour

Water Content 3.46 5.20 7.13 %w/w

Ash Content 2.49 6.89 10.82 %w/w

Protein Content 73.87 59.97 54.35 %w/w

Fat Content 5.99 14.94 12.31 %w/w

Crude Fiber 3.17 2.69 3.45 %w/w

Carbohydrate 11 10.31 11.94 %w/w

Amino Acid %w/w

Aspartic acid 7.96 6.59 5.04 %w/w

Glutamic acid 13.15 9.56 8.18 %w/w

Serine 2.56 2.62 1.42 %w/w

Histidine 1.38 1.14 0.88 %w/w

Glycine 3.21 3.18 3.71 %w/w

Threonine 3.06 2.89 2.48 %w/w

Arginine 5.91 4.18 2.74 %w/w

Alanine 4.06 3.75 4.89 %w/w

Tyrosine 2.71 2.54 2.04 %w/w

Methionine 2.67 1.63 1.27 %w/w

Valine 3.34 3.37 3.21 %w/w

Phenylalanine 3.12 2.76 2.43 %w/w

I-leucine 3.93 3.37 3.12 %w/w

Leucine 6.33 4.87 5.18 %w/w

Lysine 5.80 4.21 4.15 %w/w

Amino Acid Total 69.20 56.66 50.75 %w/w

Fatty Acid** %w/w

Undecanoic acid, C11:0 0.02 - - %w/w

Capric acid, C10:0 - - - %w/w

Lauric Acid, C12:0 0.39 0.04 0.04 %w/w

Tridecanoic Acid, C13:0 0.11 0.03 - %w/w

Myristic Acid, C14:0 0.61 2.51 0.42 %w/w

Myristoleic Acid, C14:1 0.09 0.07 0.16 %w/w

Pentadecanoic Acid, C15:0 0.18 0.32 0.26 %w/w

Palmitic Acid, C16:0 8.11 11.68 4.74 %w/w

Palmitoleic Acid, C16:1 0.25 3.94 1.02 %w/w

Heptadecanoic Acid, C17:0 0.39 0.98 1.25 %w/w

Cis-10-Heptadecanoic Acid, C17:1 0.02 0.10 0.23 %w/w

Stearic Acid, C18:0 2.83 4.15 2.66 %w/w

Elaidic Acid, C18:1n9t 0.03 0.11 0.14 %w/w

Oleic Acid, C18:1n9c 1.16 2.94 3.60 %w/w

Linoleic Acid, C18:2n6c 0.26 0.58 1.03 %w/w

Arachidic Acid, C20:0 0.09 0.10 0.15 %w/w

Cis-11-Eicosenoic Acid, C20:1 0.47 0.14 0.43 %w/w

Linolenic Acid, C18:3n6 0.14 0.14 - %w/w

Linolenic Acid, C18:3n3 - 0.25 0.24 %w/w

Cis-11,14-Eicosedienoic Acid, C20:2 0.14 0.36 - %w/w

Heneicosanoic Acid, C21:0 - - 0.13 %w/w

Behenic Acid, C22:0 0.11 0.06 0.26 %w/w

Cis-8,11,14-Eicosetrienoic Acid, C20:3n6 0.05 0.21 0.39 %w/w

Arachidonic Acid, C20:4n6 2.19 1.93 2.47 %w/w

Tricosanoic Acid, C23:0 0.02 0.03 0.07 %w/w

Cis-13,16-Docosadienoic Acid, C22:2 0.06 0.03 0.11 %w/w

Lignoceric Acid, C24:0 0.11 0.14 0.08 %w/w

Cis-5,8,11,14,17-Eicosapentaenoic Acid, C20:5n3 3.49 9.02 1.48 %w/w

Nervonic Acid, C24:1 0.08 - - %w/w

Cis-4,7,10,13,16,19-Docosahexaenoic Acid, C22:6n3 13.96 4.81 0.25 %w/w

Fatty Acid Total 35.22 44.65 22.87 %w/w

DISCUSSION OF RESULTS

The shrimp gonads growth can be improved by increasing the quality and quantity of feed, by providing feed containing high protein. Fresh feed capable to improve vanamei shrimp gonads growth is sea worms (Nereis sp) or earthworms (Lumbricus sp), and 30% oysters/shellfish biomass provided every day. But these natural organisms are expensive, and the product quality is not available throughout the year. Processed dry feed offers many advantages, such as reliable supply, controlled nutrient content, reducing maintenance media contamination and avoiding pathogens, which are very important for the production of special pathogen-free nauplii (SPF) (Shaobo Du, 2004).

Vanamei shrimp is an introduced shrimp species that has undergone domestication. It has produced prospective offspring and broodstocks through breeding activities in Indonesia. Shrimp gonad growth could be improved by increasing the quality and quantity of feed, such as providing high protein-containing feed.

Based on proximate analysis result, amino acids and fatty acids from all the ingredients could be processed into feed. It is capable to to improve prospective vanamei shrimp gonad growth. Based on Amino acids and fatty acids profile, squid has the highest protein content of 73.87%. Shellfish, Sea worm (Nereis sp.), and Earthworms (Lumbricus sp) possess 59.97%, 54.35%, and 44.22% protein content respectively.

Important nutrients needed for shrimp growth are carbohydrates, fats, proteins, minerals and vitamins other than water. The most important component is proteins because the growth rate directly depends on the quality and quantity of the protein given. Shrimp in the early stages require high percentage feed. Initially, the minimum amount of protein needed is an average of 45%. After the shrimp grow, the protein needs gradually decrease to 35% (Pascual, 1989). Wouters et al. (2001a) reported that the feed protein content formulated in their study was around 50%. Nevertheless, it was relatively low compared to the protein level contained in the fresh feed.

According to Limsuwatthanathamrong et al. (2012), fatty acids are an important factor that must be considered when providing feed for shrimp during gonad maturation process. Fat content has long been known to have a large influence on marine fish and crustaceans reproductive growth. One lipid class, high-unsaturated fatty acids (HUFA), has proven to be very important and suitable for reproduction in some marine species and has received considerable research attention (Wouters et al, 2002)

Crustaceans have long been recognized as having limited ability to synthesize high-unsaturated fatty acids (HUFA) and do not have the ability to synthesize sterols. Therefore shrimp do not have a definitive dietary fat requirement but require enough fat to meet their needs for certain nutrients, such as HUFA, phospholipids, sterols, and energy. Fats, such as phospholipids, triglycerides, and cholesterol, are the main sources of energy contained in the feed. These components are involved in several important processes such as molting, growth, and reproduction. Cholesterol is an important component of animal tissues diet. It plays a major role in the structure of cell membranes and is a precursor to sex hormones, bile acids, and vitamin D. Crustaceans cholesterol are known as the most important food source known as the sterol. It is used for development, growth, reproductive survival, and a precursor to many hormones important for the initiation of metamorphosis and molting processes (Teshima and Kanazawa, 1971).

Vanamei shrimp tend to be carnivores compared to omnivores that eat small crustaceans and Polychaeta (Hendrajat, 2003).

Chamberlain and Lawrences (1981) reported that some natural foods used for penaeid shrimp gonads development were shellfish, squid, types of crustaceans and worms.

Natural food eligible to be utilized as feed are those sustainably available, possess high nutritional content and does not disease. Nevertheless, there's limited stock of sea worms as it is dependent on nature. Sea worms' usage could be suppressed by combining sea worms with other natural feeds to produce high quality larval production (Sabrina.et al., 2014). Fresh squid (Loligo pealeii) contains 10 fatty acids. Saturated and unsaturated fatty acid levels include C15: 0 (Pentadekanoate), C17: 0 (Margarat), C18: 0 (Stearate), C20: 0 (Arachidate),

C16: 1 A9 (Palmitoleate), C18: 1 A9 (Oleate), C18: 2 9,12 (Linoleic), C20: 4 ,85,8,11,14 (Arachidonate), C24: 1 A15 (Nervonate), C27: 1 A5 (Cholesterol). (Johanis & Hanoch, 2013).

The results of the fatty acid profile analysis on squid flour, shellfish flour, and nereis flour exhibited a high content of unsaturated fatty acids (ARA: 2.19; 1.93; 2.47), (EPA: 3.14; 9.02; 1.48), (DHA: 13.96; 4.81; 0.25). Oysters and sea worms are natural feeds possessing complete nutritional content to increase gonad growth of vanamei shrimp prospective broodstock. Nevertheless, its natural availability is very limited and depends on the season. The increasing need for vanamei shrimp broodstock feeds encourages artificial feed alternatives possessing nutritional content in accordance with vanamei shrimp gonads growth and development. (Wyban et al., 1991).

CONCLUSION

Based on proximate analysis result, amino acids and fatty acids from each ingredient can be used as constituent ingredients for artificial feed formulations. These are in accordance with vanamei shrimp broodstock reproductive organ development. Therefore the feed could be used as an alternative substitute for fresh feed.

SUGGESTIONS

Further research is needed on other local ingredients as compilers of artificial feed formulations for vanamei shrimp gonads growth.

ACKNOWLEDGMENTS

This work has been conducted in the Integrated Laboratory of Bogor Agricultural University, Bogor, Indonesia.

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