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Apparent digestibility of protein, amino acids and energy in rainbow trout (Oncorhynchus mykiss) fed a fish meal based diet extruded at different temperatures
Aquaculture 211 (2002) 215 – 225 www.elsevier.com/locate/aqua-online
Apparent digestibility of protein, amino acids and energy in rainbow trout (Oncorhynchus mykiss) fed a fish meal based diet extruded at different temperatures M. Sørensen a, K. Ljøkjel b, T. Storebakken a,*, K.D. Shearer c, A. Skrede b a AKVAFORSK, Institute of Aquaculture Research AS, N-1432 A˚s Norway Department of Animal Science, Agricultural University of Norway, N-1432 A˚s Norway c Northwest Fisheries Science Center, NOAA/NMFS. 2725 Montlake Blvd E. Seattle, WA 98112 USA b
Received 5 July 2001; received in revised form 9 November 2001; accepted 12 November 2001
Abstract A fish meal/wheat flour based dry ingredient mix was extruded at three different temperatures (100, 125 and 150 jC), by varying extrusion conditions according to two different methods. Employing method 1, the temperature of the conditioner, torque, screw speed, pressure, feed rate and process water of the extruder were varied. Whereas in method 2, conditioner temperature and screw speed were kept constant, and the variation in process water input was restricted. The diets, which contained yttrium oxide as an inert maker, were fed to three replicate groups of rainbow trout. Feces for digestibility determination were obtained by stripping. The results showed that the differences in extrusion temperature caused no significant differences in apparent digestibilities of crude protein, individual amino acids or energy. The apparent digestibility of cysteine was significantly higher for diets produced by method 1 than by method 2. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rainbow trout; Extrusion-temperature; Digestibility
1. Introduction Most modern salmonid diets are produced by extrusion, which is a process where the feed is subject to mixing, shearing and heating under high pressure before the extrudate * Corresponding author. Tel.: +47-64-94-95-00; fax: +47-64-94-95-02. E-mail address: [email protected] (T. Storebakken). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 8 7 - 0
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finally is forced through a die. The feed may undergo reactions during processing that could be beneficial if the nutritional value is improved, or detrimental if nutrients are destroyed or become resistant to digestion. Reactions taking place in the feed during extrusion are largely determined by shear-forces, temperature, moisture, residence time and pH. In addition, the reactions depend on the type of reactants present, such as water, lipids, carbohydrates and proteins. Depending on the protein source, heat denaturing occurs over a temperature range of 25– 100 jC (Hultin, 1986). Denaturing is loss of quaternary, tertiary or secondary structure of the proteins while the primary structure remains intact. Thus, it causes conformational changes without affecting the amino acid composition of the protein. Moderate heating of protein-rich feed ingredients may be beneficial for the nutritional value of the feed proteins, since unfolded protein is often more readily digested than native protein (Camire, 1991). Vegetable protein sources may also contain heat labile anti-nutrients such as protease inhibitors and lectins, which are inactivated during heat treatment (Van der Poel et al., 1990). Damage to proteins during heat processing is a function of temperature, time, moisture and the presence of reducing substances (Papadopoulos, 1989). The amino acids most likely to be degraded during excessive heat treatment are arginine, cysteine, lysine, serine and threonine (Pickford, 1992). Amino acids with reactive side chains such as lysine, arginine, tryptophan and histidine may link to reducing agents present in feed (Bender, 1978), as exemplified by the Maillard reaction between lysine and reducing sugars. Cysteine reacts readily during heat treatment to form disulphide bonds between cysteine units (Bender, 1978). When overheating fish meal, increased cross-linking between proteins occurs (Opstvedt et al., 1984), causing reduced digestibility of nearly all amino acids, especially cysteine (Andorsdo`ttir, 1985; Ljøkjel et al., 2000). With harsher heat treatment, when the water activity is low, and pro-oxidants like fish lipids are present, cysteine and methionine may be oxidized into their respective sulfones and sulfoxides (Opstvedt et al., 1984). Heating can also lead to racemization of amino acids, with loss in biological activity when the biologically active L-form is converted into the inactive Dform (Hurrell, 1984). Shear is the working and mixing action that homogenizes and heats the conveyed product (Riaz, 2000). Shear is the single factor varying most extensively among different extruders, depending on screw speed, screw configuration and barrel housing. In a shear field, weak hydrogen bonds keeping proteins together are broken, and the denatured protein aligns as an extended chain in order to attain the smallest possible profile (Ledward and Mitchell, 1988; Phillips, 1989). Since denatured proteins are more easily digested compared to the native globular structures, shear forces play an important role in changing the nutritive value of proteins (Bhattacharya and Hanna, 1988; Marsman et al., 1993). The present experiment was conducted in parallel with a mink trial (Ljøkjel and Skrede, 2000). The results obtained with mink showed that the amino acid composition of the feed was not affected by extrusion. However, the true digestibilities of crude protein and total amino acids were slightly reduced by extrusion. Furthermore, digestibility of crude protein and total amino acids decreased slightly by increasing extrusion temperature from 100 to 125 jC, but not by a further increase to 150 jC. The results from the experiment with mink also indicate that different conditioning and extrusion conditions used to achieve the target temperatures, especially total time of heat treatment, influenced the digestibility of
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protein and individual amino acids more than the extrusion temperature. The objective of the present experiment was to investigate the effects of extrusion temperatures on the digestibility of proteins, amino acids and energy in rainbow trout.
2. Materials and methods 2.1. Production of extruded diets Six diets were produced by processing the same dry ingredient mix (Table 1) at three different extrusion temperatures (100, 125 and 150 jC), achieved basically by varying different extrusion conditions referred to as method 1 and method 2 (Table 2). The dry ingredients were mixed in three separate batches, due to limited capacity of the mixer. ˚ s, Norway. The dry Feed production was carried out at the Center for Feed Technology, A ingredients were mixed in a Dinnissen (Pegasus Menger 400 l, Sevenum, Holland) twin shaft high-speed mixer for 5 min and conditioned in a Miltenz single shaft (501S, Millband Technology, Auckland, New Zealand) pre-conditioner with a temperature range of 38 – 73 jC. The conditioned feed mash was fed into a five head Bu¨hler (Ex 50/134 L, Uzwil, Switzerland) twin-screw extruder, with a 90 kW engine, L/D 20:1 and 4 mm die. Steam and water jackets on the barrel were used to stabilize the temperature. Processing temperature was measured behind the die using a thermo-couple. The feed was dried to approximately 92% dry matter in a Miltenz (VC010 Gas) counterflow drier. Fish oil (33 – Table 1 Formulation and composition of the diets Formulation, (kg feed)
1
a
Fish meal , g Wheat flourb, g Fish oilc, g Vitamin mixd, g Mineral mixe, g Inert markerf, mg Composition Dry matter (DM), g kg In DM, kg 1 Crude protein, g Crude fat, g Starch, g Ash, g Gross energy, MJ a
500 308 190 1 0.5 100
1
MeanFS.E.M., n=6 940F38 413F30 238F7 206F12 76F2 24F0.5
Norse LT-94, Norsildmel, Bergen, Norway. Milled 0 – 2%>1.11 mm, 55 – 75%<0.15 mm; falling number>200, Regal Mølle, Oslo, Norway. c NorSalmOil, Nordsildmel, Bergen, Norway. d Refstie et al. (1997). e Minerals, mg kg 1 feed: CuSO4, 48; KI, 2.0; MnSO4, 109.4; Na2SeO3, 0.4; ZnSO4, 257.1. Minerals supplied by Sigma, St Louis, MO, USA. f Yttrium oxide, Y2O3,>99.9% purity, Sigma. b
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Table 2 Extrusion parameters in production of the experimental dietsa Production method
Target temp1, jC
Die temp2, jC
Cond. temp3, jC
Torque, %
RPM4, %
Pressure, Bar
Feeding rate5, %
Water6, 1h 1
1 1 1 2 2 2
100 125 150 100 125 150
101 123 151 99 129 150
42 65 59 38 39 39
28 35 40 29 34 38
85 95 93 85 85 85
20 28 26 18 29 26
16 25 32 16 21 22
39 27 45 33 35 39
Temp1=Target temperature behind the die. Temp2=Mean achieved temperature measured behind the die. Temp3=Temperature in conditioner. RPM4=Revolutions per minute. Feeding rate5=Amount of dry feed blend fed to the extruder. Water6=Water added in to the extruder, 1 h 1. a Mean of five observations.
38 jC) was added using a Dinnisen vacuum coater at 0.2 Bar. Before bagging, the product was cooled in a Mu¨nch counterflow cooler (Mu¨nch-Edelstahl, Hilden, Germany). The target was to achieve the planned extrusion temperatures, and in order to stabilize the temperature rotation rate (rpm), torque (motor load), die-pressure, conditioning temperature, ingredient flow rate and liquid addition varied. According to method 1, the temperature of the conditioner, torque, screw speed, pressure, feed rate and process water of the extruder were varied (Table 2). With reference to method 2, conditioner temperature and screw speed were kept constant, and the range of variation in process water input was restricted. 2.2. Digestibility study The digestibility study was carried out with rainbow trout (Oncorhynchus mykiss) at ˚ s. the Department of Agricultural Engineering, the Agricultural University of Norway, A The experiment was conducted over a period of 4 weeks including 1 week of adaptation to the feed and experimental facilities. Prior to the experiment, the fish were fed a commercial diet, Ewos Aura 4 mm (Ewos, Florø, Norway) while the fish were stocked in one large tank. One week before the start up of the digestibility study, fish weighing 0.3– 0.5 kg were randomly distributed to fiberglass tanks, 20-kg trout/tank. The tanks were supplied with freshwater (8– 9 jC). The oxygen content of the water, measured in the tanks, averaged 7.5 mg l 1. The fish were fed a daily ration of 10 g feed kg 1 body weight, in two daily meals, using electrically driven disc feeders. In order to avoid overestimation of digestibility due to nutrient leakage from partly dissolved feeds, feed that was not consumed within 2 min was collected using a small-meshed dip net. At samplings, fish were caught with a dip net and anesthetized with MS-222 (60 mg l 1, in water). Collection of feces was carried out as described by Austreng (1978), using yttrium oxide (Y2O3) as the inert marker (Austreng et al., 2000). Feces (pooled by tank) were collected after 1 week of adaptation to the feed. Then the feeds were fed to trout in
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another tank and adapted to the new diet for 1 week before collection of feces. Thereafter, a final rotation and adaptation was carried out, and feces were collected for the third replicate. The experiment was originally designed with nine tanks and nine feeds (three extrusion temperaturesthree production methods). However, inclusion of the inert marker in feed for one of the production methods was heterogeneous, and these data were excluded from the statistical analysis. Thus, the statistical analysis includes data from six feeds (two production methodsthree temperatures) carried out in three replicates. 2.3. Chemical analyses Feed samples (0.5 kg) were homogenized in a coffee grinder. Feces were freeze-dried, homogenized with a pestle and mortar, and scales removed with forceps. Diets and feces were analysed for dry matter (to constant weight at 105 jC), ash (pre-combustion on a hot plate, followed by 550 jC to constant weight), crude protein (semi-micro-Kjeldahl, Kjeltec-Auto System, Tecator, Ho¨gena¨s, Sweden), crude fat by petroleum ether extraction, HCl-hydrolysis, and re-extraction (Fosstec, Tecator), and gross energy (Parr 1271 Oxygen Bomb Calorimeter, Parr, Moline IL, USA). Starch in feed was determined as glucose after hydrolysis by a-amylase (Novo Nordisk, Bagsvaerd, Denmark) and amylo-glucosidase (Boehringer Mannheim, Mannheim, Germany), followed by glucose determination by the ‘‘Gluc-DH method’’ (Merck, Darmstadt, Germany). Amino acids were determined by separation with ion exchange chromatography followed by post column reaction with ninhydrin and photometric detection at 570 nm (440 nm for proline) as described by Ljøkjel et al. (2000). Yttrium oxide and mineral elements were analyzed by inductivity coupled plasma (ICP) mass spectrometry as described by Refstie et al. (1997) and Shearer (1984), respectively. 2.4. Calculations and statistical analysis Apparent digestibility of protein, amino acids, energy and ash, and apparent absorption of mineral elements were calculated as: 100 (100 MF (MD) 1 ND (NF) 1), where M and N are marker and nutrient concentrations; while D and F represent diet and feces. Analysis of variance was conducted using the General Linear Models (GLM) procedure of the SAS computer software (SAS, 1990) using the following model: Yijklm=l+ai+bj+jk+cl+eijklm Yijklm=Observation number ijklm l=Overall mean a=Fixed effect of extrusion temperature, i=1,. . .,3 bj=Fixed effect of production method, j=1,2 jk=Fixed effect of tank, k=1,. . .,6 cl=Fixed effect of fecal collection day (replicate), l=1,. . .,3 eijklm=Random effect The results are presented as meansFstandard error means (S.E.M.).
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3. Results The planned extrusion temperatures, measured behind the die, were achieved independent of methods employed, except for the 125 jC-treatment using method 2, which was 4 jC higher than planned (Table 2). Feed rate was used to raise the temperature from 100 to 125 jC and thereafter from 125 to 150 jC, and rate of water addition to the barrel was used to stabilize the target temperature. From Table 2, it can be seen that water addition relative to the feeding rate using method 2 was slightly higher compared to the feeds produced by method 1. Torque and pressure both varied due to the different combinations of feeding rate and water additions at the different temperatures. Thus, the lowest torque and pressure occurred at 100 jC independent of methods. The proximate chemical composition of the diets (Table 1) and amino acid composition (Table 3) did not differ significantly among diets. Thus, composition values are pooled for all six diets in the tables. The fish appeared to be well adapted to the facilities and experimental diets when the experiment started. However, removal of uneaten feed from the tanks twice a day and repeated stripping appeared to stress the fish. Although feed intake was not recorded, visual observation of the uneaten feed indicated a reduction in appetite during the last part of the experiment. Dark coloration and increased mortality (2.5%) during the last 10 days of the experiment also indicated that handling might have stressed the fish.
Table 3 Amino acid composition of the dietsa Amino acids, g per 16 g N Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val Nonessential AA Ala Asp Cys Glu Gly Pro Ser Tyr a
Mean valueFS.E.M., n=6 feeds.
5.81F0.07 2.57F0.03 4.94F0.11 8.15F0.16 7.87F0.21 3.18F0.07 4.39F0.09 4.26F0.07 1.28F0.03 5.70F0.05
6.62F0.14 9.68F0.02 1.24F0.03 17.33F0.30 5.94F0.08 5.08F0.16 4.96F0.11 3.63F0.06
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Extrusion temperature did not affect the digestibilities of energy, ash, protein or amino acids significantly (Table 4). The apparent digestibility of crude protein, ash and energy did not differ significantly between the two feed production methods (Table 5). The only amino acid digestibility that differed significantly between production methods was that of cysteine, which was higher for feeds produced by method 1 compared to method 2. No significant differences were seen for digestibility of macronutrients between repeated collections of feces, but significant differences occurred for individual amino acids (Table 6). The digestibilities of cysteine, arginine and tryptophan were significantly lower for the two first fecal strippings than for the last strippings of feces. The digestibility of phenylalanine was significantly lower for the first than for the two subsequent fecal strippings, and the digestibility of histidine was lower for the first stripping than for the third stripping, whereas it was intermediate for the second stripping.
Table 4 Apparent digestibility of energy, ash, crude protein and amino acids in rainbow trout fed extruded diets produced at different temperaturesa Extrusion temperature, jC
100
125
150
Digestibility, % Energy Ash Crude protein
89.0F0.3 42.1F1.6 90.6F0.3
89.7F0.5 45.7F1.3 90.3F0.4
89.3F0.7 45.1F1.6 90.3F0.3
Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val
91.9F0.5 87.7F0.6 91.3F0.7 91.7F0.6 92.0F0.5 91.6F0.6 89.3F0.6 88.5F0.6 84.4F0.6 90.6F0.6
91.7F0.6 86.9F0.9 91.2F0.5 91.7F0.5 91.5F0.6 91.4F0.4 89.0F0.7 87.9F0.6 84.1F0.8 90.1F0.5
92.3F0.4 87.6F0.7 91.7F0.5 92.0F0.4 92.3F0.4 92.2F0.5 89.6F0.5 88.6F0.6 85.8F0.5 90.6F0.5
Nonessential Ala Asp Cys Glu Gly Pro Ser Tyr
91.4F0.6 80.2F1.1 75.6F1.9 93.9F0.3 83.4F1.1 90.4F0.3 87.9F0.8 87.1F0.8
91.2F0.6 79.5F1.0 76.3F1.6 93.5F0.4 83.6F1.0 90.2F0.7 87.8F0.8 86.0F1.3
91.7F0.5 81.2F0.9 77.3F1.2 93.8F0.4 85.3F0.6 90.7F0.4 88.6F0.6 87.0F0.8
a MeanFS.E.M., n=6 observations. No significant ( P<0.05) differences were seen among extrusion temperatures.
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Table 5 Apparent digestibility of energy, ash, crude protein and amino acids in rainbow trout fed extruded diets produced by the two production methods1 Production day
Method 1
Method 2
Digestibility, % Energy Ash Crude protein
89.2F0.4 43.8F1.5 90.1F0.2
89.5F0.5 44.8F1.1 90.7F0.2
Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val
92.1F0.3 87.9F0.5 91.7F0.4 92.2F0.3 92.1F0.4 92.4F0.4 89.7F0.3 88.9F0.4 84.8F0.5 90.7F0.4
92.0F0.4 87.1F0.6 91.2F0.5 91.5F0.4 91.8F0.4 91.3F0.3 89.1F0.5 88.0F0.5 84.8F0.7 90.3F0.4
Nonessential AA Ala Asp Cys Glu Gly Pro Ser Tyr
91.6F0.4 81.1F0.7 78.2F0.4a 94.0F0.3 84.7F0.5 90.7F0.3 88.8F0.5 87.1F0.7
91.3F0.4 79.8F0.8 75.0F1.3b 93.5F0.3 83.7F0.5 90.3F0.4 87.6F0.6 86.5F0.8
1 MeanFS.E.M., n=9 observations. Different superscripts (a, b) indicate significant ( P<0.05) differences between 2 production days.
No significant differences in apparent absorption of mineral elements ascribed to extrusion temperature, production method, stripping day, or tank were found. The apparent mineral absorptions are thus not presented.
Table 6 Apparent digestibility of amino acids determined on different fecal collection days1 Collection
1
2
3
Digestibility, % Arg Cys His Phe Trp
90.7F0.3a 72.2F1.6a 85.2F0.4a 87.1F0.3a 83.6F0.5a
91.9F0.2a 76.8F1.0a 88.8F0.3ab 89.7F0.3b 84.6F0.7a
93.0F0.2b 78.7F0.4b 87.5F0.4b 90.2F0.2b 86.2F0.5b
1 MeanFS.E.M., n=6 tanks. Different superscripts (a, b) indicate significant ( P<0.05) differences among fecal collection days.
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4. Discussion The lack of effect of extrusion temperature on apparent amino acid digestibilities in rainbow trout is to some extent in contrast to observations in mink. For the latter species, increasing extrusion temperature resulted in a slight, but significant difference in true digestibility of crude protein and individual amino acids (Ljøkjel and Skrede, 2000). Previous findings have shown that trout react to excessive heat treatment of feed ingredients by a reduction in overall digestibility of amino acids, especially cysteine due to the formation of disufide bonds (Opstvedt et al., 1984). This is in keeping with the response in mink (Ljøkjel et al., 2000) and Atlantic salmon (Ando`rsdo`ttir, 1985). The digestibility coefficients for protein and individual amino acids from different protein sources have also been ranked similarly when true digestibility coefficients were compared in mink and rainbow trout (Skrede et al., 1980), and by comparing true digestibilities in mink with apparent digestibilities in Atlantic salmon (Skrede et al., 1998). The trout in the present experiment and the mink used by Ljøkjel and Skrede (2000) were fed the same diets, thus interaction with different dietary ingredients was not a factor explaining the differences in protein and amino acid digestibilities between the two species. One plausible explanation might be that a marginal response in trout digestibility to different extruder temperatures could have been masked by endogenous losses of protein and amino acids. Another possible interpretation may be that the mink are more sensitive to heatinduced changes of amino acid digestibilities than the trout. The digestibility of cysteine was significantly lower in feeds produced by method 2, where water input was restricted and extrusion parameters were kept constant, compared to feeds produced by method 1, while no such difference was seen in the mink experiment (Ljøkjel and Skrede, 2000). Different experimental designs were applied in the two digestibility experiments. Ljøkjel and Skrede (2000) utilized diets from three production methods, while the trout in the present experiment were only fed the diets from two methods. Furthermore, the trout experiment was designed with three groups of trout behind each mean value, while the experiment with mink was done with four replicates. The standard errors ranged from 0.16 to 0.31 for true digestibilities of individual amino acids in mink, except from the digestibility of cysteine, which had a pooled S.E.M. at 0.68 (Ljøkjel and Skrede, 2000). The low standard errors for the digestibilities obtained with trout (Table 4), however, do not indicate that effects of increasing extrusion temperature were masked by lower test-power. Based on the main differences in processing conditions among the two production methods, the higher digestibility in rainbow trout of cysteine in the feed produced by method 1, may be ascribed to either increased feeding rate or more water added during feed processing, or a combination of the two. Increased feeding rate of the feed mixture contributes to increased torque, less viscous extrudate, and higher pressure in the extruder. It is more likely that the increased water addition was the main factor enhancing cysteine digestibility, since water acts as a lubricant during extrusion, thus reducing shear force. Formation of disulfide bridges and heat-induced oxidation of cysteine and methionine also become significant factors when fish meals are subjected to excessive drying (Opstvedt et al., 1984). In keeping with this, Obaldo et al. (1999; 2000) observed higher growth rate in shrimp with increasing level of extrudate moisture during dry extrusion and wet extrusion of shrimp diets. An overall recommendation is to maintain
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the moisture content at 25– 30% during extrusion of fish feed and pet food in order to prevent destruction of heat labile nutrients (Rokey, 1994). The apparent digestibility figures for five of the amino acids increased as the experiment progressed. The observed reduction in feed intake at the end of the experiment due to handling stress is a more plausible explanation for this than the adaptation to protein hydrolysis or amino acid absorption, or a reduction in endogenous amino acid excretion. Increased feed intake causes faster passage rate through the gastrointestinal tract, and may cause reduced protein digestibility when the trout are fed few and large daily meals (VensCappell, 1978), as in the present experiment. The lack of effects of the experimental parameters on the apparent absorption of ash and mineral elements indicates that variation in the extrusion parameters does not affect intestinal uptake of mineral elements from a fish meal based diet in rainbow trout. In conclusion, the lack of detectable destruction of amino acids and the digestibility results showed that extrusion represents a mild heat treatment of fish feed, even when the extrusion temperature is as high as 150 jC. Short retention time in the extruder and high moisture are the most plausible explanations to this. The results also indicate that the process variables other than the extrusion temperature, such as the amount of process water, may contribute more to the digestibility of nutrients than the temperature in the extruder.
Acknowledgements The experiment and the participation of Mette Sørensen were financially supported by The Norwegian Research Council (Grant No. 108917/10), and an internal grant from AKVAFORSK. Kari Ljøkjel was supported by an internal grant from the Agricultural University of Norway.
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Ljøkjel, K., Skrede, A., 2000. Effect of feed extrusion temperatures on digestibility of protein, amino acids and starch in mink. Scientifur 24 (4 IV-A), 3 – 6. Ljøkjel, K., Harstad, O.M., Skrede, A., 2000. Effect of heat treatment of soybean meal and fish meal on amino acid digestibility in mink and dairy cows. Anim. Feed Sci. Technol. 84, 83 – 95. Marsman, G.J.P., Gruppen, H., Poel, A.F.B., 1993. Effect of extrusion on the in-vitro digestibility of toasted and untoasted soybean meal. In: Poel, A.F.B.V.D., Huisman, J., Saini, H.S. (Eds.), Recent Advances of Research in Antinutritional Factors in Legumes Seeds. Pudoc, Wageningen, The Netherlands, pp. 461 – 465. Obaldo, L.G., Dominy, W.G., Ryu, G.H., 1999. Growth, survival, and diet utilization of Pacific white shrimp, Penaeus vannamei fed dry extruded diets with different levels of shear and moisture. J. Appl. Aquacult. 9, 47 – 58. Obaldo, L.G., Dominy, W.G., Ryu, G.H., 2000. Extrusion processing and its effect on aquaculture diet quality and shrimp growth. J. Appl. Aquacult. 10, 41 – 54. Opstvedt, J., Miller, R., Hardy, R.W., Spinelli, J., 1984. Heat-induced changes in sulfhydryl groups and disulfide bonds in fish protein and their effect on protein and amino acid digestibility in rainbow trout (Salmo gairdneri). J. Agric. Food Chem. 32, 929 – 935. Papadopoulos, M.C., 1989. Effect of processing on high-protein feedstuffs: a review. Biol. Wastes 29, 123 – 138. Phillips, R.D., 1989. Effect of extrusion cooking on the nutritional quality of plant proteins. In: Phillips, R.D., Finley, J.W. (Eds.), Protein Quality and the Effects of Processing. Marcel Dekker, New York, USA, pp. 218 – 246. Pickford, J.R., 1992. Effects of processing on the stability of heat labile nutrients in animal feeds. In: Garnsworthy, P.C., Haresign, W., Cole, D.J.A. (Eds.), Recent Advances in Animal Nutrition. Redwood Press, Melksham, UK, pp. 177 – 192. Refstie, S., Helland, S.J., Storebakken, T., 1997. Adaptation to soybean meal in diets for rainbow trout, Oncorhynchus mykiss. Aquaculture 153, 263 – 272. Riaz, M.N., 2000. Introduction to extruders and their principles. In: Riaz, M.N. (Ed.), Extruders in Food Applications. Technomic Publishing, Lancaster, UK, pp. 1 – 23. Rokey, G.J., 1994. Petfood and fishfood extrusion. In: Frame, N.D. (Ed.), The Technology of Extrusion Cooking. Blackie Academic & Professional, London, UK, pp. 144 – 190. SAS, 1990. Statistical Analysis System User’s Guide, vols. 1 and 2. Version 6, 4th edn. SAS Institute, Cary, NC, USA. Shearer, K.D., 1984. Changes in elemental composition of hatchery-reared rainbow trout, Salmo gairdneri, associated with growth and reproduction. Can. J. Fish. Aquat. Sci. 41, 1592 – 1600. ˚ ., Austreng, E., 1980. Digestibility of amino acids in raw fish flesh and meat-and-boneSkrede, A., Krogdahl, A meal for the chicken, fox, mink and rainbow trout. Z. Tierphysiol., Tierernaehr. Futtermittelkd. 43, 92 – 101. Skrede, A., Berge, G.M., Storebakken, T., Herstad, O.M., Aarstad, K.G., Sundstøl, F., 1998. Digestibility of bacterial protein grown on natural gas in mink, pig, chicken and Atlantic salmon. Anim. Feed Sci. Technol. 76, 103 – 116. Van der Poel, T.F.B., Blonk, J., van Zuilichem, D.J., van Oort, M.G., 1990. Thermal inactivation of lectins and trypsin inhibitor activity during steam processing of dry beans (Phaseolus vulgaris) and effects on protein quality. J. Sci. Food Agric. 53, 215 – 228. Vens-Cappell, B., 1978. Die Abha¨ngigkeit der Verdaulichkeit des Rohproteins, der Verdaulichkeut der Trochensubstanz und der FutterverWertung von der Futterungsintensita¨t bei der Regenbogenforelle (S. gairdneri Richardson). Fisher Teichwirt. 29, 126 – 133.
Apparent digestibility of protein, amino acids and energy in rainbow trout (Oncorhynchus mykiss) fed a fish meal based diet extruded at different temperatures M. Sørensen a, K. Ljøkjel b, T. Storebakken a,*, K.D. Shearer c, A. Skrede b a AKVAFORSK, Institute of Aquaculture Research AS, N-1432 A˚s Norway Department of Animal Science, Agricultural University of Norway, N-1432 A˚s Norway c Northwest Fisheries Science Center, NOAA/NMFS. 2725 Montlake Blvd E. Seattle, WA 98112 USA b
Received 5 July 2001; received in revised form 9 November 2001; accepted 12 November 2001
Abstract A fish meal/wheat flour based dry ingredient mix was extruded at three different temperatures (100, 125 and 150 jC), by varying extrusion conditions according to two different methods. Employing method 1, the temperature of the conditioner, torque, screw speed, pressure, feed rate and process water of the extruder were varied. Whereas in method 2, conditioner temperature and screw speed were kept constant, and the variation in process water input was restricted. The diets, which contained yttrium oxide as an inert maker, were fed to three replicate groups of rainbow trout. Feces for digestibility determination were obtained by stripping. The results showed that the differences in extrusion temperature caused no significant differences in apparent digestibilities of crude protein, individual amino acids or energy. The apparent digestibility of cysteine was significantly higher for diets produced by method 1 than by method 2. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rainbow trout; Extrusion-temperature; Digestibility
1. Introduction Most modern salmonid diets are produced by extrusion, which is a process where the feed is subject to mixing, shearing and heating under high pressure before the extrudate * Corresponding author. Tel.: +47-64-94-95-00; fax: +47-64-94-95-02. E-mail address: [email protected] (T. Storebakken). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 8 7 - 0
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finally is forced through a die. The feed may undergo reactions during processing that could be beneficial if the nutritional value is improved, or detrimental if nutrients are destroyed or become resistant to digestion. Reactions taking place in the feed during extrusion are largely determined by shear-forces, temperature, moisture, residence time and pH. In addition, the reactions depend on the type of reactants present, such as water, lipids, carbohydrates and proteins. Depending on the protein source, heat denaturing occurs over a temperature range of 25– 100 jC (Hultin, 1986). Denaturing is loss of quaternary, tertiary or secondary structure of the proteins while the primary structure remains intact. Thus, it causes conformational changes without affecting the amino acid composition of the protein. Moderate heating of protein-rich feed ingredients may be beneficial for the nutritional value of the feed proteins, since unfolded protein is often more readily digested than native protein (Camire, 1991). Vegetable protein sources may also contain heat labile anti-nutrients such as protease inhibitors and lectins, which are inactivated during heat treatment (Van der Poel et al., 1990). Damage to proteins during heat processing is a function of temperature, time, moisture and the presence of reducing substances (Papadopoulos, 1989). The amino acids most likely to be degraded during excessive heat treatment are arginine, cysteine, lysine, serine and threonine (Pickford, 1992). Amino acids with reactive side chains such as lysine, arginine, tryptophan and histidine may link to reducing agents present in feed (Bender, 1978), as exemplified by the Maillard reaction between lysine and reducing sugars. Cysteine reacts readily during heat treatment to form disulphide bonds between cysteine units (Bender, 1978). When overheating fish meal, increased cross-linking between proteins occurs (Opstvedt et al., 1984), causing reduced digestibility of nearly all amino acids, especially cysteine (Andorsdo`ttir, 1985; Ljøkjel et al., 2000). With harsher heat treatment, when the water activity is low, and pro-oxidants like fish lipids are present, cysteine and methionine may be oxidized into their respective sulfones and sulfoxides (Opstvedt et al., 1984). Heating can also lead to racemization of amino acids, with loss in biological activity when the biologically active L-form is converted into the inactive Dform (Hurrell, 1984). Shear is the working and mixing action that homogenizes and heats the conveyed product (Riaz, 2000). Shear is the single factor varying most extensively among different extruders, depending on screw speed, screw configuration and barrel housing. In a shear field, weak hydrogen bonds keeping proteins together are broken, and the denatured protein aligns as an extended chain in order to attain the smallest possible profile (Ledward and Mitchell, 1988; Phillips, 1989). Since denatured proteins are more easily digested compared to the native globular structures, shear forces play an important role in changing the nutritive value of proteins (Bhattacharya and Hanna, 1988; Marsman et al., 1993). The present experiment was conducted in parallel with a mink trial (Ljøkjel and Skrede, 2000). The results obtained with mink showed that the amino acid composition of the feed was not affected by extrusion. However, the true digestibilities of crude protein and total amino acids were slightly reduced by extrusion. Furthermore, digestibility of crude protein and total amino acids decreased slightly by increasing extrusion temperature from 100 to 125 jC, but not by a further increase to 150 jC. The results from the experiment with mink also indicate that different conditioning and extrusion conditions used to achieve the target temperatures, especially total time of heat treatment, influenced the digestibility of
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protein and individual amino acids more than the extrusion temperature. The objective of the present experiment was to investigate the effects of extrusion temperatures on the digestibility of proteins, amino acids and energy in rainbow trout.
2. Materials and methods 2.1. Production of extruded diets Six diets were produced by processing the same dry ingredient mix (Table 1) at three different extrusion temperatures (100, 125 and 150 jC), achieved basically by varying different extrusion conditions referred to as method 1 and method 2 (Table 2). The dry ingredients were mixed in three separate batches, due to limited capacity of the mixer. ˚ s, Norway. The dry Feed production was carried out at the Center for Feed Technology, A ingredients were mixed in a Dinnissen (Pegasus Menger 400 l, Sevenum, Holland) twin shaft high-speed mixer for 5 min and conditioned in a Miltenz single shaft (501S, Millband Technology, Auckland, New Zealand) pre-conditioner with a temperature range of 38 – 73 jC. The conditioned feed mash was fed into a five head Bu¨hler (Ex 50/134 L, Uzwil, Switzerland) twin-screw extruder, with a 90 kW engine, L/D 20:1 and 4 mm die. Steam and water jackets on the barrel were used to stabilize the temperature. Processing temperature was measured behind the die using a thermo-couple. The feed was dried to approximately 92% dry matter in a Miltenz (VC010 Gas) counterflow drier. Fish oil (33 – Table 1 Formulation and composition of the diets Formulation, (kg feed)
1
a
Fish meal , g Wheat flourb, g Fish oilc, g Vitamin mixd, g Mineral mixe, g Inert markerf, mg Composition Dry matter (DM), g kg In DM, kg 1 Crude protein, g Crude fat, g Starch, g Ash, g Gross energy, MJ a
500 308 190 1 0.5 100
1
MeanFS.E.M., n=6 940F38 413F30 238F7 206F12 76F2 24F0.5
Norse LT-94, Norsildmel, Bergen, Norway. Milled 0 – 2%>1.11 mm, 55 – 75%<0.15 mm; falling number>200, Regal Mølle, Oslo, Norway. c NorSalmOil, Nordsildmel, Bergen, Norway. d Refstie et al. (1997). e Minerals, mg kg 1 feed: CuSO4, 48; KI, 2.0; MnSO4, 109.4; Na2SeO3, 0.4; ZnSO4, 257.1. Minerals supplied by Sigma, St Louis, MO, USA. f Yttrium oxide, Y2O3,>99.9% purity, Sigma. b
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Table 2 Extrusion parameters in production of the experimental dietsa Production method
Target temp1, jC
Die temp2, jC
Cond. temp3, jC
Torque, %
RPM4, %
Pressure, Bar
Feeding rate5, %
Water6, 1h 1
1 1 1 2 2 2
100 125 150 100 125 150
101 123 151 99 129 150
42 65 59 38 39 39
28 35 40 29 34 38
85 95 93 85 85 85
20 28 26 18 29 26
16 25 32 16 21 22
39 27 45 33 35 39
Temp1=Target temperature behind the die. Temp2=Mean achieved temperature measured behind the die. Temp3=Temperature in conditioner. RPM4=Revolutions per minute. Feeding rate5=Amount of dry feed blend fed to the extruder. Water6=Water added in to the extruder, 1 h 1. a Mean of five observations.
38 jC) was added using a Dinnisen vacuum coater at 0.2 Bar. Before bagging, the product was cooled in a Mu¨nch counterflow cooler (Mu¨nch-Edelstahl, Hilden, Germany). The target was to achieve the planned extrusion temperatures, and in order to stabilize the temperature rotation rate (rpm), torque (motor load), die-pressure, conditioning temperature, ingredient flow rate and liquid addition varied. According to method 1, the temperature of the conditioner, torque, screw speed, pressure, feed rate and process water of the extruder were varied (Table 2). With reference to method 2, conditioner temperature and screw speed were kept constant, and the range of variation in process water input was restricted. 2.2. Digestibility study The digestibility study was carried out with rainbow trout (Oncorhynchus mykiss) at ˚ s. the Department of Agricultural Engineering, the Agricultural University of Norway, A The experiment was conducted over a period of 4 weeks including 1 week of adaptation to the feed and experimental facilities. Prior to the experiment, the fish were fed a commercial diet, Ewos Aura 4 mm (Ewos, Florø, Norway) while the fish were stocked in one large tank. One week before the start up of the digestibility study, fish weighing 0.3– 0.5 kg were randomly distributed to fiberglass tanks, 20-kg trout/tank. The tanks were supplied with freshwater (8– 9 jC). The oxygen content of the water, measured in the tanks, averaged 7.5 mg l 1. The fish were fed a daily ration of 10 g feed kg 1 body weight, in two daily meals, using electrically driven disc feeders. In order to avoid overestimation of digestibility due to nutrient leakage from partly dissolved feeds, feed that was not consumed within 2 min was collected using a small-meshed dip net. At samplings, fish were caught with a dip net and anesthetized with MS-222 (60 mg l 1, in water). Collection of feces was carried out as described by Austreng (1978), using yttrium oxide (Y2O3) as the inert marker (Austreng et al., 2000). Feces (pooled by tank) were collected after 1 week of adaptation to the feed. Then the feeds were fed to trout in
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another tank and adapted to the new diet for 1 week before collection of feces. Thereafter, a final rotation and adaptation was carried out, and feces were collected for the third replicate. The experiment was originally designed with nine tanks and nine feeds (three extrusion temperaturesthree production methods). However, inclusion of the inert marker in feed for one of the production methods was heterogeneous, and these data were excluded from the statistical analysis. Thus, the statistical analysis includes data from six feeds (two production methodsthree temperatures) carried out in three replicates. 2.3. Chemical analyses Feed samples (0.5 kg) were homogenized in a coffee grinder. Feces were freeze-dried, homogenized with a pestle and mortar, and scales removed with forceps. Diets and feces were analysed for dry matter (to constant weight at 105 jC), ash (pre-combustion on a hot plate, followed by 550 jC to constant weight), crude protein (semi-micro-Kjeldahl, Kjeltec-Auto System, Tecator, Ho¨gena¨s, Sweden), crude fat by petroleum ether extraction, HCl-hydrolysis, and re-extraction (Fosstec, Tecator), and gross energy (Parr 1271 Oxygen Bomb Calorimeter, Parr, Moline IL, USA). Starch in feed was determined as glucose after hydrolysis by a-amylase (Novo Nordisk, Bagsvaerd, Denmark) and amylo-glucosidase (Boehringer Mannheim, Mannheim, Germany), followed by glucose determination by the ‘‘Gluc-DH method’’ (Merck, Darmstadt, Germany). Amino acids were determined by separation with ion exchange chromatography followed by post column reaction with ninhydrin and photometric detection at 570 nm (440 nm for proline) as described by Ljøkjel et al. (2000). Yttrium oxide and mineral elements were analyzed by inductivity coupled plasma (ICP) mass spectrometry as described by Refstie et al. (1997) and Shearer (1984), respectively. 2.4. Calculations and statistical analysis Apparent digestibility of protein, amino acids, energy and ash, and apparent absorption of mineral elements were calculated as: 100 (100 MF (MD) 1 ND (NF) 1), where M and N are marker and nutrient concentrations; while D and F represent diet and feces. Analysis of variance was conducted using the General Linear Models (GLM) procedure of the SAS computer software (SAS, 1990) using the following model: Yijklm=l+ai+bj+jk+cl+eijklm Yijklm=Observation number ijklm l=Overall mean a=Fixed effect of extrusion temperature, i=1,. . .,3 bj=Fixed effect of production method, j=1,2 jk=Fixed effect of tank, k=1,. . .,6 cl=Fixed effect of fecal collection day (replicate), l=1,. . .,3 eijklm=Random effect The results are presented as meansFstandard error means (S.E.M.).
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3. Results The planned extrusion temperatures, measured behind the die, were achieved independent of methods employed, except for the 125 jC-treatment using method 2, which was 4 jC higher than planned (Table 2). Feed rate was used to raise the temperature from 100 to 125 jC and thereafter from 125 to 150 jC, and rate of water addition to the barrel was used to stabilize the target temperature. From Table 2, it can be seen that water addition relative to the feeding rate using method 2 was slightly higher compared to the feeds produced by method 1. Torque and pressure both varied due to the different combinations of feeding rate and water additions at the different temperatures. Thus, the lowest torque and pressure occurred at 100 jC independent of methods. The proximate chemical composition of the diets (Table 1) and amino acid composition (Table 3) did not differ significantly among diets. Thus, composition values are pooled for all six diets in the tables. The fish appeared to be well adapted to the facilities and experimental diets when the experiment started. However, removal of uneaten feed from the tanks twice a day and repeated stripping appeared to stress the fish. Although feed intake was not recorded, visual observation of the uneaten feed indicated a reduction in appetite during the last part of the experiment. Dark coloration and increased mortality (2.5%) during the last 10 days of the experiment also indicated that handling might have stressed the fish.
Table 3 Amino acid composition of the dietsa Amino acids, g per 16 g N Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val Nonessential AA Ala Asp Cys Glu Gly Pro Ser Tyr a
Mean valueFS.E.M., n=6 feeds.
5.81F0.07 2.57F0.03 4.94F0.11 8.15F0.16 7.87F0.21 3.18F0.07 4.39F0.09 4.26F0.07 1.28F0.03 5.70F0.05
6.62F0.14 9.68F0.02 1.24F0.03 17.33F0.30 5.94F0.08 5.08F0.16 4.96F0.11 3.63F0.06
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Extrusion temperature did not affect the digestibilities of energy, ash, protein or amino acids significantly (Table 4). The apparent digestibility of crude protein, ash and energy did not differ significantly between the two feed production methods (Table 5). The only amino acid digestibility that differed significantly between production methods was that of cysteine, which was higher for feeds produced by method 1 compared to method 2. No significant differences were seen for digestibility of macronutrients between repeated collections of feces, but significant differences occurred for individual amino acids (Table 6). The digestibilities of cysteine, arginine and tryptophan were significantly lower for the two first fecal strippings than for the last strippings of feces. The digestibility of phenylalanine was significantly lower for the first than for the two subsequent fecal strippings, and the digestibility of histidine was lower for the first stripping than for the third stripping, whereas it was intermediate for the second stripping.
Table 4 Apparent digestibility of energy, ash, crude protein and amino acids in rainbow trout fed extruded diets produced at different temperaturesa Extrusion temperature, jC
100
125
150
Digestibility, % Energy Ash Crude protein
89.0F0.3 42.1F1.6 90.6F0.3
89.7F0.5 45.7F1.3 90.3F0.4
89.3F0.7 45.1F1.6 90.3F0.3
Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val
91.9F0.5 87.7F0.6 91.3F0.7 91.7F0.6 92.0F0.5 91.6F0.6 89.3F0.6 88.5F0.6 84.4F0.6 90.6F0.6
91.7F0.6 86.9F0.9 91.2F0.5 91.7F0.5 91.5F0.6 91.4F0.4 89.0F0.7 87.9F0.6 84.1F0.8 90.1F0.5
92.3F0.4 87.6F0.7 91.7F0.5 92.0F0.4 92.3F0.4 92.2F0.5 89.6F0.5 88.6F0.6 85.8F0.5 90.6F0.5
Nonessential Ala Asp Cys Glu Gly Pro Ser Tyr
91.4F0.6 80.2F1.1 75.6F1.9 93.9F0.3 83.4F1.1 90.4F0.3 87.9F0.8 87.1F0.8
91.2F0.6 79.5F1.0 76.3F1.6 93.5F0.4 83.6F1.0 90.2F0.7 87.8F0.8 86.0F1.3
91.7F0.5 81.2F0.9 77.3F1.2 93.8F0.4 85.3F0.6 90.7F0.4 88.6F0.6 87.0F0.8
a MeanFS.E.M., n=6 observations. No significant ( P<0.05) differences were seen among extrusion temperatures.
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Table 5 Apparent digestibility of energy, ash, crude protein and amino acids in rainbow trout fed extruded diets produced by the two production methods1 Production day
Method 1
Method 2
Digestibility, % Energy Ash Crude protein
89.2F0.4 43.8F1.5 90.1F0.2
89.5F0.5 44.8F1.1 90.7F0.2
Essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val
92.1F0.3 87.9F0.5 91.7F0.4 92.2F0.3 92.1F0.4 92.4F0.4 89.7F0.3 88.9F0.4 84.8F0.5 90.7F0.4
92.0F0.4 87.1F0.6 91.2F0.5 91.5F0.4 91.8F0.4 91.3F0.3 89.1F0.5 88.0F0.5 84.8F0.7 90.3F0.4
Nonessential AA Ala Asp Cys Glu Gly Pro Ser Tyr
91.6F0.4 81.1F0.7 78.2F0.4a 94.0F0.3 84.7F0.5 90.7F0.3 88.8F0.5 87.1F0.7
91.3F0.4 79.8F0.8 75.0F1.3b 93.5F0.3 83.7F0.5 90.3F0.4 87.6F0.6 86.5F0.8
1 MeanFS.E.M., n=9 observations. Different superscripts (a, b) indicate significant ( P<0.05) differences between 2 production days.
No significant differences in apparent absorption of mineral elements ascribed to extrusion temperature, production method, stripping day, or tank were found. The apparent mineral absorptions are thus not presented.
Table 6 Apparent digestibility of amino acids determined on different fecal collection days1 Collection
1
2
3
Digestibility, % Arg Cys His Phe Trp
90.7F0.3a 72.2F1.6a 85.2F0.4a 87.1F0.3a 83.6F0.5a
91.9F0.2a 76.8F1.0a 88.8F0.3ab 89.7F0.3b 84.6F0.7a
93.0F0.2b 78.7F0.4b 87.5F0.4b 90.2F0.2b 86.2F0.5b
1 MeanFS.E.M., n=6 tanks. Different superscripts (a, b) indicate significant ( P<0.05) differences among fecal collection days.
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4. Discussion The lack of effect of extrusion temperature on apparent amino acid digestibilities in rainbow trout is to some extent in contrast to observations in mink. For the latter species, increasing extrusion temperature resulted in a slight, but significant difference in true digestibility of crude protein and individual amino acids (Ljøkjel and Skrede, 2000). Previous findings have shown that trout react to excessive heat treatment of feed ingredients by a reduction in overall digestibility of amino acids, especially cysteine due to the formation of disufide bonds (Opstvedt et al., 1984). This is in keeping with the response in mink (Ljøkjel et al., 2000) and Atlantic salmon (Ando`rsdo`ttir, 1985). The digestibility coefficients for protein and individual amino acids from different protein sources have also been ranked similarly when true digestibility coefficients were compared in mink and rainbow trout (Skrede et al., 1980), and by comparing true digestibilities in mink with apparent digestibilities in Atlantic salmon (Skrede et al., 1998). The trout in the present experiment and the mink used by Ljøkjel and Skrede (2000) were fed the same diets, thus interaction with different dietary ingredients was not a factor explaining the differences in protein and amino acid digestibilities between the two species. One plausible explanation might be that a marginal response in trout digestibility to different extruder temperatures could have been masked by endogenous losses of protein and amino acids. Another possible interpretation may be that the mink are more sensitive to heatinduced changes of amino acid digestibilities than the trout. The digestibility of cysteine was significantly lower in feeds produced by method 2, where water input was restricted and extrusion parameters were kept constant, compared to feeds produced by method 1, while no such difference was seen in the mink experiment (Ljøkjel and Skrede, 2000). Different experimental designs were applied in the two digestibility experiments. Ljøkjel and Skrede (2000) utilized diets from three production methods, while the trout in the present experiment were only fed the diets from two methods. Furthermore, the trout experiment was designed with three groups of trout behind each mean value, while the experiment with mink was done with four replicates. The standard errors ranged from 0.16 to 0.31 for true digestibilities of individual amino acids in mink, except from the digestibility of cysteine, which had a pooled S.E.M. at 0.68 (Ljøkjel and Skrede, 2000). The low standard errors for the digestibilities obtained with trout (Table 4), however, do not indicate that effects of increasing extrusion temperature were masked by lower test-power. Based on the main differences in processing conditions among the two production methods, the higher digestibility in rainbow trout of cysteine in the feed produced by method 1, may be ascribed to either increased feeding rate or more water added during feed processing, or a combination of the two. Increased feeding rate of the feed mixture contributes to increased torque, less viscous extrudate, and higher pressure in the extruder. It is more likely that the increased water addition was the main factor enhancing cysteine digestibility, since water acts as a lubricant during extrusion, thus reducing shear force. Formation of disulfide bridges and heat-induced oxidation of cysteine and methionine also become significant factors when fish meals are subjected to excessive drying (Opstvedt et al., 1984). In keeping with this, Obaldo et al. (1999; 2000) observed higher growth rate in shrimp with increasing level of extrudate moisture during dry extrusion and wet extrusion of shrimp diets. An overall recommendation is to maintain
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the moisture content at 25– 30% during extrusion of fish feed and pet food in order to prevent destruction of heat labile nutrients (Rokey, 1994). The apparent digestibility figures for five of the amino acids increased as the experiment progressed. The observed reduction in feed intake at the end of the experiment due to handling stress is a more plausible explanation for this than the adaptation to protein hydrolysis or amino acid absorption, or a reduction in endogenous amino acid excretion. Increased feed intake causes faster passage rate through the gastrointestinal tract, and may cause reduced protein digestibility when the trout are fed few and large daily meals (VensCappell, 1978), as in the present experiment. The lack of effects of the experimental parameters on the apparent absorption of ash and mineral elements indicates that variation in the extrusion parameters does not affect intestinal uptake of mineral elements from a fish meal based diet in rainbow trout. In conclusion, the lack of detectable destruction of amino acids and the digestibility results showed that extrusion represents a mild heat treatment of fish feed, even when the extrusion temperature is as high as 150 jC. Short retention time in the extruder and high moisture are the most plausible explanations to this. The results also indicate that the process variables other than the extrusion temperature, such as the amount of process water, may contribute more to the digestibility of nutrients than the temperature in the extruder.
Acknowledgements The experiment and the participation of Mette Sørensen were financially supported by The Norwegian Research Council (Grant No. 108917/10), and an internal grant from AKVAFORSK. Kari Ljøkjel was supported by an internal grant from the Agricultural University of Norway.
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