Aus dem Institut für Tierzucht und Tierhaltung der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel ___________________________________________________________________________

RAPESEED PROTEIN PRODUCTS AS FISH MEAL REPLACEMENT IN FISH NUTRITION Dissertation zur Erlangung des Doktorgrades der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Master of Science HANNO SLAWSKI aus Neustadt in Holstein

Kiel, 2011 ___________________________________________________________________________ Dekanin: Prof. Dr. K. Schwarz Erster Berichterstatter: Prof. Dr. C. Schulz Zweiter Berichterstatter: Prof. Dr. A. Susenbeth Tag der mündlichen Prüfung: 14.07.2011 ___________________________________________________________________________ Die Dissertation wurde mit dankenswerter finanzieller Unterstützung aus dem Europäischen Fischereifond und dem Zukunftsprogramm Fischerei des Landes Schleswig-Holsteins angefertigt

Gedruckt mit Genehmigung der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel

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Table of Contents General Introduction ................................................................................................................... 1 Chapter 1: Replacement of fish meal with rapeseed protein concentrate in diets fed to common carp (Cyprinus carpio L.) ............................................................................................ 4 Chapter 2: Replacement of fish meal with rapeseed protein concentrate in diets fed to wels catfish (Silurus glanis L.) ......................................................................................................... 16 Chapter 3: Austausch von Fischmehl durch Rapsproteinkonzentrat in Futtermitteln für Steinbutt (Psetta maxima L.) .................................................................................................... 34 Chapter 4: Total fish meal replacement with rapeseed protein concentrate in diets fed to rainbow trout (Oncorhynchus mykiss W.) ................................................................................ 47 Chapter 5: Replacement of fish meal with albumin and globulin rapeseed protein fractions in diets fed to rainbow trout (Oncorhynchus mykiss W.) ............................................................. 69 Chapter 6: Total fish meal replacement with canola protein isolate in diets fed to rainbow trout (Oncorhynchus mykiss W.) .............................................................................................. 85 General Discussion ................................................................................................................. 102 General Summary ................................................................................................................... 111 Zusammenfassung .................................................................................................................. 114 Danksagung ............................................................................................................................ 117 Lebenslauf .............................................................................................................................. 118

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List of Tables Table 1.1

Proximate composition and amino acid profiles of fish meal and rapeseed protein concentrate and concentration of antinutritional factors detected in RPC

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Table 1.2

Formulation, amino acid profiles and proximate composition of experimental diets for common carp

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Table 1.3

Growth response, feed efficiencies and survival of carp fed experimental diets

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Table 1.4

Proximate whole body composition of carp fed the experimental diets

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Table 2.1

Nutrient composition and essential amino acid profiles of fish meal and rapeseed protein concentrate and concentration of antinutritional factors detected in RPC

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Table 2.2

Formulation, essential amino acids composition and proximate composition of experimental diets

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Table 2.3

Growth response, feed intake, feed efficiencies, condition factor and survival of wels catfish fed experimental diets

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Table 2.4

Proximate whole body composition of wels catfish fed the experimental diets

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Table 2.5

Blood haematocrit content and blood serum values of wels catfish fed experimental diets

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Table 3.1

Nährstoff- und Aminosäurenzusammensetzung von Fischmehl und Rapsproteinkonzentrat

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Table 3.2

Formulierung der Versuchsfuttermittel

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Table 3.3

Nährstoffund Versuchsfuttermittel

Table 3.4

Ganzkörperzusammensetzung Fütterungsperiode

Aminosäurenzusammensetzung

der

39

der

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Table 3.5

Wachstumsparameter und Futterverwertung der Steinbutt nach dem Fütterungsversuch

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Table 4.1

Proximate and amino acid composition of fish meal and rapeseed protein concentrate and concentration of antinutritional factors determined

51

IV

der

Steinbutt

nach

Table 4.2

Formulation of experimental diets

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Table 4.3

Proximate and amino acid composition of experimental diets

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Table 4.4

Growth response, feed efficiencies and survival of rainbow trout fed experimental diets

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Table 4.5

Proximate whole body composition of rainbow trout fed experimental diets

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Table 4.6

Blood parameters of trout fed experimental diets

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Table 5.1

Nutrient composition and amino acid profiles of fish meal, albumin concentrate and globulin concentrate

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Table 5.2

Formulation and nutrient composition and amino acid profiles of experimental diets used in the digestibility trial

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Table 5.3

Formulation, proximate nutrient composition and amino acid composition of experimental diets for rainbow trout

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Table 5.4

Apparent digestibility coefficients

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Table 5.5

Growth performance, feed intake and feed efficiencies of rainbow trout fed experimental diets

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Table 5.6

Proximate whole body composition of rainbow trout fed experimental diets

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Table 6.1

Nutrient composition and essential amino acid profiles of fish meal and canola protein isolate

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Table 6.2

Formulation, nutrient composition and essential amino acid profiles of experimental diets used in the digestibility trial

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Table 6.3

Formulation, proximate composition and essential amino acid profiles of experimental diets

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Table 6.4

Apparent digestibility coefficients

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Table 6.5

Growth response, feed intake and feed efficiencies of rainbow trout fed experimental diets

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Table 6.6

Proximate whole body composition of rainbow trout fed experimental diets

89

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General Introduction In 2009 aquaculture production hit a landmark: half of all fish and shellfish destined for human consumption were cultured, and production of farmed fish eclipsed that of wild caught fish. But, the increased aquaculture production also accounted for 68 % of the worldwide fish meal consumption (Naylor et al. 2009). However, fish meal, the most important source of protein in fish feeds, is a limited resource with an annual production volume between 5 to 6.5 Mio t (FAO 2004). Tremendous price increases for fish meal together with environmental concerns therefore force the aquaculture sector to find alternative protein sources to be included in fish feeds. Presently, most relevant alternatives are protein concentrates derived from vegetables. Among them, soybean protein concentrates have become a commonly accepted fish feed ingredient and fish meal alternative (Gatlin et al. 2007). While soybean ranks as number one oilseed worldwide (222.2 Mio t/a), protein products derived from rapeseed, which ranks as number three oilseed worldwide (61.6 Mio t/a) (FAO 2010), are less commonly used as fish feed ingredients. However, simple oilcakes or rapeseed meals with increased protein content produced from oilcakes that were de-oiled with organic solvents have been widely tested as protein sources in feeding trials with several fish species. Experiments with rainbow trout (Burel et al. 2000a,c; Shafaeipour et al. 2008), Nile tilapia (Davies et al. 1990), common carp (Dabrowski and Kozlowska 1981) and turbot (Burel et al. 2000a,b) have shown, that the nutritional quality of simple rapeseed products is below that of fish meal although they contained a well balanced amino acid profile. Particularly antinutritional factors (ANF) determine the quality of rapeseed products for fish nutrition. The most prominent ANF in rapeseed products are glucosinolates, phytic acid, phenolic constituents and indigestible carbohydrates (Francis et al. 2001). By several processing techniques the level of antinutrients in rapeseed products can be reduced and their value for fish nutrition can be improved. Dehulling of seeds and utilisation of high temperatures and organic solvents during oil extraction as well as sieving of meal decrease content of glucosinolates, phytate, fibre, cellulose, hemicellulose, sinapin and tannins (Fenwick et al. 1986; Anderson-Haferman et al. 1993). Protein extraction from meals by methanol-ammoniatreatment or ethanol-treatment will increase protein level and effectively remove glucosinolates, phenolic compounds, soluble sugars, such as sucrose, and some oligosaccharides (Naczk and Shahidi 1990; Chabanon et al. 2007). In different countries, rapeseed protein products of high quality were produced for application in animal nutrition.

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However, these products were made for test purposes in small volumes until their potential as protein source in animal nutrition is clarified. Besides nutritive quality, their costs of production must decrease to make rapeseed protein products available at a competitive price compared to other protein sources, especially fish meal. In the present study, different protein protein products derived from rapeseed (including canola) were tested as fish meal replacement in diets for several fish species. A high quality rapeseed protein concentrate (RPC) with a protein content of 71 % was evaluated as fish meal replacement in diets for common carp (chapter 1), wels catfish (chapter 2), turbot (chapter 3) and rainbow trout (chapter 4). Based on the results presented in chapter 4, in chapter 5 the potential of two rapeseed protein concentrates partitioned in albumin and globulin fractions as fish meal alternatives was evaluated in a digestibility study and a consecutive growth trial with rainbow trout. Compared to the RPC, the fractionized protein concentrates were produced under lower cost and time effort. In chapter 6 a canola protein isolate with a crude protein content of 81 % was evaluated as fish meal alternative in diets for rainbow trout. The nutritional quality of the raw material was determined in a digestibility experiment followed by a growth trial.

References Anderson-Hafermann, J.C., Zhang, Y., Parsons, C.M., 1993. Effects of processing on the nutritional quality of canola meal. Poultry Science 72, 326-333. Burel, C., Boujard, T., Tulli, F., Kaushik, S.J., 2000a. Digestibility of extruded peas, extruded lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima). Aquaculture 188, 285–298. Burel, C., Boujard, T., Kaushik, S.J., Boeuf, G., van der Geyten, S., Mol, K.A., Kühn, E.R., Quinsac, A., Krouti, M., Ribaillier, D., 2000b. Potential of plant-protein sources as fish meal substitutes in diets for turbot (Psetta maxima): growth, nutrient utilisation and thyroid status. Aquaculture 188, 363-382. Burel, C., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der Geyten, S., Kühn, E.R., 2000c. Dietary low glucosinolate rapeseed meal affects thyroid status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). Brit. J. Nutr. 83, 653–664. Chabanon, G., Chevalot, I., Framboisier, X., Chenu, S., Marc, I., 2007. Hydrolysis of rapeseed protein isolates: Kinetics, characterization and functional properties of hydrolysates. Process Biochem. 42, 1419–1428.

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Dabrowski, K., Kozlowska, H., 1981. Rapeseed meal in the diet of common carp reared in heated waters. I. Growth of fish and utilization of the diet. In: K. Tiews (ed.). Aquaculture in Heated Effluents and Recirculation Systems. Heenemann, Hamburg, pp. 263-274. Davies, S.J., McConnel, S., Bateson, R.I., 1990. Potential of rapeseed meal as an alternative protein source in complete diets for tilapia (Oreochromis mossambicus Peters). Aquaculture 87, 145-154. Food and Agriculture Organization (FAO), United Nations (2004). FAO Fisheries Department, Fishery Information, Data and Statistics Unit. Fishstat Plus: Universal software for Fishery Statistical Time series, version 2.30 (www.fao.org). FAO (2010): http://faostat.fao.org/site/567/default.aspx#ancor Fenwick, G.R., Spinks, E.A., Wilkinson, A.P., Henry, R.K., Legoy, M.A., 1986. Effect of processing on the antinutrient content of rapeseed. J. Sci. Food Agr. 37, 735-741. Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture 199, 197–227. Gatlin III, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, Å., Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D., Souza, E.J., Stone, D., Wilson, R., Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research 38, 551-579. Naczk, M., Shahidi, F., 1990. Carbohydrates of canola and rapeseed. In: F. Shahidi (ed.). Canola, Rapeseed: Production, Chemistry, Nutrition & Processing Technology. Van Nostrand Reinhold, New York, pp211-220. Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin III., D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. PNAS 106, 15103-15110. Shafaeipour, A., Yavari, V., Falahatkar, B., Maremmazi, J.G.H., Gorjipour, E., 2008. Effects of canola meal on physiological and biochemical parameters in rainbow trout (Oncorhynchus mykiss). Aquaculture Nutrition 14, 110–119.

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Chapter 1: Replacement of fish meal with rapeseed protein concentrate in diets fed to common carp (Cyprinus carpio L.)

H. Slawski1,2, H. Adem3, R.-P. Tressel3, K. Wysujack4, U. Koops4 and C. Schulz1,2

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Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum

Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel, D-24098 Kiel

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Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg

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Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg

Published in: The Israeli Journal of Aquaculture (2011) 63, 605-611. 4

Abstract The potential of rapeseed protein concentrate (RPC) as fish meal alternative in diets for common carp (initial average weight 26.7 ± 0.8 g) was evaluated. Triplicate groups of fish were fed isonitrogenous (40.4 ± 0.2 % crude protein) and isocaloric (21.4 ± 0.1 kJ g-1) experimental diets with 0% (R0), 33% (R33), 66% (R66) or 100 % (R100) of fish meal replaced with rapeseed protein concentrate. At the end of the 56 days feeding period, growth parameters and feed efficiencies were not significantly different between fish fed on diet R0 and R33. Diets R66 and R100 led to reduced diet intake and feed efficiencies resulting in lower growth performances. It appears that diet taste and amino acid profiles were negatively affected by high inclusion levels of rapeseed protein concentrate resulting in reduced diet acceptance and protein value. It is concluded, that the used rapeseed protein concentrate can effectively replace 33 % of fish meal in diets for carp without using palatability enhancers or amino acid supplements.

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Introduction Wide availability, high protein content and a desirable amino acid profile have caused an interest in rapeseed products as fish meal alternative in fish feeds. Rapeseed and canola products have been tested as protein sources in diets for several fish species, including rainbow trout (Thiessen et al. 2004), Coho salmon (Higgs et al. 1979), Chinook salmon (Satoh et al. 1998), tilapia (Yigit and Olmez 2009), channel catfish (Lim et al. 1998), silver perch (Booth and Allan 2003), carp (Dabrowski and Kozlowska 1981), red sea bream (Glencross et al. 2004), and turbot (Burel et al. 2000ac). It was found, that the nutritional quality of rapeseed products largely depends on their levels of antinutritional factors. Prominent antinutritional factors in rapeseed are glucosinolates, phytic acid, phenolic constituents (e.g. tannins), and indigestible carbohydrates (Francis et al. 2001). Several processing techniques have been adapted to reduce the level of antinutrients in rapeseed in order to improve its value for fish nutrition. Dehulling of seeds and utilisation of high temperatures and organic solvents (hexane) during oil extraction as well as sieving of meal decrease content of glucosinolates, phytate, fibre, cellulose, hemicellulose, sinapin and tannins (Anderson-Haferman et al. 1993; Mawson et al. 1993, 1994ab, 1995; Leming et al. 2004) and increase protein level in meals (Mwachireya et al. 1999). In addition, protein extraction from meals by methanol-ammonia-treatment or ethanol-treatment will further increase protein level and effectively remove glucosinolates, phenolic compounds, soluble sugars, such as sucrose, and some oligosaccharides (Naczk and Shahidi 1990; McCurdy and March 1992; Chabanon et al. 2007) but will also increase levels of non-digestible fibre (Mwachireya et al. 1999). In the present study liquid water extractions combined with ultrafiltration were used to further increase the protein concentration of the final product and at the same time deposit non-digestible fibres. The resulting rapeseed protein concentrate (RPC) contained 71 % crude protein. Momentarily, rapeseed and canola protein products of similar quality are being produced in different countries for application in animal nutrition. However, these products are produced for test purpose until their potential as protein source in animal nutrition is clarified. Besides nutritive quality, their costs of production will have to become low enough to make rapeseed and canola protein concentrates available at a competitive price compared to other protein sources, e.g. fish meal. As basic trial in a series of consecutive feeding trials in order to optimize the produced RPC for application in fish feeds, the product was tested as fish meal replacement in pelleted diets, using juvenile

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common carp as model species. By this, we intended to determine the fundamental native limitations of our RPC as fish feed ingredient.

Materials and methods Diet preparation and experimental procedures Four experimental diets were formulated to replace fish meal with rapeseed protein concentrate (RPC) at 0, 33, 66, or 100 % (designated as R0, R33, R66 and R100, respectively). Vitamins and minerals were added to diets to meet the dietary requirements of carp (NRC, 1993). Diets were manufactured to give pellets 4 mm in diameter (L 14-175, AMANDUS KAHL, Reinbek, Germany). The diets were formulated to be isonitrogenous (40.4 ± 0.2 % crude protein) and isocaloric (21.4 ± 0.1 kJ g−1). Since this is the first trial in a series of consecutive feeding trials investigating our RPC as fish meal replacement, we intended to highlight direct effects on feed quality resulting from dietary RPC incorporation. Therefore diets were formulated without palatability enhancers or crystalline amino acids. Diet formulations, nutritional compositions and amino acid profiles are given in Table 1.1. Solvent extracted RPC was obtained from PPM, Magdeburg, Germany. For oil extraction, rapeseed was cold pressed and residual oil removed by a hexane treatment. Glucosinolates were extracted with an ethanol solution. Liquid water extraction as well as dia- and ultrafiltration of proteins followed by spray drying provided a protein concentrate with 71 % crude protein content (Table 2.1). The growth trial was conducted at the Johann Heinrich von Thünen Institute of Fisheries Ecology, Ahrensburg, Germany. In the growth trial, common carp (Cyprinus carpio L.) was used as model fish. In its juvenile stage, common carp has a high dietary protein requirement (Fine et al. 1996) making this relatively modest fish an ideal model species for fish meal replacement studies. Juvenile common carp that had been hatched in the institute were used. One week before the experiment started 12 fish were stocked in each of twelve experimental tanks (70 L; bottom surface 480 cm2), being part of a freshwater recirculation system. Tanks were provided with water at 1 L min-1 (temperature: 23.8 ± 0.7 °C; O2: 6.5 ± 0.7 mg L-1; pH: 7.0 ± 0.7; NH4+: