SBA 5512

FISH NUTRITION

LAB REPORT 1 PROTEIN NUTRIENT CONTENT ANALYSIS

Lecturer:

ASSOC. PROF. DR. ROSSITA SHARPAWI

Prepared by:

MUHAMMAD HILMY BIN ABDUL JAMIL GSK 1275 MSC AQUACULTURE INSTITUTE AQUACULTURE TROPICA

1.0

Introduction

Global human consumption of protein is significantly produced by aquaculture industry. In 2007 only, 43% consumed protein globally was from aquaculture and is forecasted to grow in response of increasing demand and population (Bostock et al., 2010). The worldwide marine aquaculture industry has experienced tremendous growth over the last several decades producing 52.5 million tons of products in 2008 worth $98.5 billion (Bostock et al., 2010).

Feeding cost dominated 50% of the production cost of aquaculture product, causing feeding become competitive edge to succeed (Vielma et al., 2000). Proteins are the major and most expensive component of formulated aqua feeds (Wilson, 2002). Protein is a basic component of fish diets, both in terms of quantity and quality, protein requirements being higher than those of other animals (Cowey, 1975).

Proteins are polymers of amino acids. It contains carbon, hydrogen, oxygen, nitrogen and sometime sulphur and phosphorus. Twenty different types of amino acids occur naturally in proteins. Proteins are important constituents of fish feed for tissue growing, source of energy and formation of other biological substances. Ingested protein is hydrolyzed to free amino acids, dipeptides, and tripeptides by digestive enzymes secreted into the gastrointestinal tract. Amino acid then will be distributed by blood to the organs and tissues to be utilized.

Protein content in an ingredient should be analyzed to create more accurate formulation. Too much protein in a diet will caused only several protein will be used and the remainder will be converted to energy. However, inadequate amount of protein will lead to reduction of growth due to withdrawal of protein from less vital tissues to support vital tissues. This will reduces fishs resistance to disease and lower the effect of immune response.

There are several methods that have been created over the past century to do protein analysis. But, the standard method that used globally is Kjehdal Method. The Kjeldahl method was developed in 1883 by a brewer named Johann Kjehdal for determining the nitrogen contents in organic and inorganic substances. Although the technique and apparatus have been modified over the years, the basic principles introduced by Johan Kjeldahl still endure today.

1.1

Objectives 1. To analyze crude protein content of fishmeal and soybean meal using Kjedahl method. 2. To compare crude protein content of both ingredients.

2.0 Material and method 2.1 Material 1. Digestion unit set 2. Distillation unit set 3. Titration unit 4. Electronic weigh 5. Sulphuric acid (H2SO4) 6. Catalyzer (1 CuSo4: 9 K2SO4) 7. Boric Acid (4%) 8. Dye (Methyl red) 9. Natrium Hydroxide, NaOH (40% w/v) 10. Standard solution 0.01N HCL 11. Volumetric flask 12. Kjedahl digestion tube 13. Conical flask 14. 100mL Beaker 15. 100mL graduated cylinder 2.2 Method 2.2.1 Ingestion

1. 0.5g sample of fishmeal and soybean meal was weighed and put in Kjedahl tube. The weigh will be recorded as W1. Another two tubes left without sample and labeled as blank. 2. All tubes placed on holder rack. 10mL of sulphuric acid was added to all tubes and was done in laminar flow. Catalyzer was placed into the tubes. The tubes shake well. 3. The tubes were transferred to heating block on ingestion set. Exhaust system was locked to Kjedahl tubes and the system activated. 4. Left for 30-60 minutes until all sample dissolve to light-green/blue solution depending on the type of sample.

5. NaOH solution (40% w/v) was prepared. 40g of NaOH powder was dissolved in volumetric flask with distilled water. Distilled water was added until reaching 100mL mark. 2.2.2 Distillation

1. Ingested sample was liquefied by adding 40mL distilled water. All solution as transferred to 100mL graduated cylinder. Kjedahl tube was rinsed with distilled water and added to cylinder. Distilled water was added until 100mL mark and recorded as V1. 2. 10mL (V2) liquefied sample placed in distillation tube and added with 8mL of NaOH (40%). The tube placed to Kjedahl distillation set. 3. 5mL Boric acid (4%) was filled in 100mL conical flask and added with 6 drops of dye. This receiver solution was red-colored. 4. Condenser end was immersed in receiving conical flask. 5. The system activated and distillation occurred for three minutes. The beaker was lowered to get the distilled product. Solution in receiving was changed from red to gray. 6. Continue with titration process using HCL. 2.2.3 Titration

1. Distilled solution was titrated with 0.01N HCL until changed to red/pink color. 2. Volume of HCL titrated was recorded and labeled as T.

2.2.4

Calculation (g)

Titrated sample weigh (W2) = W1 x V2 V1 = W1 x V2 V1 Where, W1 = Digested sample (g) V2 = Distilled sample solution volume (ml) V1 = Liquefied sample volume (ml)

x 1000 (mg)

% N in sample

= (T-B) x N x 14.007 W2

x 100 x 100

% Protein in sample Where,

= %NxF

T = HCL volume used in titration (ml) N = HCL Normality W2 = Titrated sample weigh (mg) F = Protein factor (6.25)

3.0 3.1
Sample

Result Fish Meal

Fish meal, W1 (g) 0.5024 0.5020 Titration value, T (ml) 28.0 17.0 Weight sample titrate, W2 (g) 50.2 50.2 Nitrogen in sample (%) Crude protein (%)

1 2

7.78 4.72

48.62 29.47

Table 1: W1, W2, T value and percentage of Nitrogen and crude protein in fish meal sample

Titration value showed too much difference between the two replicates. This lead to larger difference in nitrogen and crude protein proportion in fish meal sample even though the value still lies in range that should be retained by fish meal. 3.2
Sample

Soybean Meal
Soybean meal, W1 (g) 0.5 0.5 Titration value, T (ml) 16.1 21.2 Weight sample titrate, W2 (g) 50.0 50.0 Nitrogen in sample (%) Crude protein (%)

1 2

4.48 5.91

28.01 36.94

Table 2: W1, W2, T value and percentage of Nitrogen and crude protein in soybean meal sample

Percentage of nitrogen in sample and crude protein in sample showed small difference. Generally, protein in soybean meal is slightly lower when compared to fish meal. This maybe because of lack in amino acid profile. Where, W1 = Digested sample (g) W2 = Titrated sample weigh (mg) T = HCL volume used in titration (ml)

4.0 4.1

Discussion Kjeldahl Method The Kjeldahl method may be broken down into three main steps: digestion, distillation,

and titration. 4.1.1 Digestion

Digestion is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. Sulphuric acid (H2SO4) act as oxidizing which digest sample. Catalyst added to speed up the reaction. Digestion converts any nitrogen in the food into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution: The general equation for the digestion of an organic sample is shown below: Organic N + H2SO4 (NH4)SO4 + H2O + CO4 + other sample matrix byproducts Fig.1: Equation for digestion 4.1.2 Distillation

The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, NaOH which converts the ammonium sulfate, (NH4)2SO4 into ammonia gas, NH3. The NH3 is recovered by distilling the reaction product.

ammonium heat ammonia sulfate gas (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O Fig.2: Equation for distillation

The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask - which contains boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion: ammonia gas NH3 + boric ammoniumacid borate complex NH4 + H2BO-3 + H3BO3 (color change occurs) Fig.3: Boric acid equation The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life. 4.1.3 Titration excess boric acid H3BO3

Titration quantifies the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution. The nitrogen content is estimated by titration of the ammonium borate formed with hydrochloric acid, HCL, using a suitable indicator to determine the end-point of the reaction.

H2BO3- + H+

H3BO3

Fig.4: Titration equation 4.2 Ingredient used 4.2.1 Fish Meal

Fishmeal is the major ingredient that been used in producing feed formulation. This is because fishmeal is considered the ideal protein as the amino acid profile is similar to the whole body, causing protein utilization and growth are maximized (Trushenski et al., 2006). However, awareness has been heighten due to negative effect on fish meal production (Naylor et al., 2000) and inflated price (Fujise and Kanmuri, 2010) 4.2.2 Soybean meal

To reduce constriction on fish meal supply, many researches has been focused to find alternative for fish meal (Luquet and Kaushik, 1978; Pfeffer, 1982; Hardy, 1995; Tacon et al.,
1998). Among the ingredient being researched is soybean meal (Storebakken et al., 2000; Swick, 2002). This is mainly because security of supply, competitive price and its amino acid profile. However, complete replacement of fish meal causing lot of negative effects on cultured fish such as due to nutritional imbalances in amino acids, presence of antinutritional factors or due to low palatability (Hardy, 1996). The solution is to partially replace fish meal, as this show minor difference in growth and feed conversion in rainbow trout (Watanabe et al., 1993) and Atlantic salmon (Opstvedt et al., 2003, Mundheim et al., 2004)

4.3

Protein contain Comparing result of protein analysis in fish meal (Table 1) and in soy bean meal (Table

2), we can see that fish meal contain higher amount of crude protein. This fit its role as the most complete ingredient to supply protein for fish to be used in feed formulation. Even though soy bean meal contain lower amount of crude protein, this is not overall bad for feed formulation as it still have provide essential amino acid for fish.

In table 1, the difference in amount of crude protein between the two samples of fish meal is caused by several reasons. First, concentration of chemical reagents. Lower concentration will only convert inadequate amount of ammonium sulphate, (NH4)2SO4 + 2NaOH to ammonia gas, NH3. This will lead to lower amount of ammonia gas that will bind to boric acid to form ammonium-boric acid, H2BO-3. Second, inaccuracy of titrated hydrochloric acid, HCL during titration procedure. This may caused by wrong normality of HCL used or too much HCL being used for titration, thus lead to error. Person operating titration procedure must have adequate skill to avoid errors. Third, the protein in fish meal has already denatured. Heating process during distillation procedure maybe cause this. However, this is not strong reason as other sample also being heated with the same temperature and length of time.

Soybean meal value of crude protein show little difference as shown in Table 2. The result, even though lower, it still in the range of crude protein contain in soybean meal.

5.0

Conclusion Protein analysis of the ingredients of fish feed can be easily been done by Kjehdahl

method. This method show high precision and good reproducibility. Generally, soybean meal have lower amount of protein when compare to fish meal due to multiple reason. Person operating protein analysis must be skilled enough to handle the chemical reagent and operate the machines.

6.0 Journal

References

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Fujise, M., Kanmuri, T., 2010. The issues surrounding aquaculture feeds and the current measures associated with them in Japan. Bull. Fish. Res. Agen. No. 31, pg 1-7, 2010

Hardy, R.W. 1995. Current issues in salmonid nutrition. Nutrition and Utilization Technology in Aquaculture, (ed by Lim, C. and Sessa, D.J.), AOAC Press, Champaign, USA, 26-35. Hardy, R.W., 1996. Alternative protein sources for salmon and trout diets. Anim. Feed Sci. Technol. 59, 7180.
Luquet, P. and Kaushii, S.J., 1978. Progr& recent dans le domaine de lalimentation proteique des salmonides:Bpargne des proteines et mat&es premieres de substitution a la farine de poisson. Piscicult. Fr., 53-54: 14-17.

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Pfeffer, E., 1982. Utilization of dietary protein by salmonid fish. Comp. Biochem. Physiol., 73B: 51-57.

Swick, R.A. 2002. Soybean meal quality: assessing the characteristics of a major aquatic feed ingredient. Global Aquaculture Advocate 5:46-49. Tacon, A.G.J., Dominy, W.G., Pruder, G.D. 1998. Global trends and challenges in aquafeeds for marine shrimp. AquaFeed International 4:28 - 35. Tacon, A., 2000. Feed ingredients for carnivorous fish species. Alternatives to fish meal and other fishery resources. FAO Fish Circ. No. 881, Rome, 35 pp.

Trushenski, J., Kasper, C., & Kohler, C. (2006). Challenges and opportunities in finfish nutrition. North American Journal of Aquaculture, 68, 122140. Vielma, J.T. Makinen, P. Ekholm and J. Koskela, 2000. Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load. Aquaculture, 183: 349-362 Watanabe, T., Pongmaneerat, J., Satoh, S., Takeuchi, T., 1993. Replacement of fish meal by alternative protein sources in rainbow trout diets. Nippon Suisan Gakkaishi 59, 1573 1579. Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J., Folke, C., Lubchenco, J., Mooney, H. and Troell, M. 2000. Effect of aquaculture on world fish supplies. Nature 405:1017-1024. Book Nutrient Requirements of Fish, 1993, Subcommittee on Fish Nutrition, National Research Council, National Academies Press, pp20-26