Aquaculture Nutrition

Transcription

1 Aquculture Nutrition ; doi: /j x 1 1,2 1 1 Ntionl Institute of Nutrition nd Sefood Reserch (NIFES), Nordnes, Bergen, Norwy; 2 Ewos AS, Bergen, Norwy Four het cogulted erly wening diets with incresing concentrtions of pepsin hydrolysed protein, were investigted with regrd to the chnge in protein qulity during feed production nd exposure to leching. Wter-soluble N, trichlorocetic cid-soluble N nd mino cid (AA) profiles were determined in finished diets nd in diets leched for 6 min. In vitro diet digestibility ws mesured nd relted to incresing inclusion of hydrolysed protein nd N lekge. Seventeen to 47% of soluble N in the feed ingredients ws mde insoluble by het denturtion during feed production, but the concentrtion of peptides nd free mino cids (FAA) were not influenced. All peptides/faa nd 70 80% of wter-soluble protein were lost fter exposure to leching. Incresed inclusion of hydrolysed protein incresed the loss of crude protein (15 30%). All turine nd 30% of histidine ws lost during leching, no other mjor chnges in AA profile were found. There ws no difference in digestibility between diets exposed to leching. However, leched diets showed reduced digestibility s compred to diets tht hd not been exposed to leching. In conclusion micro-bound type diets s used in this study hve low efficiency in delivering soluble N to fish lrve nd should be crefully considered for this purpose. KEY WORDS: fish lrve, formulted diet, in vitro digestibility, protein hydrolyste, protein qulity Received 27 Februry 2008, ccepted 2 June 2008 Correspondence: A. Nordgreen, Ntionl Institute of Nutrition nd Sefood Reserch (NIFES), Post-box 2029, Nordnes, 5817 Bergen, Norwy. E-mil: ndres.nordgreen@nifes.no Mrine fish lrve hve gret growth potentil (Houde 1989; Kmler et l. 1992; Conceico et l. 1997) nd even in cold wter species such s the Atlntic cod (Gdus morhu) specific growth rtes exceeding 25% dy )1 hve been reported (Otterlei et l. 1999). The lrvl body is len nd growth is therefore minly the result of protein deposition (Houlihn et l. 1995). Accordingly, both high qulity nd quntity of dietry protein re required. At n erly stge lrve lck functionl stomch (Govoni et l. 1986; Pittmn et l. 1990; Segner et l. 1994) nd my hve low digestive enzyme ctivity (Cousin et l. 1987; Kvåle et l. 2007). Lrvl bility to digest complex protein hs thus been questioned (Tonheim et l. 2004). Tube feeding showed tht lrvl Atlntic Hlibut (Hippoglossus hippoglossus) hd higher bsorption efficiency of hydrolysed protein s compred to intct soluble protein (Tonheim et l. 2005). Stomchless fish lrve fed formulted diets hve reduced growth nd survivl compred to lrve fed live feed (Howell et l. 1998; Chu & Zmbonino-Infnt 2001; Kolkovski et l. 2001). The high concentrtion of wter-soluble nitrogen (N) (copepods 54 ± 2%, rotifers 56.9 ± 0.8% nd Artemi 54 ± 0.4% of totl N, respectively, Tonheim et l. 2007; Srivstv et l. 2006; Crvlho et l. 2003) nd high concentrtion of low moleculr weight N (Crvlho et l. 2003; Tonheim et l. 2007) in live feed compred to formulted diets, is suggested explntion for the reduced growth nd survivl reported from trils with stomchless fish lrve fed on formulted feeds (Crvlho et l. 2004). Crvlho et l. (2003) showed tht nerly 90% of the soluble N in rotifers nd Artemi ws less thn 500 D in size. To mimic the free N pool of live feed nd get improved growth nd survivl, free mino cids (FAA) or hydrolysed protein hve been dded to formulted feeds for erly stge lrve (Crvlho et l. 1997, 2004; Chu et l. 1999; Hmre et l. 2001; Kvåle et l. 2002). Although results re not lwys consistent, low inclusion levels of hydrolysed protein in diets hve been shown to improve survivl nd growth (Zmbonino-Infnte et l. 1997; Chu et l. 1999; Crvlho et l. 2004) while higher inclusion levels hve shown negtive effects on lrvl performnce (Sprus urt L., Kolkovski &... Ó 2008 Blckwell Publishing Ltd

2 Tndler 2000; Cyprinus crpio, Crvlho et l. 1997, 2004; Dicentrrchus lbrx, Chu et l. 1999; H. hippoglossus Kv le 2007). Supplementtion of 40% pepsin hydrolysed protein to het cogulted diet (Hmre et l. 2001) improved survivl rtes in cod (G. morhu) lrve, compred to lower levels of supplementtion (Kv le 2007). On the other hnd, Atlntic Hlibut (H. hippoglossus) juveniles fed identicl experimentl diets showed declining survivl rte s the inclusion level of hydrolysed protein incresed (Kv le 2007). It is suggested tht the reduced survivl in hlibut fed incresed levels of hydrolysed protein is cused by their typicl slow feeding behviour (Stoss et l. 2004; Kv le 2007). Feed will therefore sty in the wter for longer time before being eten compred to species with more ggressive feeding behviour. The loss due to leching of wtersoluble nitrogenous compounds will be correspondingly higher nd proportionl to the inclusion level of hydrolysed protein. This will consequently result in lowered levels of both totl protein nd wter-soluble N in consumed feed prticles, which will in turn ffect the mount of digestible protein nd mino cids vilble for the lrve. Loss of low moleculr weight nutrients such s FAA nd peptides re severe in lmost ll tested formulted lrvl diets supplemented with such hydrolysed protein (Lopez Alvrdo et l. 1994; Kv le et l. 2006). The loss of FAA ws s high s 90% within less thn 2 min in diets tested by Crvlho et l. (2004). Experimentl diets hve been developed which to some extent cn reduce the loss of low moleculr weight nutrients during feeding (Villmr & Lngdon 1993; Lopez Alvrdo et l. 1994; Yufer et l. 2002; Onl & Lngdon 2005). However, nerly ll studies conducted to investigte the nutritionl effect of hydrolysed protein on lrve of different fish species hve been crried out with micro-bound or ground diets tht ll hve in common rpid leching of soluble compounds (see review by Lngdon 2003). In ddition to high loss during feeding, wter-soluble protein is susceptible to loss or modifiction during production, such s denturtion (de Wet 1983; Boye et l. 1997; Mohmmed et l. 2000), crosslinking (Jones et l. 1974; Lngdon 1989; Yufer et l. 1996), Millrd rections (Plks et l. 1985; Deng et l. 2005) nd leching (Nordgreen et l. 2007). The feed production process cn therefore significntly chnge protein qulity nd reduce the diet digestibility nd thereby msk ny biologicl effects of incresed supplementtions of hydrolysed protein. When investigting the effect of such supplementtions to formulted diets, it is thus of gret importnce to investigte the effect of production process nd leching on the remining protein qulity. The present study ws designed to investigte chnges in protein qulity due to production process nd leching in four diets with incresing levels of hydrolysed protein. A het cogulted diet ws used (Hmre et l. 2001), tht hd previously been used for wening of cod nd Atlntic hlibut lrve in severl studies (Hmre et l. 2001, 2002, 2003, 2005; Kv le et l. 2002; Moren et l. 2004). The diet hd lso been used in severl studies to investigte possible effects of hydrolysed protein on performnce of Atlntic hlibut lrve nd Atlntic cod lrve (Kvåle et l. 2002; Kvåle 2007). To understnd the chnges in protein qulity nd biovilbility during production nd leching of diets, the present study investigted chnges in crude protein, wter-soluble N, trichlorocetic cid (TCA)-soluble N (FAA nd peptides), mino cid profile nd in vitro digestibility. Four het cogulted diets bsed on fresh cod fillet nd incresing concentrtions of pepsin hydrolysed cod fillet s the mjor protein sources were produced t the Ntionl Institute of Nutrition nd Sefood Reserch (NIFES), Bergen, Norwy, s described by Hmre et l. (2001). Different inclusion levels of pepsin hydrolysed cod fillet represented 0, 15, 30 nd 45% of diet totl protein content (Tble 1). Feed ingredients were blended in high speed cutter to thick dough nd finger thick strings of the feed blend were fed onto conveyor belt. The feed ws first heted Tble 1 Composition of the formulted diets investigted in this study (g kg )1 dry weight) Diets Ingredients (dw) g kg )1 gkg )1 gkg )1 gkg )1 Suprex whet Squid mntle Cod fillet Pepsin hydrolysed cod fillet Fish oil Lecithin Vitmin mixture Minerls Suprex whet. 2 Fresh frozen squid mntle. 3 Fresh cod. 4 See description in Mterils nd methods. 5 Epx A/S, Lysker, Norwy. 6 Soy lecithin, Norsk Medisinldepot, Bergen, Norwy. 7 As recommended by NRC (1993) except for lf-tocopheryl cette supplemented t 200 mg kg )1 nd vitmin C t 400 mg kg )1. 8 As recommended by NRC (1993)....

3 using electromgnetic energy nd then dried in wrm ir tunnel dryer over night (with immobile conveyor; 60 C). Therefter pellets were crushed nd sieved. The size frction sieved through 600 lm sieve nd collected on 300 lm sieve ws studied. Pepsin hydrolysis ws crried out ccording to the method described by Kv le et l. (2002). Fresh cod fillet ws minced nd the ph ws lowered to 4.2 by ddition of 6 M HCl. A solution consisting of pepsin (7.5 g kg )1 mince; Sigm- Aldrich Corp., St Louis, MO, USA), glycerol (93 ml kg )1 mince; Merck, Drmstdt, Germny) nd distilled wter (173 ml kg )1 mince) ws mixed into the minced fish solution nd incubted t room temperture for 24 h under continuous shking. Therefter the ph ws rised to 7.2 by ddition of 5 M NOH. The hydrolysed solution ws frozen ()30 C) until use. The enzyme ws inctivted by het during feed production. Prticle size ws evluted by tking pictures under the microscope (Olympus BX 51, Tokyo, Jpn) t 40 mgnifiction. The width nd length of minimum of 90 feed prticles of ech diet produced were mesured with Olympus DP-soft. Centrifuge 5415 C) for 9 min. Finlly 350 ll of superntnt ws dded to tin cpsules (Smooth wll, Round bse; Elementl Micronlysis Limited) nd nlysed for totl N by totl combustion. To investigte the difference in leching between the four diets, five prllel smples with mg of ech diet were suspended in tubes contining 1.5 ml of phosphte buffer for 6 min. Tubes were rotted slowly for 6 min to keep feed in suspension. Tube contents were then filtered onto 0.65 lm N free Durpore membrne filter (25 mm). Filters were wrpped in tin foil cones (Elementl Micronlysis Limited) nd nlysed for crude protein (N 6.25) by totl combustion. The loss of N ws clculted by dividing the N content in the leched smples with the nlysed N content in the preleched smples. To clculte the remining concentrtion of TCA soluble N fter leching the diet ws lyophilized before 30 mg of the ech diet (N = 4) were weighed into 1.8 ml Eppendorf tubes, precipitted with TCA nd nlysed s described bove. Crude protein (N 6.25), wter-soluble N nd trichlorocetic cid (TCA) soluble N were mesured in ll rw mterils nd the concentrtions of the different N frctions in the rw mteril blends were clculted bsed on results from Tonheim et l. (2007). Crude protein, wter-soluble N nd TCA soluble N were lso nlysed in the four diets. For nlysis of wter-soluble N, diets were crushed nd mg ws weighed into 1.8 ml Eppendorf tubes. A volume of 1 ml of 80 mm phosphte buffer (ph 8) ws dded nd tubes where shken vigorously on shking tble for 5 h. Tubes were then centrifuged t 8000 rpm (Eppendorf Centrifuge 5415 C, Hmburg, Germny) for 9 min. The superntnt ws removed nd the pellet ws wshed nd centrifuged twice in phosphte buffer. The pellet ws trnsferred to evporting tinfoil cups (5 ml, Elementl Micronlysis Limited, Cmbridge, UK) nd residues in the tube were rinsed into the tinfoil cups. The tinfoil cups were dried t 65 C overnight before nlysing for crude protein (N 6.25) by totl combustion. TCA soluble N ws nlysed by weighing 30 mg of ech of the four diets (n = 4) into 1.8 ml Eppendorf tubes. A volume of 1.1 ml of phosphte buffer ws dded nd tubes were shken vigorously before 250 ll of TCA (40%) ws dded to ech smple. Smples were incubted t 4 C until the next dy before centrifugtion t 8000 rpm (Eppendorf... The protocol for in vitro digestion used in the present study ws bsed on the methods described by Hsu et l. (1977) nd Sterlee et l. (1979) nd ws performed ccording to the modified method described by Tonheim et l. (2007). Diet smples equivlent to 20 mg of crude protein were suspended in 1 ml of phosphte buffer in 1.8 ml Eppendorf tubes. A mixture of trypsin (type IX, bovine pncres) chymotrypsin (type II, bovine pncres) nd bcteril protese (type XIV, strepomyces griseus), ll obtined from Sigm Aldrich, MO, USA were dded to ech tube to finl concentrtions of 73, 145 nd 64 lg ml )1 phosphte buffer, respectively. Digestion ws performed t room temperture (22 C). Smples were plced on shking tble t low speed during the digestion period. Undigested protein ws precipitted nd the in vitro digestion terminted by dding 250 ll of TCA (40%). For ech nlysed diet nd tretment four prllel tubes were nlysed t 0 h, ½ h, 1 h nd 12 h, respectively. All tubes were centrifuged nd the crude protein content in the superntnt (350 ll), contining proteolysis products, i.e. FAA nd smll peptides, ws nlysed nd regrded s representing digested protein. Ech smple of superntnt (350 ll) ws dded to tin cpsule (Smooth wll, Round bse; Elementl Micronlysis Limited) nd nlysed for totl N by totl combustion. Crude protein (N 6.25) in the

4 precipitte, contining intct protein nd lrge peptides, ws regrded s representing undigested protein. To remove wter-soluble N from the four diets prior to in vitro digestibility, 1 g of ech diet ws suspended in 10 ml phosphte buffer nd shken for 2 min. Tubes were centrifuged t 3220 g for 30 s nd the superntnt ws removed. This ws repeted once; the feed ws in solution for pproximtely 6 min to equl the leching study. The leched diets were frozen nd freeze dried nd sub smple ws nlysed for N content before the in vitro digestibility studies. Chnge in mino cid (AA) profile due to leching nd in vitro digestibility ws nlysed with the diet contining 30% hydrolysed protein (Tble 2). The diet ws leched ccording to the method described bove. The preleched nd leched diets were nlysed in triplicte. To investigte the AA profile of the digested diet, the preleched nd leched diets were in vitro digested in triplicte for 12 h ccording to method described. Smples of the superntnts were nlysed for AA content. N ws determined by totl combustion using nitrogen nlyser (Leco FP-528, St Joseph, MI, USA). Crude protein ws clculted s N Totl AA profile ws nlysed ccording to the method described by Cohen et l. (1989). Smples were hydrolysed in 6 M HCl for 22 h t 110 C. The hydrolysed solution contining free AA ws then nlysed using the Wters HPLC nlyser system (Pico Tg) fter prederivtiztion with phenyl isothiocynte (PITC) using norleucine s internl stndrd. AA were detected by UV t 254 nm nd identified by retention time. Tryptophn is degrded during cidic hydrolysis nd is therefore not nlysed. protein content (N 6.25), wter-soluble N nd TCA soluble N dt were nlysed by T-test. The effects of the production process on wter-soluble protein content nd TCA soluble content in different diets were nlysed by regression nlysis long with one-wy nlysis of vrince (ANOVA; Sokl & Rohlf 1969) followed by TukeyÕs HSD multiple comprison test. Chnge in AA profile due to production process, leching nd in vitro digestibility ws nlysed by one-wy nlysis of vrince (ANOVA; Sokl & Rohlf 1969) followed by TukeyÕs multiple comprison test. The effect of incresed inclusion of hydrolysed protein on prticle size ws nlysed by regression nlysis long with one-wy ANOVA. Mens with P vlues less thn 0.05 were considered significntly different. All sttisticl nlyses were performed using STATISTICA 7.1 (Sttsoft Inc., Tuls, OK, USA). Pepsin hydrolysis of the cod fillet led to 91% increse in wter-soluble N (Tble 2) nd 122% increse in TCA soluble N (Tble 2). Due to the crushing nd sieving process most of the prticles were rod shped (Fig. 1) nd it ws therefore necessry to mesure both width nd length of the prticles. The prticles were nerly twice s long s they were wide nd hd men length 593 ± 184 lm nd men width of 320 ± 81 lm. There ws no significnt correltion (R 2 = 0.006, P = 0.35) between incresed hydrolysed protein nd prticle size nd no significnt difference (ANOVA, P > 0.05) in prticle size between ny of the diets. Dt re expressed s mens ± stndrd devition. To investigte the effect of pepsin hydrolysis on chnge in crude Tble 2 Anlysis of the different N frctions of minced cod fillet before nd fter pepsin hydrolysis (mens ± SD, n =3) Wter-soluble N % of tot N TCA soluble N % of tot N Cod fillet 34 (n = 1) 18 ± 2.4 Pepsin hydrolysed cod fillet 65 ± 3 40 ± 4.2 Figure 1 Smple of diet contining 15% hydrolysed protein. The picture is tken with differentil interference contrst (DIC) (Olympus BX 51; mgnifiction 40 )....

5 There ws negtive correltion (R 2 = 0.91, P < , b = )0.96) between concentrtion of hydrolysed protein nd crude protein (N 6.25) in the four diets (Fig. 2) with 5% reduction in crude protein from the diet contining 0% hydrolysed protein to the diet with 45% hydrolysed protein (Fig. 2). Leching ws positively correlted (R 2 = 0.96, P = 0.000, b = 0.982) with inclusion of hydrolysed protein. Loss of crude protein rnged from 14.9 ± 0.5% to 30.3 ± 0.4% of totl crude protein from the diet contining 0% hydrolysed protein to the diet contining 45% hydrolysed protein (Fig. 2). The clculted increse in wter-soluble N in the rw ingredients, with n increse of hydrolysed protein from 0 to 45% hydrolysed protein, ws 38% (Fig. 3). Incresed inclusion of hydrolysed protein ws positively correlted (R 2 = 0.89, P < , b = 0.943) with concentrtion of wter-soluble N in the diet (Fig. 3), but there ws significnt decrese (R 2 = 0.73, P < , b = )0.86) in reltive loss (%) of wter-soluble N with inclusion of hydrolysed protein during feed production (Fig. 3). The loss of wter-soluble N due to het denturtion rnged from 46.8 ± 2.7% to 17.4 ± 2.4% Crude protein ( %) Crude protein (produced diet) Loss of crude protein (leched 6 min) Totl weight loss (leched 6 min) b b Hydrolyzed protein in diet (%) Figure 2 Grey br is concentrtion of crude protein (N 6.25) in for four diets with incresing inclusion of pepsin hydrolysed protein rnging from 0 to 45% of the totl protein (n = 3). The white spotted brs re loss of crude protein (N 6.25) in the sme diets fter 6 min leching (n = 5). The blck spotted brs re loss (%) of totl weight fter 6 min leching (n = 4). The weight loss from the diet contining 0 nd 45% hydrolysed protein hs n = 1 nd sttistic nlysis is therefore not performed. Otherwise error brs represent stndrd devitions. Significnt differences between the diets re shown by different letters (ANOVA, followed by Tukey HSD, P < 0.05).... c c b d d % wter-soluble N of totl N Rw ingredients Produced diet Leched diet (6 min) b b Hydrolyzed protein in diet (%) Figure 3 Concentrtion of wter-soluble N in rw ingredients (clculted), produced diet (n = 4) nd diets leched for 6 min (n =5) for four diets with inclusion of pepsin hydrolysed protein rnging from 0 to 45% of the totl protein. Error brs represent stndrd devitions. Significnt differences between the diets re shown by different letters (ANOVA, followed by Tukey HSD, P < 0.05). of wter-soluble N from the diet contining 0% hydrolysed protein to the diet contining 45% hydrolysed protein (Fig. 3). Totl loss of wter-soluble N due to leching ws positively correlted with concentrtion of hydrolysed protein (Fig. 3), nd the remining concentrtion of wter-soluble N rnged from 3.0 ± 0.5% wter-soluble N for the diet contining 0% hydrolysed protein to 11.0 ± 0.5% wter-soluble N for the diet contining 30% hydrolysed protein (Fig. 3). There ws no correltion between concentrtion of hydrolysed protein nd reltive loss of wter-soluble N by leching nd the reltive loss of wter-soluble N rnged from minimum of 70% to mximum of 83% of totl soluble N in the respective diets. The rw ingredients hd 77% increse in TCA soluble protein from the diet contining 0% hydrolysed protein to the diet contining 45% hydrolysed protein (Fig. 4). The concentrtion of TCA soluble N ws not ffected by feed production (Fig. 4) nd the produced diets hd significnt increse (R 2 = 0.9 P < , b = 0.949) in TCA soluble N from 10.6 ± 0.6% of totl N to 21.6 ± 0.9% of totl N (Fig. 4). There ws n lmost complete loss of TCA soluble N due to 6 min leching nd no significnt difference (P>0.05) in concentrtion of retined TCA soluble N between diets (Fig. 4). There ws significnt increse (ANOVA, P < 0.05) in digestibility with time for both the leched nd unleched c d d c

6 %TCA soluble N of totl N Rw ingredients Produced diet Leched diet (6 min) b Hydrolized protein in diet (%) Figure 4 Concentrtion of TCA soluble N in diets with incresing inclusion of pepsin hydrolysed protein rnging from 0 to 45% of the totl protein. The concentrtion of TCA soluble N is investigted in the rw ingredients (clculted) the produced diets (n = 4) nd in the diets fter 6 min leching (n = 4). Error brs represent stndrd devitions. Significnt differences between the diets re shown by different letters (ANOVA, followed by Tukey HSD, P < 0.05). diets (Fig. 5). The unleched diets ll hd significntly higher (ANOVA, P < 0.05) digestibility t ll investigted time intervls thn the leched diets (Fig. 5). Incresed digestibility correlted with incresed concentrtion of hydrolysed protein for time 0 h, ½ h nd 1 h for the unleched diets nd there ws no significnt difference in digestibility between the four diets t 12 h in vitro digestibility (Fig. 5). For the leched diets, no significnt differences (ANOVA, P 0.05) in digestibility between the diets t ny of the investigted time intervls were found (Fig. 5). % TCA soluble N b c c 0 % 15 % 30 % 45 % Not-leched b b b c 0h 1/2 h 1h 12 h 0 h 1/2 h 1h 12 h c Digestion (h) Leched c Figure 5 In vitro digestibility of protein in four diets with incresing concentrtion of hydrolysed protein rnging from 0 to 45% of the totl protein. In vitro digestibility ws investigted for both not leched nd leched (6 min) diets. The digestibility ws investigted t four time intervls from 0 h to 12 h (n = 4). Error brs represent stndrd devitions. Significnt differences between the diets t ech time intervl re shown by different letters (ANOVA, followed by Tukey HSD, P < 0.05). There were significnt chnges in AA profile due to leching for 6 min, but the only severe chnge ws 100% loss of turine nd 30% loss of histidine (Fig. 6). No significnt (P 0.05) differences in AA profile were found compring the preleched diet with the digested preleched diet nd the leched diet with the digested leched diet (Fig. 6). The significnt difference in the AA profile between the in vitro digested preleched nd leched diet ws consequence of the chnges occurring during the leching process (Fig. 6). These experiments were performed to evlute chnges in protein qulity s consequence of production procedures nd leching in het cogulted diet (Hmre et l. 2001) with incresing inclusion levels of prehydrolysed protein. The results clerly indicted tht both the production process nd leching cn drmticlly chnge the protein qulity nd could possibly msk the biologicl effects of including high concentrtion of hydrolysed protein. Different lrvl diets hve shown to hve different leching rtes (Lopez Alvrdo et l. 1994; Kv le et l. 2006), the findings of this study re vlid for micro-bound type diets, but my differ with other diet production technologies such s microencpsulted diets. Considering the concentrtion of soluble N in the rw ingredients, there were reltively smll differences in soluble N between the four diets fter exposure to wter for 6 min. For TCA soluble N there were no differences between the four diets fter exposure to wter. This shows the difficulties in ccomplishing feeding studies to find the optiml concentrtion of hydrolysed protein for different species of mrine fish lrve nd could explin the vrying results in feeding trils with formulted diets contining different concentrtions of hydrolysed protein, s suggested by Kv le et l. (2006). Fresh cod fillet hs reltively high initil concentrtion of soluble N compred to commonly used feed ingredients such s het processed fish mel (Tonheim et l. 2007). Pepsin hydrolysis of cod fillet leds to doubling in concentrtion of both wter-soluble N, from 34 (n = 1) to 65 ± 3%, nd TCA soluble N, from 18 ± 2.4 to 40 ± 4.2% of totl N. Tonheim et l. (2007) showed tht 12 h pncretic hydrolysis of cod fillet gve product with 79 ± 9.1% TCA soluble N while pepsin + pncretic (24 h + 12 h) gve product...

7 Figure 6 AA profile of the het cogulted diet contining 30% hydrolysed protein before nd fter exposure to leching for 6 min nd the AA profile of the in vitro digested TCA precipitted superntnt of the not leched nd leched diet. Error brs represent stndrd devitions (n = 3) Diet Leched diet In vitro dig. diet In vitro dig. leched diet ASP GLU HYP SER GLY HIS TAU ARG THR ALA PRO TYR VAL MET ILE LEU PHE LYS with 86 ± 2.7% TCA soluble N. This shows tht different degrees of hydrolysis leds to lrge chnges in product qulity which might ffect fish lrve during feeding trils. Due to possibly lrge differences in protein qulity between hydrolysed protein sources, it is of importnce to describe the qulity of hydrolysed protein frctions nd the totl protein of diets used in feeding trils. Due to the high initil concentrtion of soluble N in cod fillet, inclusion of 45% hydrolysed protein led only to 37% increse in wtersoluble N in the rw ingredient mixture in this diet, compred to the diet contining 0% hydrolysed protein. However, there ws 104% increse in TCA soluble N. Inclusion of pure peptides nd FAA mixtures could be used insted of hydrolysed mrine ingredients (Dbrowski et l. 2003; Zhng et l. 2006). Although there might be better control of the concentrtion nd qulity of the soluble N frction by using pure peptides nd FAA mixtures, use of hydrolysed mrine products (rndom peptide mixtures), such s fish fillet used in this study, is chep nd effective wy of producing product with good AA profile. When hydrolysing the min protein source in the diet, insted of dding incresing mounts of different hydrolysed protein source, the diet will keep the sme AA profile only differing in solubility of the N frction. This is in ccordnce with Dbrowski et l. (2003), who rgued tht the sme source of protein hd to be fed in both intct nd hydrolysed forms to investigte if inclusion of protein hydrolystes leds to enhnced growth in lrvl fish. A protein bound het cogulted diet is bound together by therml denturtion of the protein. A diet with incresed hydrolysed protein will contin less protein which is ble to bind nd contribute to mtrix formtion nd might... therefore result in decresed stbility of the feed prticles. The binding properties of the four diets were not investigted, however het cogulted diets contining similr concentrtions of hydrolysed protein hve been used previously without detecting problems with the prticle stbility (Kvåle, pers. comm, NIFES, Bergen, Norwy). Crushing nd sieving led to rod shped prticles with shorter distnce to the centre of the prticles nd lrger surfce re compred to round prticles with the sme volume. Increse of surfce to volume rtio cn led to n incresed leching rte (Lee & Rosenberg 2000, 2001; Kv le et l. 2006). However, rod shped prticles cn mke it possible for mrine fish lrve to swllow lrger prticles tht might compenste for the incresed leching due to the incresed surfce re. As expected there ws reduction in soluble N due to therml denturtion, but the concentrtion of TCA soluble N did not seem to be ffected by het tretment. However, the concentrtion of TCA soluble N in the rw ingredients ws clculted from the pooled ingredients (Tonheim et l. 2007) so no sttistics could be run. Incresing inclusion levels of hydrolysed protein incresed the totl content of wtersoluble N from the ingredients nd reltively higher percentge of this wter-soluble N remined wter soluble fter feed production s compred to feeds with lower inclusion levels of hydrolysed protein. The most likely reson for this is tht hydrolysed protein prticipted to lesser extent in mtrix binding due to reduced moleculr size, nd thus remins unbound nd wter soluble fter prticle formtion. Loss of soluble N due to het denturtion ws reduced from 45 ± 4% in the diet contining 0% hydrolyste to 17 ± 2% in the diet contining 45% hydrolyste. The incresed loss of

8 soluble N due to het denturtion from the diets contining zero or low levels of pepsin hydrolysed cod is due to the higher concentrtion of het unstble intct soluble protein. Use of protein s the binder cn be desirble in order to reduce the concentrtion of binders with low or nonnutritionl qulities or to reduce the concentrtion of crbohydrtes for species with low tolernce such s hlibut (Hjertnes et l. 1991; Hmre et l. 2003). However, different processes tht cuse protein mtrix formtion such s het or crossbinding (Jones 1980) might lso negtively ffect the digestibility. For diets exposed to het during feed production it is necessry to include soluble N in the form of FAA, peptides nd thermlly stble proteins such s csein, to void reductions in the concentrtion of soluble N due to denturtion. This study confirms the high loss of wter-soluble N frctions when formulted diets with smll prticle size re submerged in wter (Lopez Alvrdo et l. 1994; Kv le et l. 2006). A 6 min leching tril is reltively long nd except for slow feeding species such s hlibut, most feed prticles would most likely be ingested within minutes. According to leching trils on het cogulted diets produced using the sme protocol (Kvåle et l. 2006), the burst relese ws over fter 5 min nd there ws still 50% retention. Bsed on these results, 6-min leching tril seemed to be n pproprite time intervl to investigte the reltive difference in leching rte between the diets. However, the present study showed higher rte of leching compred to studies on het cogulted diets produced using the sme protocol (Kvåle et l. 2006). Kv le et l. (2006) investigted leching with the use of rdioctively mrked lge extrct. The 14 C-lbelled lge extrct contined only 8% protein nd presumbly lrge content of 14 C-lbelled crbohydrtes. The reduction of soluble 14 C-lbelled lge extrct due to het tretment during production ws not investigted; denturing of the protein s shown in this study or severe Millrd rection of the crbohydrtes (Deng et l. 2005) during production could explin the reduced leching rte. In the leching studies by Kv le et l. (2006) diets were submerged in 3% NCl in comprison to this study where diets were leched in phosphte buffer. The leching my therefore be overestimted due to stronger osmotic grdient thn with sltwter s leching medi. However, to better compre chnges in concentrtion of soluble N in rw ingredients (Tonheim et l. 2007), produced diets, leched diets nd finlly in vitro digested diets, it ws of importnce to use the sme buffer to better compre results. The choice of leching medi will presumbly not ffect the reltive difference in leching rte between diets. Loss of soluble N from diets with high concentrtions of hydrolysed protein will not only cuse shift in qulity of the remining protein, but lso to lrge extent ffect protein quntity. Soluble N lost from the diet contining 45% hydrolysed protein ccounted for 30% of the totl protein frction. As erlier hypothesized by Kv le et l. (2006) mongst others, it might be the reduction in dietry crude protein levels tht led to the reduced survivl of Atlntic hlibut juvenile fed incresed concentrtions of hydrolysed protein (Kvåle 2007). Depending on hydrolysis method there my be significnt chnge in AA profile between soluble nd insoluble frction. For diets contining lrge concentrtion of soluble N this my led to chnge in the AA profile of ingested diet, compred the diet s formulted, due to leching. For the investigted diet contining 30% hydrolysed protein there were no mjor chnges in AA profile due to leching except 32% loss of histidine nd 100% loss of turine. Histidine is n essentil AA nd high loss my led to mlnutrition; studies by Argo et l. (2004) nd Svedr et l. (2007) suggests tht histidine my be the first limiting AA when fish lrve re fed on rotifers. Turine is not relly n mino cid s it lcks the crboxylic group, nd is not incorported in protein. Turine cn thus only be ingested in the free form nd not s prt of dietry protein. Turine will therefore be lost rpidly from most formulted diets s reported for protein encpsulted diet tht lso hd 100% loss fter exposure to wter (Nordgreen et l. 2007). Turine is described s non-essentil nutrient, however, there re severl indictions tht turine is essentil during erly development of fish. Enrichment of live feed with turine leds to incresed growth of Jpnese flounder (Prlichtys olivceus) (Tkeuchi et l. 2001; Chen et l. 2002, 2004, 2005; Kim et l. 2003, 2005; Mtsunri et l. 2003) nd red se brem (Pgrus mjor) (Chen et l. 2004). In ddition to complete loss by exposure to leching, the initil concentrtion of turine (4.7 mg g )1 dw) ws one qurter of tht found in copepods (18 mg g )1 dw, vn der Meeren et l. 2008) nd 0.28 times tht in Artemi (13 mg g )1 dw, vn der Meeren et l. 2008). However, the concentrtion ws times higher thn in rotifers ( mg g )1 dw, vn der Meeren et l. 2008; Srivstv et l. 2006). Kim et l. (2005) found tht juvenile Jpnese flounder required t lest 15 mg g )1 turine in the diet, confirming tht formulted diets bsed on mels need...

9 extr ddition of turine (Kim et l. 2005; Nordgreen et l. 2007). However, the turine requirement for Jpnese flounder estblished by Kim et l. (2005) might be overestimted due to leching. Het processing during feed production cn decrese both the in vivo nd in vitro protein digestibility of mrine protein sources significntly (Ynes et l. 1970; Opstvedt et l. 1984; Ln & Pn 1993; Grci-Orteg et l. 2000). The in vitro digestibility of the rw ingredients used in this study ws unfortuntely not mesured. On the other hnd, the individul digestibility of the protein sources in vitro ws reported by Tonheim et l. (2007), using n identicl btch of enzymes nd protocol with this study. The results indicte tht there is decrese in digestibility from pproximtely 80% for the pooled ingredients (Tonheim et l. 2007) to 65% for the finished diets, suggesting reduction in protein digestibility due to the production process. As shown in this study, pepsin hydrolysis of fish fillet led to doubling in both TCA nd wter-soluble N. However 35% of the hydrolysed product ws still insoluble protein. The complete hydrolysed product with its remining insoluble N ws included in the formulted diet s in previous studies (Kvåle et l. 2002; Kvåle 2007). It is likely tht the N frction of the rw mteril tht will be solubilized by enzymtic hydrolysis is the sme frction tht would first be digested by mrine fish lrve. A high inclusion of hydrolysed protein, with subsequent leching during feeding, my therefore led to n ingested diet with not only reduced concentrtion of totl protein, but lso hving lower qulity of the remining protein compred to formulted diet using the sme protein source unhydrolysed. This could explin the lower digestibility in the leched diets compred to the diets tht hd not been exposed to leching. When high inclusion of hydrolysed protein is required, seprtion of the insoluble frction before inclusion into formulted diet should be considered. The FAA pool in live feed (Frolov et l. 1991; Hmre et l. 2002; Crvlho et l. 2003; Hellnd et l. 2003; vn der Meeren et l. 2008; Srivstv et l. 2006) hs been suggested to be one of the resons for incresed growth of lrve fed these feeds, compred to lrve fed formulted diets. However, lter studies hve lso suggested tht lrger soluble nitrogenous compounds in live feed orgnisms could be s beneficil s FAA for incresing growth. More thn 85% of... the soluble N in Artemi nd rotifers ws lrger thn 500 D (Crvlho et l. 2003) nd studies hve indicted improved digestibility (Tonheim et l. 2007) nd growth with the use of wter-soluble proteins compred to insoluble proteins (Crvlho et l. 2004). There ws lower loss of wter-soluble N thn TCA soluble N in the present study. This is in ccordnce with Kv le et l. (2006) who showed tht incresed size of the soluble N frction led to significnt reduction in leching rte. The use of higher moleculr weight soluble N, rther thn FAA nd smll peptides, should be considered in order to reduce the leching rte from formulted diets. For diets tht hd not been exposed to leching there ws decresing difference in digestibility between diets with increse in time of digestibility. The difference in digestibility t ½ h nd 1 h is most likely logicl consequence of initil difference in TCA soluble N between diets. At 12 h there were no significnt differences between diets, indicting tht incresed concentrtion of hydrolysed protein did not led to incresed digestibility of the non-tca soluble N frction. The benefit of using pepsin hydrolystes in lrvl diets my therefore be the incresed frction of highly vilble peptides nd FAA nd not n incresed frction of prtly digested non-tca soluble N. However, this hypothesis needs to be confirmed by further studies. The diets tested in the present study performed similrly with regrds to digestibility in vitro, to commercilly vilble MiniproÔ fter both 1 h nd 12 h but better thn protein encpsulted diet tht ws digested to only 50% fter 12 h (Tonheim et l. 2007). Diets exposed to leching prior to digestibility hd significntly lower digestibility t ll time intervls compred to diets tht were not leched. At ½ h nd 1 h digestion the lower digestibility is most likely consequence of loss of TCA soluble N. After 12 h, on the other hnd, difference in digestibility might be due to difference in protein qulity. Similr digestion of diets exposed to leching t ll time intervls, confirms suggestions by Kvåle et l. (2006) tht leching cn chnge the qulity of the ingested diets. Reduction in crude protein due to leching ws suggested by Kv le et l. (2006) to be possible reson for the lower survivl of hlibut juvenile fed diets with incresed inclusion levels of hydrolysed protein. This is not possible to evlute when using in vitro digestibility methods. The protocol for in vitro digestion used in the present study ws bsed on methods described by Hsu et l. (1977) nd Sterlee et l. (1979) nd hs been thoroughly discussed by Chong et l. (2002) nd Tonheim et l. (2007). Chong et l. (2002) found good correltion in reltive digestibility between in vitro nd in vivo mesurements using juvenile discus fish

10 (Symphysodon equifscit) lthough there were some differences in bsolute digestibility. Thus, if iming to investigte true digestion of feed or feed ingredients, in vivo mesurements in the trget species should be performed. As discussed by severl uthors it is difficult to estimte digestibility of different protein sources in smll fish lrve. Tube feeding of rdio-lbelled protein to mrine fish lrve (Rønnestd et l. 2001; Tonheim et l. 2004, 2005) hs proved to be good method to evlute protein digestibility in such smll nimls. The method cn, however, only be used when tube feeding soluble model proteins (Tonheim et l. 2004) nd insoluble dietry proteins cn therefore not be investigted by this method. Micro-bound type diets s the one used in this study hve low efficiency in delivering soluble N to fish lrve nd should be crefully considered for this purpose. However, it should be emphsized tht the results in this study re vlid for micro-bound type diets, but my differ with other production technologies. This study clerly indictes tht feed production nd leching cn hve significnt effects on protein qulity nd tht optiml level of inclusion of hydrolysed protein in formulted diets for mrine fish lrve my be difficult to determine. The optimum levels my hve to be set for every individul diet, protein source nd dpted to species feeding behviour. The severe chnges in protein qulity during feed production nd leching show the importnce of considering both the protein sources used nd the type of diet produced when effects of hydrolysed proteins re investigted. Production process, leching rte, nd type nd qulity of the hydrolysed product used, hve to be considered. Audil Kv le PhD t Ntionl Institute of Nutrition nd Sefood Reserch (NIFES) is gretly cknowledges for discussions nd suggestions on methodologicl issues. A specil thnk to Joseph Mlimn (NIFES) for AA nlysis nd feed mnufcturing. This study ws prt of project finnced by the Norwegin Reserch Council (Project No /120). Argo, C., Conceico, L.E.C., Fyhn, H.J. & Dinis, M.T. (2004) Estimted mino cid requirements during erly ontogeny in fish with different life styles: gilthed sebrem (Sprus urt) nd Seneglese sole (Sole seneglensis). Aquculture, 242, Boye, J.I., M, C.-Y. & Hrdwlker, V.R. (1997) Therml denturizing nd cogultion of proteins. In: Food Proteins nd Their Applictions (Dmodrn, S. & Prf, A. eds), pp Mrcel Dekker, New York. Chu, C.L. & Zmbonino-Infnt, J.L. (2001) Substitution of live food by formulted diets in mrine fish lrve. Aquculture, 200, Chu, C.L., Infnte, J.L.Z., Quzuguel, P. & Le Gll, M.M. (1999) Protein hydrolyste vs. fish mel in compound diets for 10-dy old se bss Dicentrrchus lbrx lrve. Aquculture, 171, Crvlho, A.P., Escffre, A.M., Teles, A.O. & Bergot, P. 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