Pigment Reduction in Corn Gluten Meal and Its Effects on Muscle ...

<strong>Pigment</strong> <strong>Reduction</strong> <strong>in</strong> <strong>Corn</strong> <strong>Gluten</strong> <strong>Meal</strong> <strong>and</strong> <strong>Its</strong> <strong>Effects</strong> on Muscle<strong>Pigment</strong>ation of Ra<strong>in</strong>bow Trout (Oncorhynchus mykiss)byPatricio J SaezA ThesisPresented toThe University of GuelphIn partial fulfillment of requirementsFor the degree ofDoctor <strong>in</strong> Philosophy<strong>in</strong>Animal <strong>and</strong> Poultry ScienceGuelph, Ontario, Canada© Patricio J Saez, May, 2013

ABSTRACTPIGMENT REDUCTION OF CORN GLUTEN MEAL AND ITS EFFECTS ONMUSCLE PIGMENTATION OF RAINBOW TROUT (Oncorhynchus mykiss)Patricio J SaezUniversity of Guelph, 2013Advisor:Professor DP Bureau<strong>Corn</strong> gluten meal (CGM) is a high prote<strong>in</strong> (60% crude prote<strong>in</strong>), highly digestible feed<strong>in</strong>gredient widely used <strong>in</strong> diets for salmonids, however its use has been related to reduction <strong>in</strong>muscle pigmentation possibly due to pigment <strong>in</strong>teraction. Therefore, laboratory scale <strong>and</strong> <strong>in</strong> vivotrials were conducted to reduce pigment level <strong>in</strong> CGM <strong>and</strong> to assess its effect on fish musclepigmentation, respectively. In the first chapter, a bench-scale study was carried out to <strong>in</strong>vestigatefactors that affect bleach<strong>in</strong>g of carotenoids <strong>in</strong> CGM, us<strong>in</strong>g white soy flake flour (WSFF) as alipoxygenase (LOX) source. Plackett-Burman <strong>and</strong> Box-Behnken designs were used to screen <strong>and</strong>optimize factors, respectively. Furthermore, a 12-week growth trial was conducted <strong>in</strong> order toassess the effects of dietary regular <strong>and</strong> pigment bleached CGM on growth <strong>and</strong> musclepigmentation of ra<strong>in</strong>bow. In the second chapter, a 24-week growth trial was carried out <strong>in</strong> orderto assess the effects of <strong>in</strong>creas<strong>in</strong>g levels of CGM on growth <strong>and</strong> muscle pigment deposition <strong>in</strong>ra<strong>in</strong>bow trout. In the third chapter, a bench-scale (10 g) corn wet mill<strong>in</strong>g procedure was used toassess the bleach<strong>in</strong>g of carotenoids from CGM dur<strong>in</strong>g steep<strong>in</strong>g. Studies from this thesisconfirmed the negative effects of CGM on fillet pigmentation <strong>and</strong> highlighted the need forevaluation of muscle quality traits such as colour <strong>in</strong> response to <strong>in</strong>clusion of new feed

<strong>in</strong>gredients. Furthermore, this thesis gives <strong>in</strong>sight on how to reduce pigments from corn glutenmeal us<strong>in</strong>g cost-effective <strong>and</strong> practical bleach<strong>in</strong>g approaches.

ACKNOWLEDGEMENTSI would like to thanks my academic advisor Dr. Dom<strong>in</strong>ique Bureau for his guidance,constructive criticism <strong>and</strong> support dur<strong>in</strong>g the conduction of my thesis project. There are notenough words to thank you Dom for the opportunity you gave me to be a part of the FishNutrition Research Laboratory.I express my utmost gratitude to Dr. El-Sayed Abdel-Aal from the Agriculture Agri-FoodCanada, Guelph, for the extraord<strong>in</strong>ary help <strong>and</strong> guidance provided for the development of mythesis project.I would also extend my gratitude to the committee member of my thesis project Dr. JimAtk<strong>in</strong>son for his guidance <strong>and</strong> support.Thanks to the additional thesis external member Dr. Am<strong>and</strong>a Wright <strong>and</strong> the thesisexternal exam<strong>in</strong>er Dr. Ronald Hardy for their valuable comments on my thesis manuscript <strong>and</strong>the time they <strong>in</strong>vest to be part of my thesis defense.I would like to thank Mrs. Iwona Rabalski from the Agriculture Agri-Food Canada,Guelph, for all her help <strong>and</strong> guidance through all the many chemical analysis I have to conduct <strong>in</strong>order to complete the studies of my thesis project. The help of Mr. Michael Burke <strong>and</strong> all thestaff from th Alma Research Station is also highly appreciated for all their help dur<strong>in</strong>g theconduction of large scale <strong>in</strong> vivo trial.Thanks to Dr. Aliro Borquez, Dr. Patricio Dantagnan <strong>and</strong> Dr. Adrian Hern<strong>and</strong>ez for alltheir important support <strong>in</strong> many aspects.iv

Thanks to all my colleagues, friends, volunteers <strong>and</strong> visit<strong>in</strong>g scientist from the FishNutrition Research Laboratory for all their help <strong>and</strong> support on every step of my thesis.At last but def<strong>in</strong>itely not least, I would like to thanks my wife Elena for all her support<strong>and</strong> love. Thanks to my parents Jose <strong>and</strong> Maria, <strong>and</strong> my family members for all their love <strong>and</strong>support. Thanks to all my friends from Chile <strong>and</strong> the ones I have met <strong>in</strong> Guelph, your friendshiphas been really important to me.v

TABLE OF CONTENTSACKNOWLEDGEMENTS ....................................................................................................... ivTABLE OF CONTENTS ........................................................................................................... viLIST OF TABLES ..................................................................................................................... ixLIST OF FIGURES ................................................................................................................... xiCHAPTER - 1 GENERAL INTRODUCTION .......................................................................... 11.1 Objectives ......................................................................................................................... 3CHAPTER - 2 LITERATURE REVIEW .................................................................................. 42.1 Introduction ...................................................................................................................... 42.2 Evolution of diet formulation for salmonid fish <strong>and</strong> flesh quality assessment ................ 62.3 Fish muscle <strong>and</strong> quality st<strong>and</strong>ards .................................................................................... 72.4 Salmonid fish fillet colour <strong>and</strong> consumers’ perception .................................................... 82.5 Economic implications of salmonid muscle pigmentation .............................................. 92.6 Muscle pigmentation <strong>in</strong> salmonid fish ........................................................................... 102.6.1 Carotenoid pigments ............................................................................................... 102.6.2 Metabolism of carotenoids <strong>in</strong> salmonid fish ........................................................... 112.7 <strong>Corn</strong> fractionation <strong>and</strong> implications <strong>in</strong> animal nutrition ................................................ 142.7.1 <strong>Corn</strong> mill<strong>in</strong>g ............................................................................................................ 142.7.2 <strong>Corn</strong> gluten meal production .................................................................................. 162.7.3 <strong>Corn</strong> gluten meal: nutritional value <strong>and</strong> use <strong>in</strong> animal diets ................................... 172.7.4 Use of corn gluten meal <strong>in</strong> diets for terrestrial animals .......................................... 172.8 Use of corn gluten meal <strong>in</strong> diets for fish ........................................................................ 192.9 <strong>Effects</strong> of dietary corn gluten meal on salmonid fish muscle pigmentation .................. 212.10 Bleach<strong>in</strong>g of yellow xanthophylls from plant prote<strong>in</strong> <strong>in</strong>gredients ............................. 22vi

2.10.1 Enzymatic treatments .............................................................................................. 222.10.2 Other enzymes ........................................................................................................ 272.10.3 Chemical reagents as pigment bleach<strong>in</strong>g agents ..................................................... 272.11 Conclusion .................................................................................................................. 28CHAPTER - 3 OPTIMIZATION OF THE REDUCTION OF CAROTENOIDS IN CORNGLUTEN MEAL FOR EVALUATION ON GROWTH AND MUSCLE PIGMENTATIONOF RAINBOW TROUT (Oncorhynchus mykiss) ................................................................... 333.1 Introduction .................................................................................................................... 343.2 Materials <strong>and</strong> methods ................................................................................................... 363.2.1 Bleach<strong>in</strong>g of carotenoids......................................................................................... 363.2.2 Fish muscle pigmentation trial ................................................................................ 373.2.3 Analytical methods ................................................................................................. 403.2.4 Statistical analysis ................................................................................................... 443.3 Results <strong>and</strong> discussion .................................................................................................... 443.3.1 <strong>Pigment</strong> bleach<strong>in</strong>g trial ........................................................................................... 443.3.2 Fish muscle pigmentation trial ................................................................................ 48CHAPTER - 4 EFFECTS OF FEEDING INCREASING LEVELS OF CORN GLUTENMEAL ON GROWTH AND MUSCLE PIGMENTATION OF RAINBOW TROUT(Oncorhynchus mykiss) .............................................................................................................. 734.1 Introduction .................................................................................................................... 754.2 Materials <strong>and</strong> methods ................................................................................................... 774.2.1 Fish husb<strong>and</strong>ry <strong>and</strong> experimental conditions .......................................................... 774.2.2 Experimental diets .................................................................................................. 784.2.3 Chemical analysis ................................................................................................... 794.2.4 Muscle colour determ<strong>in</strong>ation .................................................................................. 814.2.5 Calculations............................................................................................................. 814.2.6 Statistical analysis ................................................................................................... 83vii

4.3 Results ............................................................................................................................ 834.4 Discussion ...................................................................................................................... 84CHAPTER - 5 BLEACHING OF CAROTENOIDS FROM CORN GLUTEN MEALTHROUGH A MODIFIED STEEPING PROCESS TO IMPROVE ITS VALUE AS ASALMONID FISH INGREDIENT: EFFECTS OF KERNEL VARIETY, HYDROGENPEROXIDE AND STEEPWATER PH .................................................................................. 1055.1 Introduction .................................................................................................................. 1075.2 Materials <strong>and</strong> methods ................................................................................................. 1095.2.1 Bench-scale maize wet mill<strong>in</strong>g ............................................................................. 1095.2.2 General analysis methods ..................................................................................... 1105.2.3 Statistical analysis ................................................................................................. 1125.3 Results .......................................................................................................................... 1125.4 Discussion .................................................................................................................... 114CHAPTER - 6 GENERAL DISCUSSION ............................................................................. 1326.1 Metabolism of carotenoids <strong>in</strong> fish ................................................................................ 1366.2 Factors other than carotenoids <strong>in</strong> corn gluten meal reduc<strong>in</strong>g astaxanth<strong>in</strong> muscledeposition ................................................................................................................................ 1376.3 Future research ............................................................................................................. 138viii

LIST OF TABLESTable 3. 1 - Factors <strong>and</strong> levels for screen<strong>in</strong>g us<strong>in</strong>g Plackett-Burman design ............................... 53Table 3. 2 - Experimental design us<strong>in</strong>g Plackett-Burman design for screen<strong>in</strong>g of factors ........... 54Table 3. 3 - Summary of variables for the Box-Behnken experimental design ............................ 55Table 3. 4 - Analysed proximate composition of regular <strong>and</strong> bio-bleached CGM ....................... 56Table 3. 5 - Formulation, analyzed proximate composition <strong>and</strong> pigment content of experimentaldiets ............................................................................................................................................... 57Table 3. 6 - Plackett-Burman design results ................................................................................. 59Table 3. 7 - Lipoxygenase activity <strong>in</strong> different <strong>in</strong>gredients .......................................................... 60Table 3. 8 - Box-Behnken experimental design ............................................................................ 61Table 3. 9 - Box-Behnken design l<strong>in</strong>ear, quadratic <strong>and</strong> <strong>in</strong>teraction effects .................................. 63Table 3. 10 - HPLC carotenoid profile of regular <strong>and</strong> bleached CGM ......................................... 64Table 3. 11 - Growth performance <strong>and</strong> fed efficiency of ra<strong>in</strong>bow trout (IBW=132 g fish -1 ) fedexperimental diets for 12 weeks ................................................................................................... 65Table 3. 12 - Reta<strong>in</strong>ed levels <strong>and</strong> retention efficiencies of nitrogen <strong>and</strong> energy by ra<strong>in</strong>bow trout(IBW=132 g fish -1 ) fed experimental diets for 12 weeks ............................................................. 66Table 3. 13 - Proximate composition of the carcass of ra<strong>in</strong>bow trout a (IBW=132 g fish -1 ) fedexperimental diets for 12 weeks ................................................................................................... 67Table 3. 14 - Fillet carotenoid concentration <strong>and</strong> colour attributes from ra<strong>in</strong>bow trout a (IBW=132g fish -1 ) fed experimental diets for 12 weeks ................................................................................ 68Table 4. 1 - Formulation, analyzed proximate composition <strong>and</strong> pigment content of experimentaldiets ............................................................................................................................................... 89Table 4. 2 - Growth performance of ra<strong>in</strong>bow trout (IBW= 549 g fish -1 ) fed experimental diets for24 weeks........................................................................................................................................ 92Table 4. 3 - Carcass chemical proximate composition of ra<strong>in</strong>bow trout b (IBW= 549 g fish -1 ) fedexperimental diets for 24 weeks ................................................................................................... 94Table 4. 4 - Reta<strong>in</strong>ed levels <strong>and</strong> retention efficiencies of nitrogen <strong>and</strong> energy by ra<strong>in</strong>bow trout(IBW= 549 g fish -1 ) fed experimental diets for 24 weeks ............................................................ 96ix

Table 4. 5 - <strong>Pigment</strong> content <strong>in</strong> muscle of ra<strong>in</strong>bow trout (IBW= 549 g fish -1 ) fed experimentaldiets for 24 weeks ......................................................................................................................... 98Table 4. 6 - Colour attributes <strong>in</strong> muscle from ra<strong>in</strong>bow trout a (IBW= 549 g fish -1 ) fedexperimental diets for 24 weeks ................................................................................................. 100Table 5. 1 - Analyzed proximate composition <strong>and</strong> pigment content of the two corn kernelvarieties used <strong>in</strong> the study ........................................................................................................... 120Table 5. 2 - Yields of different fractions <strong>and</strong> mass balance of dry matter obta<strong>in</strong>ed us<strong>in</strong>g thelaboratory scale corn wet mill<strong>in</strong>g under different steep<strong>in</strong>g conditions ....................................... 121Table 5. 3 - Total starch <strong>and</strong> crude prote<strong>in</strong> content <strong>in</strong> starch <strong>and</strong> gluten fractions ..................... 123Table 5. 4 - Carotenoids profile <strong>in</strong> corn gluten meal fraction produced under experimentalconditions .................................................................................................................................... 125Table 5. 5 - Mass balance of total starch <strong>and</strong> crude prote<strong>in</strong> obta<strong>in</strong>ed us<strong>in</strong>g the laboratory-scalecorn wet mill<strong>in</strong>g .......................................................................................................................... 127Table 5. 6 - Mass balance of carotenoids obta<strong>in</strong>ed <strong>in</strong> the laboratory-scale corn wet mill<strong>in</strong>g ..... 129Table 5. 7 - Significance of s<strong>in</strong>gle effect as well as of two-way <strong>and</strong> three-way <strong>in</strong>teraction offactors on carotenoids mass balance ........................................................................................... 131x

LIST OF FIGURESFigure 2. 1 - Molecular structure of some important carotenoids founds <strong>in</strong> nature...................... 29Figure 2. 2 - Schematic representation of the corn wet mill<strong>in</strong>g process. ...................................... 30Figure 2. 3 - Schematic representation of the dry corn mill<strong>in</strong>g process. ...................................... 31Figure 2. 4 - Schematic representation of the mode of action of lipoxygenase dur<strong>in</strong>g lipidoxidation (Taylor <strong>and</strong> Morris 1983). a) Penta-1,4-cis-diene system with<strong>in</strong> l<strong>in</strong>oleic acid, b)Intermediate peroxy radical responsible for carotenoid pigments co-oxidation, c) hydroperoxide,the f<strong>in</strong>al product of the enzyme-catalyzed lipid oxidation............................................................ 32Figure 3. 1 - Typical trend for spectrophotometric measurement of lipoxygenase activityobta<strong>in</strong>ed for WSFF <strong>in</strong> the present study. ...................................................................................... 69Figure 3. 2 - Bleach<strong>in</strong>g pattern of carotenoids observed over time (120 m<strong>in</strong>) for the optimumfactor comb<strong>in</strong>ation, PS = 90 m<strong>in</strong>, RT = 15°C, BP = 600 ppm <strong>and</strong> SO = 5% <strong>in</strong>clusion. .............. 70Figure 3. 3 - Response surface plot show<strong>in</strong>g the effect of SO <strong>and</strong> BP on CGM carotenoidsbleach<strong>in</strong>g, keep<strong>in</strong>g PS fixed at 90 m<strong>in</strong> at three different RT: 20, 15 <strong>and</strong> 10°C for A, B <strong>and</strong> C,respectively. .................................................................................................................................. 71Figure 3. 4 - Growth curve of ra<strong>in</strong>bow trout fed the experimental diets for 12 weeks. D1 (Diet 1,Control), D2 (Diet 2, regular CGM), D3 (Diet 3, Bleached CGM. .............................................. 72Figure 4. 1 - Growth curves of ra<strong>in</strong>bow trout fed the experimental diets for 24 weeks. ............ 102Figure 4. 2 - Muscle pigment concentration to graded levels of corn gluten meal <strong>in</strong> ra<strong>in</strong>bow troutfed the experimental diets for 24 weeks. Larger markers on each trend l<strong>in</strong>e represent resultsobta<strong>in</strong>ed by diets where <strong>in</strong>clusion of corn gluten meal was at expense of soy prote<strong>in</strong> concentrateonly (0, 9 <strong>and</strong> 18% CGM <strong>in</strong>clusion). .......................................................................................... 103Figure 4. 3 - Muscle colour attributes to graded levels of corn gluten meal <strong>in</strong> ra<strong>in</strong>bow trout fedthe experimental diets for 24 weeks. Larger markers on each trend l<strong>in</strong>e represent results obta<strong>in</strong>edby diets where <strong>in</strong>clusion of corn gluten meal was at expense of soy prote<strong>in</strong> concentrate only (0, 9<strong>and</strong> 18% CGM <strong>in</strong>clusion). .......................................................................................................... 104xi

CHAPTER - 1 GENERAL INTRODUCTIONAquaculture, a fast grow<strong>in</strong>g food supply<strong>in</strong>g <strong>in</strong>dustry, produced about 64 million MT offish <strong>in</strong> 2011, account<strong>in</strong>g for nearly 50% of consumed fish food globally (FAO, 2012). Productionis expected to <strong>in</strong>crease up to 60% by 2020. These production levels are currently achieved by theculture of more than 300 different species, with various carp species dom<strong>in</strong>at<strong>in</strong>g globalaquaculture production. In 2010, salmonid fish, Atlantic salmon <strong>and</strong> ra<strong>in</strong>bow trout (Salmo salar<strong>and</strong> Oncorhynchus mykiss, respectively) production share represented a 3.5% of the worldaquaculture production of fish, crustaceans <strong>and</strong> molluscs valued at US$1.4 billion (FAO, 2012).Flesh quality <strong>in</strong> f<strong>in</strong>al products from salmonid fish is determ<strong>in</strong>ed by a comb<strong>in</strong>ation ofseveral factors <strong>in</strong>clud<strong>in</strong>g colour. This quality trait is positively correlated to product price asconsumers associate colour with freshness, higher quality <strong>and</strong> better flavour (Alfnes et al., 2006).In the wild, salmonid fish obta<strong>in</strong> carotenoid from prey they consume. In salmonid fish culture,premixes conta<strong>in</strong><strong>in</strong>g synthetic <strong>and</strong> naturally occurr<strong>in</strong>g pigments, ma<strong>in</strong>ly astaxanth<strong>in</strong>, must be<strong>in</strong>cluded <strong>in</strong> the feed as salmonids are not able to synthetize carotenoids de novo. <strong>Pigment</strong>premixes are very expensive <strong>and</strong> carotenoid supplementation represents up to 15% of f<strong>in</strong>al feedcosts (Bjerkeng, 2000; Choubert et al., 2009).Formulation of diets for salmonid fish has evolved <strong>in</strong> order to <strong>in</strong>crease <strong>in</strong>clusion levels ofcost-effective prote<strong>in</strong> <strong>in</strong>gredients due to the better underst<strong>and</strong><strong>in</strong>g of nutritional requirements.Large amounts of research have been conducted <strong>in</strong> order to assess the effects of different<strong>in</strong>gredients on growth performance, <strong>and</strong> nutrient utilization <strong>in</strong> the last five decades. Howevernegligible efforts have been conducted on the effects of these <strong>in</strong>gredients on f<strong>in</strong>al productquality.1

Ingredients commonly <strong>in</strong>cluded <strong>in</strong> formulation for fish are fish meal, poultry by-productmeal, meat <strong>and</strong> bone meal, soybean meal, soy prote<strong>in</strong> concentrates <strong>and</strong> corn gluten meal (Alexiset al., 1985; Cha et al., 2000; de Francesco et al., 2004). <strong>Corn</strong> gluten meal, is a high prote<strong>in</strong>(~60% crude prote<strong>in</strong>), highly digestible by product of the corn wet mill<strong>in</strong>g process<strong>in</strong>g that iswidely utilized <strong>in</strong> diets for many fish species. <strong>Corn</strong> wet mill<strong>in</strong>g is an <strong>in</strong>dustrial process aim<strong>in</strong>g tofractionate corn kernel ma<strong>in</strong> constituents (i.e. starch, prote<strong>in</strong>, germ <strong>and</strong> fiber) <strong>and</strong> furtherprocess/ref<strong>in</strong>e them <strong>in</strong> order to produce high quality products for food, feed <strong>and</strong> <strong>in</strong>dustrialpurposes. Dur<strong>in</strong>g wet mill<strong>in</strong>g, naturally occurr<strong>in</strong>g yellow pigments from corn rema<strong>in</strong> with theprote<strong>in</strong> fraction. Consequently corn gluten meal conta<strong>in</strong>s substantial amount of yellowxanthophylls (200 – 550 mg kg -1 ), ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> (Skonberg et al., 1998; Park etal., 1997).Anecdotal evidence from fish farmers <strong>in</strong>dicates that high <strong>in</strong>clusion levels of CGM havebeen related to reduction <strong>in</strong> muscle pigmentation <strong>in</strong> salmonid fish. Results from few scientificstudies suggest that yellow xanthophylls from CGM might <strong>in</strong>duce ‘yellowish’ appearance <strong>in</strong>fillets but also a reduction of astaxanth<strong>in</strong> muscle deposition (Skonberg et al., 1998). Fillets fromAtlantic salmon presented a significant reduction <strong>in</strong> pigmentation (measured us<strong>in</strong>g SalmoFancolorimetric analysis) <strong>in</strong> response to graded levels of a vegetable prote<strong>in</strong> blend (CGM <strong>and</strong> full fatsoybean meal <strong>in</strong> a 2:1 ratio) <strong>in</strong> diets supplemented with astaxanth<strong>in</strong> (Mundheim et al., 2004).Higher yellowish hue was determ<strong>in</strong>ed <strong>in</strong> fillets from ra<strong>in</strong>bow trout fed a high CGM diet with nopigment supplementation compared with those from fish fed a high CGM <strong>and</strong> supplementedsynthetic canthaxanth<strong>in</strong> (Skonberg et al., 1998).Given the lipid soluble nature <strong>and</strong> the similarities of molecular structure shared byastaxanth<strong>in</strong> <strong>and</strong> yellow xanthophylls from corn gluten meal, an <strong>in</strong>teraction/competition among2

these pigments have been hypothesized to occur before/dur<strong>in</strong>g <strong>in</strong>test<strong>in</strong>al absorption, transportwith<strong>in</strong> bloodstream <strong>and</strong>/or muscle deposition.<strong>Reduction</strong> of yellow pigments from corn gluten meal has been assessed us<strong>in</strong>g differentapproaches <strong>in</strong>clud<strong>in</strong>g lipoxygenase-catalyzed oxidation (Cha et al., 2000; Gél<strong>in</strong>as et al., 1998;Park et al., 1997). Lipoxygenases (LOX) are nonheme iron-conta<strong>in</strong><strong>in</strong>g dioxygenases (EC1.13.11.12) that catalyze the oxygenation of polyunsaturated fatty acids conta<strong>in</strong><strong>in</strong>g a cis-cis-1, 4-pentadiene system. More than 60% of total yellow carotenoids pigments content was bleachedwhen 5% of soy flour as LOX source was mixed with wet CGM <strong>in</strong> an aqueous medium at pH 6.5(Park et al., 1997). However, none of these studies have evaluated the <strong>in</strong>clusion of pigmentbleachedcorn gluten meal on muscle astaxanth<strong>in</strong> deposition <strong>in</strong> salmonid fish.1.1 ObjectivesThe ma<strong>in</strong> objectives of this thesis were 1) to assess the effect of dietary corn gluten meal <strong>and</strong>treated corn gluten meal on astaxanth<strong>in</strong> deposition <strong>and</strong> muscle colour attributes <strong>in</strong> ra<strong>in</strong>bow trout(O. mykiss). And 2) to develop practical, laboratory-scale (10-20 g) process<strong>in</strong>g methods for thereduction of carotenoid content <strong>in</strong> corn gluten meal for its use <strong>in</strong> formulated diets for ra<strong>in</strong>bowtrout (O. mykiss).3

CHAPTER - 2 LITERATURE REVIEW2.1 IntroductionThe aquaculture <strong>in</strong>dustry is fac<strong>in</strong>g major challenges to manage production cost <strong>and</strong> toachieve high quality st<strong>and</strong>ards of their products. As a result of the high cost <strong>and</strong> volatile supplyof fish meal <strong>and</strong> oil, formulation of aquaculture feeds has evolved <strong>in</strong> order to <strong>in</strong>crease the<strong>in</strong>clusion levels of more cost-effective <strong>in</strong>gredients. Numerous scientific studies have addressedthe effects of new feed <strong>in</strong>gredients on growth <strong>and</strong> feed utilization; nonetheless there is a lack ofresearch focus<strong>in</strong>g on how these raw materials might affect muscle quality traits.The characteristic red or p<strong>in</strong>k colour <strong>in</strong> the muscle of salmonid fish is the result ofcarotenoid, ma<strong>in</strong>ly astaxanth<strong>in</strong>, deposition with<strong>in</strong> muscle fibers. This quality attribute is closelyrelated to the economic value of this product. Fish must obta<strong>in</strong>ed astaxanth<strong>in</strong> from dietarysources as they are unable to synthesize it de novo. While wild fish obta<strong>in</strong> astaxanth<strong>in</strong> from preythey <strong>in</strong>gest. Formulated diets for farmed salmonid fish must be supplemented with natural orsynthetic pigment. Astaxanth<strong>in</strong> premixes are very expensive <strong>and</strong> supplementation accounts for15% - 20% of f<strong>in</strong>al feed cost.One of the most commonly utilized <strong>in</strong>gredients for aquaculture feeds is corn gluten meal, aprote<strong>in</strong>-rich (60% crude prote<strong>in</strong>) by-product of the corn wet mill<strong>in</strong>g process. <strong>Corn</strong> wet mill<strong>in</strong>g isan <strong>in</strong>dustrial process aim<strong>in</strong>g to split the corn kernels major constituents (starch, prote<strong>in</strong>, germ<strong>and</strong> fiber) <strong>in</strong>to fairly purified fractions <strong>and</strong> to further ref<strong>in</strong>ed them for feed, food <strong>and</strong> <strong>in</strong>dustrialpurposes. Dur<strong>in</strong>g wet mill<strong>in</strong>g, most of the naturally occurr<strong>in</strong>g pigments <strong>in</strong> corn kernels rema<strong>in</strong>4

embedded with<strong>in</strong> the gluten fraction; consequently this commodity conta<strong>in</strong>s substantial amountof pigments (200 – 550 mg kg -1 ), ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>.Negative effects related to dietary corn gluten meal on muscle astaxanth<strong>in</strong> deposition <strong>and</strong>colour attributes are supported by anecdotal evidence from fish farmers along with a few studies.<strong>Reduction</strong> <strong>in</strong> colour attributes was determ<strong>in</strong>ed <strong>in</strong> fillets from Atlantic salmon (Salmo salar) feddiets formulated to conta<strong>in</strong> <strong>in</strong>creas<strong>in</strong>g levels of a plant prote<strong>in</strong> blend (CGM <strong>and</strong> full fat soybeanmeal <strong>in</strong> a 2:1 ratio) <strong>and</strong> 64 mg kg -1 of astaxanth<strong>in</strong> after an 11-week feed<strong>in</strong>g trial (Mundheim etal., 2004). High values <strong>in</strong> yellowness (based on tristimulus colour analysis) were determ<strong>in</strong>ed <strong>in</strong>fillets from ra<strong>in</strong>bow trout fed a high CGM diet (22.5%) compared to those from fish fed a highCGM diet (22.5%) supplemented with 100 mg kg -1 canthaxanth<strong>in</strong> for 12 weeks (Skonberg et al.,1998).<strong>Reduction</strong> <strong>in</strong> astaxanth<strong>in</strong> deposition <strong>and</strong> colour attributes <strong>in</strong> muscle from salmonid fish feddiets conta<strong>in</strong><strong>in</strong>g CGM has been associated with the substantial amount of yellow carotenoidsconta<strong>in</strong>ed <strong>in</strong> this commodity. Even though the metabolic pathways of carotenoids <strong>in</strong> fish are notfully understood, <strong>in</strong>teractions <strong>and</strong>/or competition between different types of pigments dur<strong>in</strong>g<strong>in</strong>test<strong>in</strong>al absorption, transport with<strong>in</strong> bloodstream <strong>and</strong>/or deposition with<strong>in</strong> muscle cells arehypothesized based on hydrophobic properties <strong>and</strong> molecular structure similarities shared byastaxanth<strong>in</strong> <strong>and</strong> yellow xanthophylls from CGM.Bleach<strong>in</strong>g of yellow pigments from CGM, for its <strong>in</strong>clusion <strong>in</strong> salmonid fish formulated diets,has been assessed us<strong>in</strong>g different practical process<strong>in</strong>g approaches (i.e. natural maturation,enzymatic oxidation <strong>and</strong> solvent extraction), with diverse results (Park et al., 1997; Gél<strong>in</strong>as et al.,1998; Cha et al., 2000). Up to 70% of the total yellow carotenoids pigments content <strong>in</strong> CGM was5

leached when 5% of soy flour as LOX source was mixed with wet CGM <strong>in</strong> an aqueous mediumat pH 6.5 (Cha et al., 2000; Park et al., 1997).The aims of this literature review are 1) to contribute to a better underst<strong>and</strong><strong>in</strong>g on the effectsof <strong>in</strong>clusion of plant-prote<strong>in</strong> <strong>in</strong>gredients, specifically corn gluten meal, on muscle quality traitssuch colour <strong>and</strong> 2) to <strong>in</strong>troduce some practical process<strong>in</strong>g methods for corn gluten meal <strong>in</strong> orderto reduce its pigment content <strong>and</strong> reduce its negative effect on muscle pigmentation of salmonidfish.2.2 Evolution of diet formulation for salmonid fish <strong>and</strong> flesh quality assessmentHistorically, aquaculture feeds have relied upon fish meal as a major prote<strong>in</strong> source due tothe high nutritive value of this commodity. Production of salmonid feed has shown a fast growth<strong>in</strong> the last decade, the annual global production of formulated diets for salmonid fish <strong>in</strong> 2006 wasestimated at about 2.6 MMT (Tacon <strong>and</strong> Metian, 2009). The steady <strong>in</strong>crease of price <strong>and</strong> variableavailability of fisheries by-products observed dur<strong>in</strong>g the last three decades had forced salmonidfeed formulators to <strong>in</strong>crease <strong>in</strong>clusion levels of more available <strong>and</strong> cost-effective feed <strong>in</strong>gredients<strong>in</strong> order to manage their production costs.Ingredients which have found wide use <strong>in</strong> aquaculture feeds are poultry by-product meal,meat <strong>and</strong> bone meal, soybean meal, soy prote<strong>in</strong> concentrates <strong>and</strong> corn gluten meal (Alexis et al.,1985; Cha et al., 2000; de Francesco et al., 2004). Numerous scientific studies assess<strong>in</strong>g theeffects of these <strong>in</strong>gredients (<strong>and</strong> many other new potentially useful ones) on growth, digestibility<strong>and</strong> nutrient retention have been published <strong>in</strong> the last decades. On the other h<strong>and</strong>, only a fewscientific publications address<strong>in</strong>g the effects of these commodities on flesh quality <strong>in</strong> salmonidfish are available. Inclusion of soy prote<strong>in</strong> concentrate <strong>and</strong> soybean meal (62 <strong>and</strong> 42% <strong>in</strong>clusion6

level, respectively) resulted <strong>in</strong> significant different values for colour attributes <strong>and</strong> texture(expressed as maximum shear strength) <strong>in</strong> fillet from ra<strong>in</strong>bow trout fed non-pigmentsupplemented experimental diets for 12 weeks (Kaushik et al., 1995). Higher hardness, lesssweetness <strong>and</strong> odour <strong>in</strong>tensity, <strong>and</strong> a trend to lower juic<strong>in</strong>ess, as well as significant differences <strong>in</strong>redness (a*) <strong>and</strong> yellowness (b*) <strong>in</strong> different sections (dorsal, ventral <strong>and</strong> caudal site) weredeterm<strong>in</strong>ed <strong>in</strong> fillets from ra<strong>in</strong>bow trout fed a diet supplemented with 70% of a plant prote<strong>in</strong>blend composed of corn gluten meal, wheat gluten meal, extruded peas <strong>and</strong> rapeseed meal (<strong>in</strong> a2:2:2:1 ratio) compared to those obta<strong>in</strong>ed from fish fed a fish meal based diet conta<strong>in</strong><strong>in</strong>g 0.01%of astaxanth<strong>in</strong> for 24 weeks (de Francesco et al., 2004).2.3 Fish muscle <strong>and</strong> quality st<strong>and</strong>ardsMuscle is the soft, contractile tissue responsible for produc<strong>in</strong>g force <strong>and</strong> caus<strong>in</strong>g motion <strong>in</strong>animals. Three different types of muscle tissue are recognized <strong>in</strong> vertebrates, skeletal or‘voluntary’ muscle controll<strong>in</strong>g locomotion, <strong>and</strong> two types of ‘<strong>in</strong>voluntary muscle, smooth found<strong>in</strong> <strong>in</strong>ternal organs (i.e. esophagus, stomach, among others) <strong>and</strong> cardiac muscle, found only <strong>in</strong> theheart.The major edible part of fish fillets is composed by ‘W’ shaped stacks of skeletal musclecalled myomeres. These muscle blocks are separated from adjacent myomeres by th<strong>in</strong> horizontal(myosepta) <strong>and</strong> vertical (myocommata) layers of connective tissue. Three different types ofskeletal muscle have been described <strong>in</strong> fish based on their metabolic <strong>and</strong> contractilecharacteristics: 1) red muscle (slow twitch or type 1 cells): composed of superficial cells with ahigh capacity for aerobic energy supply, 2) white muscle (fast twitch or type 2b cells): formed by7

deep <strong>and</strong> fast glycolytic cells, <strong>and</strong> 3) p<strong>in</strong>k muscle (located between red <strong>and</strong> white muscle),shar<strong>in</strong>g metabolic properties of red <strong>and</strong> white muscle (Fauconneau et al., 1997).Myofibers (myocytes, muscle cells or muscle fibers) are cyl<strong>in</strong>drical mult<strong>in</strong>ucleated cellsarranged with<strong>in</strong> myomeres <strong>in</strong> clusters of cells called fascicles. Myofibers are composed of longcontractile prote<strong>in</strong> bundles (conta<strong>in</strong><strong>in</strong>g act<strong>in</strong> <strong>and</strong> myos<strong>in</strong> complexes) called myofribrils. Act<strong>in</strong><strong>and</strong> myos<strong>in</strong> are organized with<strong>in</strong> myofibrils <strong>in</strong> units of repetition named sarcomeres.Flesh quality is determ<strong>in</strong>ed by a comb<strong>in</strong>ation of muscle characteristics <strong>in</strong>clud<strong>in</strong>g fleshfirmness <strong>and</strong> colour (Anderson, 2000). Flesh quality traits dramatically <strong>in</strong>fluence the price of thef<strong>in</strong>al product, thus achiev<strong>in</strong>g high st<strong>and</strong>ards <strong>in</strong> flesh quality will result <strong>in</strong> premium price. On theother h<strong>and</strong>, fillets present<strong>in</strong>g poor physical characteristic will be downgraded (i.e. reduction ofthe economic value of the product) at process<strong>in</strong>g plants (Johnston et al., 2006).2.4 Salmonid fish fillet colour <strong>and</strong> consumers’ perceptionThe conspicuous red to p<strong>in</strong>k colour <strong>in</strong> fillets from salmonid fish is an important qualityparameter. Colour has been positively related to consumer purchas<strong>in</strong>g decision at sell<strong>in</strong>g po<strong>in</strong>ts.Consumers associate colour with characteristics such as age, orig<strong>in</strong>, expected flavour/texture <strong>and</strong>freshness (Anderson, 2000).<strong>Reduction</strong> of expected colour <strong>in</strong> muscle from salmonid fish negatively affects its appeal <strong>and</strong>reduces its economic value as consumers relate this treat with a more expensive product. There isa clear preference from consumers for redder fillets compare to pale ones. Consumers’will<strong>in</strong>gness to pay (WTP) was positively correlated to fillet redness <strong>in</strong> a real choice experiment,8

ased on a sensory panel evaluat<strong>in</strong>g colour variation (expressed as SalmoFan TM scores, rang<strong>in</strong>gfrom score 21 to 29). Fillets with colour scores below 23 were identified as hard to sell at anyprice (Alfnes et al., 2006).2.5 Economic implications of salmonid muscle pigmentationAstaxanth<strong>in</strong> is by far the most expensive commodity <strong>in</strong>cluded <strong>in</strong> formulated diets forsalmonid fish, its price may vary from 1500 to 2000 USD kg -1 (Bjerkeng, 2008). <strong>Pigment</strong>supplementation generally represents up to 10-20% of the feed <strong>in</strong>gredients’ cost (Bjerkeng,2000; Choubert et al., 2009), however these expensive pigments are poorly utilized by fish.Astaxanth<strong>in</strong> flesh deposition rate is <strong>in</strong> general very low (

observed <strong>in</strong> ra<strong>in</strong>bow trout with an <strong>in</strong>itial body weight of 550 g fed a commercial diet conta<strong>in</strong><strong>in</strong>g60 mg kg -1 of astaxanth<strong>in</strong> for 56 days (Ytrestoyl <strong>and</strong> Bjerkeng 2007).Differences of 30 to 50% have been reported between digested astaxanth<strong>in</strong> (ADC) <strong>and</strong>astaxanth<strong>in</strong> muscle deposition <strong>in</strong> ra<strong>in</strong>bow trout (958 g, f<strong>in</strong>al body weight) fed a diet conta<strong>in</strong><strong>in</strong>g52 mg of astaxanth<strong>in</strong> per kg after a 6 weeks feed<strong>in</strong>g trial (No <strong>and</strong> Storebakken, 1991).2.6 Muscle pigmentation <strong>in</strong> salmonid fish2.6.1 Carotenoid pigmentsCarotenoids are hydrophobic, isoprene derivatives, pigments bio-synthetized by plants,algae <strong>and</strong> photosynthetic bacteria; more than 600 of these compounds have been identified,characterized <strong>and</strong> isolated from natural sources.On a molecular basis, all carotenoids are characterized by a C 40 carbon skeleton. Thiscarbon backbone conta<strong>in</strong>s 3 to 13 conjugated double bounds (polyene), <strong>and</strong> may be cyclized atone or both ends. Figure 2.1 shows the structures of some important carotenoids found <strong>in</strong> nature.Carotenoids are classified <strong>in</strong>to two major groups accord<strong>in</strong>g to their chemical composition: (1)carotenes, when only carbon <strong>and</strong> hydrogen atoms are present <strong>in</strong> their structure, <strong>and</strong> (2)xanthophylls, when one or more oxygen molecules are conta<strong>in</strong>ed with<strong>in</strong> their moleculararrangement (Brenna <strong>and</strong> Berardo, 2004).<strong>Pigment</strong>s are widely distributed <strong>in</strong> nature <strong>and</strong> are responsible for the yellow, orange <strong>and</strong>red colouration <strong>in</strong> many fruits, birds, fish <strong>and</strong> crustacea. Animals are unable to synthesise10

carotenoids de novo, hence they must rely upon dietary sources <strong>in</strong> order to access them (Furr <strong>and</strong>Clark, 1997; Yonekura <strong>and</strong> Nagao, 2007).2.6.2 Metabolism of carotenoids <strong>in</strong> salmonid fishCarotenoids are lipid soluble compounds, consequently they are metabolized (i.e.digested, absorbed <strong>and</strong> transported) accord<strong>in</strong>g to the matabolic pathways followed by lipidmolecules (Castenmiller <strong>and</strong> West, 1998; Rajas<strong>in</strong>gh et al., 2006).Carotenoids are solubilized <strong>in</strong> lipid droplets after be<strong>in</strong>g released from feed with<strong>in</strong> thestomach. Lipid aggregates are further emulsified by liver-secreted bile <strong>in</strong> the duodenum.Emulsified lipids are then hydrolysed (by pancreatic lipases) <strong>in</strong>to free fatty acids <strong>and</strong>monoglycerols, which rema<strong>in</strong> associated with carotenoids, other lipids <strong>and</strong> bile acids <strong>in</strong>structures called mixed micelles. Polar xanthophylls are found <strong>in</strong> the surface of lipid emulsions<strong>and</strong> mixed micelles. On the other h<strong>and</strong>, less polar carotenes are located with<strong>in</strong> the hydrophobiccore from these assemblies (Borel et al., 1996; Furr <strong>and</strong> Clarck, 1997). Molecules positionedwith<strong>in</strong> surface of this lipid structures can be spontaneously transferred as opposed to componentattached to the core, which need digestion before be<strong>in</strong>g released (Borel et al., 1996).Carotenoids with<strong>in</strong> micelles are absorbed, along with other lipids, by <strong>in</strong>test<strong>in</strong>al cells(enterocytes) through simple diffusion. With<strong>in</strong> the enterocyte, carotenoids are aggregated <strong>in</strong>totriglyceride-rich lipoprote<strong>in</strong>s called chylomicrons <strong>and</strong> released <strong>in</strong>to the lymph <strong>and</strong> eventually<strong>in</strong>to the blood stream for deliver to peripheral tissue <strong>and</strong> liver.11

Chylomicron constituents are removed by lipoprote<strong>in</strong> lipase <strong>and</strong> distributed <strong>in</strong> peripheraltissue. Chylomicron remnants are then taken up by the liver <strong>and</strong> rema<strong>in</strong><strong>in</strong>g carotenoids are eithermetabolized or assembled <strong>in</strong>to lipoprote<strong>in</strong>s <strong>and</strong> sent aga<strong>in</strong> to the bloodstream toward peripheraltissue (Jackson et al., 2008).The liver has been reported as a major site for metabolism <strong>and</strong> excretion of carotenoids <strong>in</strong>fish. The occurrence of high levels of 14 C-labelled canthaxanth<strong>in</strong> (96 h after a s<strong>in</strong>gle forcefeed<strong>in</strong>g) <strong>and</strong> no detectable levels of canthaxanth<strong>in</strong> <strong>in</strong> bile from ra<strong>in</strong>bow trout (400 g fish -1 )suggests that digested canthaxanth<strong>in</strong> was metabolized <strong>in</strong>to different compounds <strong>in</strong> the liver <strong>and</strong>excreted with<strong>in</strong> the bile (Hardy et al., 1990). Similarly, biliary excretion of cantaxanth<strong>in</strong> wasreported for Atlantic salmon (1,000 – 1,400 g fish -1 ) fed diets supplemented with 14 C-labelled<strong>and</strong> regular canthaxanth<strong>in</strong> (Torrissen <strong>and</strong> Ingebrigtsen, 1992).Carotenoids not metabolized or excreted by the liver are re-<strong>in</strong>corporated <strong>in</strong>to lipoprote<strong>in</strong>sbefore be<strong>in</strong>g released from the liver <strong>in</strong>to the blood stream (Rajas<strong>in</strong>gh et al., 2006). Astaxanth<strong>in</strong>has been reported to b<strong>in</strong>d to high density lipoprote<strong>in</strong>s <strong>in</strong> ra<strong>in</strong>bow trout (250 g fish -1 ) fed a dietsupplemented with 80 mg kg -1 of astaxanth<strong>in</strong> for 1 month (Choubert et al., 1995). Canthaxanth<strong>in</strong>was bound to three serum lipoprote<strong>in</strong> classes although was transported ma<strong>in</strong>ly by low densitylipoprote<strong>in</strong>s <strong>in</strong> ra<strong>in</strong>bow trout (195 g fish -1 ) fed a diet supplemented with 80 mg kg -1ofcanthaxanth<strong>in</strong> for 6 weeks (Salvador et al., 2009). In Atlantic salmon, astaxanth<strong>in</strong> was reportedto be transported by both high <strong>and</strong> low density lipoprote<strong>in</strong>s <strong>and</strong> bond to a serum prote<strong>in</strong>hypothesised to be album<strong>in</strong> (Aas et al., 1999).Astaxanth<strong>in</strong> is deposited <strong>in</strong> fish flesh <strong>in</strong> its free form (Schiedt et al., 1985). A subcellularfractionation study conducted <strong>in</strong> muscle samples from wild chum <strong>and</strong> sockeye salmon12

(Oncorhynchus keta <strong>and</strong> Oncorhynchus nerka, respectively), <strong>and</strong> farmed coho salmon(Oncorhynchus kisutch), demonstrated that carotenoids exist <strong>in</strong> myofibril filaments of musclecells, be<strong>in</strong>g part of a pigment-actomyos<strong>in</strong> complex (Henmi et al., 1987). Carotenoids areattached to the prote<strong>in</strong> surface through a weak hydrophobic bond. It was proposed that the β-ionone r<strong>in</strong>g from the pigment molecule is bound to the bottom of a hydrophobic hole <strong>in</strong> thehydrophobic b<strong>in</strong>d<strong>in</strong>g site on the actomyos<strong>in</strong> prote<strong>in</strong> complex, <strong>and</strong> that the pigment-prote<strong>in</strong>comb<strong>in</strong>ation strength is determ<strong>in</strong>ed by the number of hydrogen bonds (Henmi et al., 1989). Morerecently, the development of a method capable of detect<strong>in</strong>g aff<strong>in</strong>ity of myofibrillar prote<strong>in</strong>s forastaxanth<strong>in</strong> <strong>in</strong> muscle from Atlantic salmon, demonstrated that α-act<strong>in</strong><strong>in</strong> was the only prote<strong>in</strong>correlat<strong>in</strong>g significantly (p

Muscle growth <strong>in</strong> ra<strong>in</strong>bow trout is characterized by an <strong>in</strong>crease <strong>in</strong> the number of fibercells (hyperplasia) <strong>and</strong> hypertrophy (enlargement of cell size). Hyperplasia reaches a plateau at~40% of fork length, whereas hypertrophy accounts for growth until fish reach sexual maturation(Johnston et al., 2006). Whether the <strong>in</strong>crease <strong>in</strong> pigment deposition rate <strong>in</strong> salmonid fish isrelated to the <strong>in</strong>tense muscle fiber hypertrophy (<strong>and</strong> the subsequent <strong>in</strong>crease <strong>in</strong> contractile prote<strong>in</strong>act<strong>in</strong> <strong>and</strong> myos<strong>in</strong>) <strong>in</strong> fish rema<strong>in</strong>s to be determ<strong>in</strong>ed.When salmonid fish reach sexual maturation, carotenoids deposited <strong>in</strong> the flesh areredistributed to both the sk<strong>in</strong> <strong>and</strong> ovaries. As a result, a significant reduction <strong>in</strong> astaxanth<strong>in</strong>muscle concentration is observed <strong>in</strong> mature fish (Leclercq et al., 2010). In chum salmon areductive metabolic pathway for astaxanth<strong>in</strong> <strong>in</strong>to zeaxanth<strong>in</strong> has been determ<strong>in</strong>ed for mature<strong>in</strong>dividuals. Carotenoids were determ<strong>in</strong>ed to be transported with<strong>in</strong> lipoprote<strong>in</strong>s with<strong>in</strong> the bloodstream (Ando <strong>and</strong> Hatano, 1988).2.7 <strong>Corn</strong> fractionation <strong>and</strong> implications <strong>in</strong> animal nutrition2.7.1 <strong>Corn</strong> mill<strong>in</strong>g<strong>Corn</strong> (Zea mays L.), after rice (Oryza sativa L.) <strong>and</strong> wheat (Triticum sp. L., respectively),is the most important cereal gra<strong>in</strong> <strong>in</strong> the world. <strong>Corn</strong> world production is about 815 millionmetric tons (USDA, 2011). This gra<strong>in</strong> is usually processed by dry mill<strong>in</strong>g, alkal<strong>in</strong>e process<strong>in</strong>g,wet mill<strong>in</strong>g or dry gr<strong>in</strong>d process. Dry mill<strong>in</strong>g <strong>and</strong> alkal<strong>in</strong>e processed corn result <strong>in</strong> ‘human grade’products such as corn flour, corn meal, tortilla <strong>and</strong> related foods. The major product of the cornwet mill<strong>in</strong>g <strong>and</strong> dry gr<strong>in</strong>d processes is ethanol, even though many high quality by-products for14

food, feed <strong>and</strong> <strong>in</strong>dustrial purposes are obta<strong>in</strong>ed through these processes. Figures 2.2 <strong>and</strong> 2.3show a schematic representation of the corn wet <strong>and</strong> dry mill<strong>in</strong>g, respectively.<strong>Corn</strong> kernels are composed of three different sections: pericarp (ma<strong>in</strong>ly fibre), endosperm(starch <strong>and</strong> prote<strong>in</strong>s) <strong>and</strong> germ (prote<strong>in</strong>s <strong>and</strong> oil). Ma<strong>in</strong> prote<strong>in</strong>s found <strong>in</strong> corn kernel arealbum<strong>in</strong>s, globul<strong>in</strong>s, glutel<strong>in</strong> <strong>and</strong> ze<strong>in</strong>. Album<strong>in</strong>s <strong>and</strong> globul<strong>in</strong>s are conta<strong>in</strong>ed <strong>in</strong> the germ,glutel<strong>in</strong> is homogenously distributed between germ <strong>and</strong> endosperm, <strong>and</strong> ze<strong>in</strong> is conta<strong>in</strong>ed ma<strong>in</strong>ly<strong>in</strong> the endosperm (Shukla <strong>and</strong> Cheryan, 2001). Xanthophyll pigments <strong>in</strong> corn kernels areembedded with<strong>in</strong> prote<strong>in</strong> complexes. The total xanthophyll content <strong>in</strong> whole corn ranges from 11to 30 mg kg -1 (Moros et al., 2002).<strong>Corn</strong> gluten meal <strong>and</strong> corn gluten feed are the by-products from the wet mill<strong>in</strong>g withhigher prote<strong>in</strong> level (around 60% <strong>and</strong> 23% of prote<strong>in</strong> content on a dry matter basis, respectively).Dur<strong>in</strong>g the wet mill<strong>in</strong>g, naturally occurr<strong>in</strong>g xanthophylls rema<strong>in</strong> attached to the gluten fraction(ma<strong>in</strong>ly ze<strong>in</strong>) which is ma<strong>in</strong>ly recovered <strong>in</strong> corn gluten meal. Consequently, this commodityconta<strong>in</strong>s high levels (224 to 550 mg kg -1 on a dry matter basis) of yellow carotenoids (Park et al.,1997; Moros et al., 2002), ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>. <strong>Corn</strong> gluten feed on the other h<strong>and</strong>,conta<strong>in</strong>s a level of yellow pigment similar to that found <strong>in</strong> corn kernels (Tucker <strong>and</strong> Hargreaves,2004).Dried distiller gra<strong>in</strong>s (DDG) <strong>and</strong> DDG plus soluble (DDGS) are the dry gra<strong>in</strong> mill<strong>in</strong>g byproductswith higher prote<strong>in</strong> content (around 23%) (Shukla <strong>and</strong> Cheryan, 2001). Xanthophyllcontent <strong>in</strong> these by-products range from 22 to 40 mg kg -1 (Li et al., 2011).15

2.7.2 <strong>Corn</strong> gluten meal production<strong>Corn</strong> gluten meal is a high prote<strong>in</strong> (~60% crude prote<strong>in</strong>, on a dry matter basis), highlydigestible, by-product of the corn wet mill<strong>in</strong>g. <strong>Corn</strong> wet mill<strong>in</strong>g is an <strong>in</strong>dustrial ref<strong>in</strong><strong>in</strong>g process<strong>in</strong> which the four major constituents of the corn kernel (i.e. starch, germ, prote<strong>in</strong> <strong>and</strong> fiber) areisolated <strong>in</strong>to relatively pure fractions <strong>and</strong> transformed <strong>in</strong>to high quality products. Use of corn bythe wet mill<strong>in</strong>g process <strong>in</strong> the U.S. has grown an average of 1.2 million bushels (unit of dryvolume, 1 bushel = 35.2 litres) per month, reach<strong>in</strong>g up to 90 - 96 million bushels from January2007 to November 2009 (O’Brien, 2009).The major by-products from the corn wet mill<strong>in</strong>g used for animal feeds are steep liquor,bran, germ <strong>and</strong> gluten meal, represent<strong>in</strong>g about 30% of the total processed corn. An averageyield from the wet mill<strong>in</strong>g process gives 56% starch, 22% corn gluten feed, 3% corn gluten meal<strong>and</strong> 2% corn oil (Davis, 2001). Figure 2.2 shows the wet mill<strong>in</strong>g process overview. Briefly, afterclean<strong>in</strong>g <strong>and</strong> removal of impurities, corn kernels are placed <strong>in</strong>to large sta<strong>in</strong>less still tanks(steep<strong>in</strong>g tanks) to be soaked <strong>in</strong> steepwater (diluted sulphur dioxide solution) for 30-50 hours at52 -54 °C. Soak<strong>in</strong>g softens the kernel <strong>and</strong> the diluted sulphurous reduces the occurrence offermentation <strong>and</strong> assists <strong>in</strong> separation of the starch <strong>and</strong> prote<strong>in</strong> bounds (Galitsky et al., 2003).Dur<strong>in</strong>g soak<strong>in</strong>g, soluble nutrients from corn are transferred <strong>in</strong>to the steep water. This water isfurther evaporated <strong>in</strong> order to concentrate those nutrients <strong>and</strong> recovered them as condensed cornfermented extractives or corn steep liquor.After steep<strong>in</strong>g is completed, kernels are coarsely ground <strong>and</strong> oil-rich germ is removed.Oil is extracted from germ <strong>and</strong> the de-oiled germ leftovers are recovered as corn germ meal.Germless corn kernels are then f<strong>in</strong>ely ground <strong>and</strong> hulls are screen removed <strong>and</strong> recovered as corngluten feed.16

Rema<strong>in</strong><strong>in</strong>g starch <strong>and</strong> gluten are centrifuged to form a two layer system (denser starch <strong>in</strong>the hypo phase <strong>and</strong> lighter prote<strong>in</strong> <strong>in</strong> the epi phase). Prote<strong>in</strong>-rich gluten is further concentrated,dried <strong>and</strong> recovered as corn gluten meal. Starch is further processed <strong>and</strong> ref<strong>in</strong>ed for feed, food<strong>and</strong>/or <strong>in</strong>dustrial processes (i.e. ethanol, sweeteners, etc) (Davis, 2001).2.7.3 <strong>Corn</strong> gluten meal: nutritional value <strong>and</strong> use <strong>in</strong> animal diets<strong>Corn</strong> gluten meal (CMG) is a high prote<strong>in</strong> (60% m<strong>in</strong>imum), highly digestible by-productof the corn wet mill<strong>in</strong>g. Prote<strong>in</strong> from corn gluten meal is composed ma<strong>in</strong>ly of ze<strong>in</strong> (68%),glutel<strong>in</strong> (27%) <strong>and</strong> small amounts of globul<strong>in</strong>s (1.2%) (Cha et al., 2000). Compared to fish meal,corn gluten meal has an imbalanced am<strong>in</strong>o acid profile with lys<strong>in</strong>e, methion<strong>in</strong>e <strong>and</strong> tryptophanbe<strong>in</strong>g the more deficient ones compared to fish meal.CGM is a rich source of vitam<strong>in</strong> B complex <strong>and</strong> E source <strong>and</strong> conta<strong>in</strong>s low amounts ofphosphorus (Skonberg et al., 1998). Dewatered or wet CGM conta<strong>in</strong>s 35 – 40% solids. Howeverbefore be<strong>in</strong>g marketed it is dried to obta<strong>in</strong> a ~90% of dry matter content (Park et al., 1997).Due to its high nutritional value, low cost <strong>and</strong> relatively good availability, CGM isgenerally utilized as a feed <strong>in</strong>gredient for a wide variety of rum<strong>in</strong>ant <strong>and</strong> mono gastric animals<strong>in</strong>clud<strong>in</strong>g fish.2.7.4 Use of corn gluten meal <strong>in</strong> diets for terrestrial animals<strong>Corn</strong> gluten meal is widely utilized <strong>in</strong> diets for terrestrial animals, ma<strong>in</strong>ly due to its highprote<strong>in</strong> content, elevated yellow pigment level <strong>and</strong> relative low cost (<strong>Corn</strong> Ref<strong>in</strong>ers Association,17

2010). However, due to its imbalanced am<strong>in</strong>o acid profile, it must be mixed with differentcommodities or supplemented with synthetic am<strong>in</strong>o acids <strong>in</strong> order to reach am<strong>in</strong>o acidrequirements (Peter et al., 2000).CGM has been commonly <strong>in</strong>cluded <strong>in</strong> formulated diets for poultry as a yellowxanthophyll pigment source <strong>in</strong> order to enhance sk<strong>in</strong> <strong>and</strong> yolk pigmentation (Sasse <strong>and</strong> Baker,1973). Carotenoid content <strong>in</strong> eggs from birds fed a CGM based diet varied from 24 to 30 µg g -1 ,<strong>and</strong> xanthophyll concentration <strong>in</strong> body tissues <strong>and</strong> egg yolk are well correlated with those present<strong>in</strong> the diet (Surai et al., 2001).Inclusion of corn gluten meal <strong>in</strong> diets for grow<strong>in</strong>g chicks as a prote<strong>in</strong> source has showedno negative effects on growth. No differences <strong>in</strong> weight ga<strong>in</strong>, feed <strong>in</strong>take, or feed efficiency(ga<strong>in</strong>:feed ratio) were observed for chicks fed an am<strong>in</strong>o acid fortified CGM based diet (18%<strong>in</strong>clusion), compared to birds fed a corn-soybean positive control diet dur<strong>in</strong>g a 12 days trial(Peter et al., 2000).In rum<strong>in</strong>ant nutrition, CGM is considered an effective source of bypass (slowlydegrad<strong>in</strong>g) prote<strong>in</strong>. A comb<strong>in</strong>ation of CGM (3%) <strong>and</strong> dehydrated alfalfa (15%) resulted <strong>in</strong> ahigher daily ga<strong>in</strong> when compared to a soybean meal (9%) based diet <strong>in</strong> grow<strong>in</strong>g steers (200 kg,<strong>in</strong>itial body weight) dur<strong>in</strong>g a 112 days growth trial (Rock et al., 1983).In dairy cows, a dietary <strong>in</strong>clusion of 6% of CGM <strong>and</strong> blood meal <strong>in</strong> a 50:50 comb<strong>in</strong>ationratio, resulted <strong>in</strong> multiparous Holste<strong>in</strong> cows (614 kg, average <strong>in</strong>itial weight) consum<strong>in</strong>g more drymatter <strong>and</strong> produc<strong>in</strong>g milk with higher prote<strong>in</strong> percentage compared to those fed a control dietbased conta<strong>in</strong><strong>in</strong>g urea (0.2%) after a four week trial period (De Gracia et al., 1989).18

CGM is <strong>in</strong>cluded <strong>in</strong> low amounts (less than 5%) <strong>in</strong> diets for sows <strong>and</strong> grow<strong>in</strong>g pigs. Drydiets for cats have are usually supplemented with corn gluten meal due to its am<strong>in</strong>o acidscomposition (Funaba et al., 2002).2.8 Use of corn gluten meal <strong>in</strong> diets for fishCGM <strong>in</strong>clusion <strong>in</strong> formulated diets for fish results <strong>in</strong> feed <strong>in</strong>take, nutrient digestibility<strong>and</strong> growth rates similar to those obta<strong>in</strong>ed with fish meal based diets. Due to its imbalancedam<strong>in</strong>o acid profile, CGM is generally blended with complementary <strong>in</strong>gredients <strong>in</strong> order to<strong>in</strong>crease limit<strong>in</strong>g am<strong>in</strong>o acid availability (especially lys<strong>in</strong>e) <strong>and</strong> reach species specificrequirements.No differences <strong>in</strong> weight ga<strong>in</strong> <strong>and</strong> higher prote<strong>in</strong> productive value (PPV) compare to afish meal based diet, were obta<strong>in</strong>ed with ra<strong>in</strong>bow trout (2 g fish -1 <strong>in</strong>itial body weight) fed a fishmeal free diet conta<strong>in</strong><strong>in</strong>g corn gluten meal (20%), poultry by-product meal (30%) <strong>and</strong> carob seedgerm meal (12%) as a prote<strong>in</strong> source <strong>and</strong> supplemented with lys<strong>in</strong>e (2%) for 197 days (Alexis etal., 1985). No significant effects (P>0.05) <strong>in</strong> f<strong>in</strong>al weight, feed conversion rate (FCR), prote<strong>in</strong>efficiency ratio (PER) or PPV were reported for ra<strong>in</strong>bow trout (30g fish -1 <strong>in</strong>itial body weight) fedgraded levels (20, 30 <strong>and</strong> 40%) of corn gluten meal <strong>in</strong> a 8-week growth trial (Moyano et al.,1991). Significantly lower (p

No differences <strong>in</strong> f<strong>in</strong>al weight, feed conversion rate or apparent prote<strong>in</strong> digestibility weredeterm<strong>in</strong>ed for Atlantic salmon (15 g fish -1 <strong>in</strong>itial body weight) fed diets supplemented up to50% with corn gluten meal <strong>and</strong> no synthetic am<strong>in</strong>o acid supplementation dur<strong>in</strong>g a 40-day trial.However, high fish meal <strong>in</strong>clusion <strong>in</strong> all experimental diets (27% <strong>in</strong> the diet with highest CGM<strong>in</strong>clusion) <strong>in</strong> this trial may expla<strong>in</strong> that Atlantic salmon lys<strong>in</strong>e requirement was reached (Menteet al., 2003).Inclusion of CGM <strong>in</strong> diets for carnivorous fish (other than salmonids), with no negativeeffects on growth parameters, has been reported to be feasible up to a certa<strong>in</strong> level. Graded levelsof CGM (11, 16 <strong>and</strong> 21%) <strong>and</strong> no am<strong>in</strong>o acid supplementation <strong>in</strong> diets for sea bream (Sparusaurata), 40g fish -1 <strong>in</strong>itial body weight, did not show significant (p>0.05) differences <strong>in</strong> f<strong>in</strong>albody weight, prote<strong>in</strong> efficiency ratio (PER), prote<strong>in</strong> productive value (PPV), f<strong>in</strong>al carcassproximate composition, liver histology or total, prote<strong>in</strong> <strong>and</strong> lipids ADC compared to fish fed afish meal based control after a 13-week growth trial (Roba<strong>in</strong>a et al., 1997).No significant differences (p>0.05) <strong>in</strong> f<strong>in</strong>al body weight, st<strong>and</strong>ard growth rate or feedefficiency were observed <strong>in</strong> turbot (Psetta maxima) 66 g fish -1 <strong>in</strong>itial body weight, fed a dietsupplemented with 20% CGM <strong>and</strong> no am<strong>in</strong>o acid supplementation compared to those fish fed afish meal based control. However, growth parameters <strong>and</strong> ADC for prote<strong>in</strong> <strong>and</strong> energy weresignificantly (p

2.9 <strong>Effects</strong> of dietary corn gluten meal on salmonid fish muscle pigmentationAnecdotal evidence from fish farmers along with a small number of scientific studies suggesta negative effect of dietary CGM on muscle astaxanth<strong>in</strong> deposition <strong>and</strong> colour attributes <strong>in</strong> filletsfrom salmonid fish. This phenomenon has been related to the high content of yellow xanthophyllcarotenoids, ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, present <strong>in</strong> this commodity (220 to 550 mg kg -1 on adry matter basis). Naturally occurr<strong>in</strong>g xanthophylls from CGM have been proposed to<strong>in</strong>teract/compete with astaxanth<strong>in</strong> dur<strong>in</strong>g pigment metabolism (digestion, absorption, transport<strong>and</strong>/or deposition) due to hydrophobic properties <strong>and</strong> molecular characteristics shared by thesecarotenoids (Aas et al., 1999; Furr <strong>and</strong> Clark, 1997; Henmi et al., 1987; Matthews et al., 2006;Salvador et al., 2007).Deposition of different types of pigments <strong>in</strong> muscle from salmonid fish has been previouslyreported. Results from a subcellular fractionation study us<strong>in</strong>g salmon muscle samples <strong>in</strong>dicatedthat actomyos<strong>in</strong> (astaxanth<strong>in</strong> b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> complex <strong>in</strong> salmonids) is able to comb<strong>in</strong>e withcarotenoids other than astaxanth<strong>in</strong> or canthaxanth<strong>in</strong>, i.e. lute<strong>in</strong>, zeaxanth<strong>in</strong> <strong>and</strong> β-carotene(Henmi et al., 1987; 1989). Zeaxanth<strong>in</strong> has been reported to occur <strong>in</strong> muscle from wild masu(Oncorhynchus masou B.), coho (Oncorhynchus kisutch W.), p<strong>in</strong>k (Oncorhynchus gorbuschaW.), sockeye (Oncorhynchus nerka W.) <strong>and</strong> chum (Oncorhynchus keta W.) salmon (Kitahara1983, 1984). On the other h<strong>and</strong>, lute<strong>in</strong> (3 mg kg -1 ) was determ<strong>in</strong>ed <strong>in</strong> muscle from ra<strong>in</strong>bow trout(360 g fish -1 ) fed an astaxanth<strong>in</strong>-free diet supplemented with 8 mg kg -1 of lute<strong>in</strong> from algal orig<strong>in</strong>(Caldophora glomerata) for 40 days (Welker et al., 2001).A significant (p

supplemented with 42% of a vegetable prote<strong>in</strong> blend (corn gluten meal <strong>and</strong> full fat soybean meal<strong>in</strong> a 2:1 ratio) <strong>and</strong> 64 mg kg -1 astaxanth<strong>in</strong> dur<strong>in</strong>g a 11-week growth trial (Mundheim et al., 2004).Ra<strong>in</strong>bow trout (320g fish -1 , body weight) fed a diet supplemented with 23% CGM <strong>and</strong> nosynthetic pigment <strong>in</strong>clusion for 12 weeks showed fillets with yellowish appearance (b* values,tristimulus color analysis) (Skonberg et al., 1998). Conversely, <strong>in</strong>clusion of 23% of syntheticlute<strong>in</strong> did not significantly affect (P>0.05) astaxanth<strong>in</strong> blood level <strong>and</strong> muscle deposition <strong>in</strong>Atlantic salmon (1260 g f<strong>in</strong>al body weight) fed a diet supplemented with 54 mg kg -1 of <strong>and</strong>astaxanth<strong>in</strong>, respectively <strong>in</strong> a 138-days study. However, a non-significant (p>0.05) tendency oflower<strong>in</strong>g both muscle <strong>and</strong> blood astaxanth<strong>in</strong> content was observed, moreover lute<strong>in</strong> content <strong>in</strong>blood <strong>and</strong> muscle showed a significant positive correlation with dietary <strong>in</strong>clusion level (Olsen<strong>and</strong> Baker, 2006).2.10 Bleach<strong>in</strong>g of yellow xanthophylls from plant prote<strong>in</strong> <strong>in</strong>gredients2.10.1 Enzymatic treatments2.10.1.1 Natural bleach<strong>in</strong>g<strong>Reduction</strong> of colour <strong>in</strong> flours by ag<strong>in</strong>g is a practice used by millers for centuries. <strong>Pigment</strong>content <strong>in</strong> flour would be bleached <strong>in</strong> approximately 2 to 3 weeks <strong>in</strong> a process called atmosphericoxidation (Saunders et al., 2008, Mercier <strong>and</strong> Gel<strong>in</strong>as, 2001). Due to the long periods of time <strong>in</strong>storage needed for the bleach<strong>in</strong>g reaction to complete, this method is less applicable on acommercial scale.22

2.10.1.2 LipoxygenasesLipoxygenases (LOX) (l<strong>in</strong>olate:oxygen oxidoreductase, EC 1.13.11.12) are nonheme,iron-conta<strong>in</strong><strong>in</strong>g enzymes that catalyze the regio <strong>and</strong> stereo selective dioxygenation ofpolyunsaturated fatty acids conta<strong>in</strong><strong>in</strong>g a (Z,Z)-1,4-pentadiene system. This oxidative reactionproduces an optically active, conjugated (Z,E) diene hydroperoxy derivative (Barone et al., 1999;Gӧkmen et al., 2002; Junqueira et al., 2007). LOX is widely distributed <strong>in</strong> plants <strong>and</strong> animals(Axelrod et al., 1981). Plant-orig<strong>in</strong> LOX have been related to flavour <strong>and</strong> odour formation, fruitripen<strong>in</strong>g, <strong>and</strong> wound repair among many other physiological functions. In animals, lipoxygenaseforms precursors for chemical messengers such as leukotrienes or lipox<strong>in</strong>s (Barone et al., 1999;Saunders et al., 2008).On a molecular basis, the lipoxygenase-catalyzed lipid oxidation is characterized by theabstraction of a stereospecific hydrogen from a poly-unsaturated fatty acid (position 3 of thepenta-1,4-diene system with<strong>in</strong> the backbone of the lipid molecule). The <strong>in</strong>termediate fatty acidradical (generated after the hydrogen withdrawal) reacts with molecular oxygen (triplet state) toform a peroxy radical product. The oxidative reaction is completed when the peroxy radical istransformed <strong>in</strong>to a peroxy anion <strong>and</strong> further protonated to give a hydroperoxide molecule.Figure 2.4 shows a schematic representation of the lipoxygenase mediated fatty acid oxidation(Taylor <strong>and</strong> Morris, 1983). The <strong>in</strong>termediate peroxy radical products formed dur<strong>in</strong>g the LOXcatalizedreactions are able to co-oxidize pigment molecules. The chromophore arrangementwith<strong>in</strong> the pigment molecule is cleaved. Consequently its chemical properties are changed <strong>and</strong>colour <strong>in</strong>tensity is reduced.23

Soybean seeds are the richest known source of lipoxygenase. Four (4) isoforms oflipoxygenase have been isolated from this raw material: L1, L2, L3a, <strong>and</strong> L3b. L3a <strong>and</strong> L3b arevery similar <strong>in</strong> their properties for determ<strong>in</strong>ation purposes both are usually referred to as L3(Axelrod et al., 1981). Different lipoxygenase isoforms differ <strong>in</strong> pH optima, substrate specificity<strong>and</strong> product formation. L1 optimum pH is ~ 9.0 <strong>and</strong> is characterized by a low capacity forbleach<strong>in</strong>g of pigment. On the other h<strong>and</strong>, L2 <strong>and</strong> L3 have a pH optimum of 6.1 <strong>and</strong> 6.5,respectively, <strong>and</strong> they show a high pigment bleach<strong>in</strong>g properties (Junqueira et al., 2007). Basedon its pigment bleach<strong>in</strong>g capacity, hereafter L3 lipoxgenase iso-enzyme is go<strong>in</strong>g to be referred aslipoxygenase (LOX).L<strong>in</strong>oleic acid is the specific substrate for lipoxygenase <strong>and</strong> its presence is essential forlipid oxidation <strong>and</strong> the subsequent co-oxidation reactions to occur. Substantial pigment oxidation(expressed as pigment disappearance rate) was observed only when a lipoxygenase crude extractfrom pepper fruit <strong>and</strong> pure l<strong>in</strong>oleic acid were simultaneously comb<strong>in</strong>ed <strong>in</strong> the reaction mediumdur<strong>in</strong>g a lipoxygenase catalyzed pigment bleach<strong>in</strong>g study. On the other h<strong>and</strong>, m<strong>in</strong>imal pigmentreduction was observed when each oxidant promoter was tested separately or when pigment autooxidation was evaluated (Jaren-Galan <strong>and</strong> M<strong>in</strong>guez-Mosquera, 1999). The rate of lipoxygenasecatalizedpigment co-oxidation is dependent on substrate concentration. At fixed l<strong>in</strong>oleic acid(l<strong>in</strong>oleate) concentration, soybean lipoxygenase showed a reduction <strong>in</strong> both conjugated dienesformation <strong>and</strong> bleach<strong>in</strong>g rate of β- carotene <strong>and</strong> chlorophyll a dur<strong>in</strong>g a pigment bleach<strong>in</strong>g assay(Cohen et al., 1985).Lipoxygenase activity is dependent on oxygen availability <strong>in</strong> the reaction medium. Verylow enzymatic activity was determ<strong>in</strong>ed <strong>in</strong> an anaerobic environment (compared to those obta<strong>in</strong>ed<strong>in</strong> aerobic conditions) when the <strong>in</strong>teraction between soybean lipoxygenase <strong>and</strong> pigments (β-24

carotene <strong>and</strong> chlorophyll a) was addressed <strong>in</strong> a study with different LOX iso-enzymes (Cohen etal., 1985).2.10.1.3 Bleach<strong>in</strong>g of yellow xanthophylls us<strong>in</strong>g lipoxygenaseResearch focus<strong>in</strong>g on the lipoxygenase-catalyzed pigment co-oxidation has beenconducted for many years, primarily due to its applications <strong>in</strong> vegetable <strong>in</strong>gredient process<strong>in</strong>g,primarily for the bakery <strong>and</strong> pasta <strong>in</strong>dustries. More than 66% of carotenoid (lute<strong>in</strong> <strong>and</strong>zeaxanth<strong>in</strong>) content was reduced <strong>in</strong> whole gra<strong>in</strong> bread wheat (Triticum aestivum) flour (<strong>in</strong>itialcarotenoid content of 1 µg g -1 ) after knead<strong>in</strong>g dur<strong>in</strong>g a bread mak<strong>in</strong>g trial, the oxygen <strong>in</strong>crease <strong>in</strong>the dough is hypothesised to facilitate the lipoxygenase catalized carotenoid oxidation.Furthermore a l<strong>in</strong>ear positive relationship (r 2 = 0.97) was determ<strong>in</strong>ed between carotenoid loss (%)dur<strong>in</strong>g knead<strong>in</strong>g <strong>and</strong> lipoxygenase activity <strong>in</strong> 3 Triticum wheat species (Leenhardt et al., 2006).A highly significant (p

2.10.1.4 Bleach<strong>in</strong>g of yellow pigment from corn gluten meal us<strong>in</strong>g lipoxygenaseWet (67% moisture basis) corn gluten meal was bleached us<strong>in</strong>g soy flour as alipoxygenase source. CGM (95%) <strong>and</strong> soy flour (5%) were mixed on a 20g scale <strong>and</strong> 75 mL ofdistilled water were added to create slurry. 48% carotenoids (determ<strong>in</strong>ed us<strong>in</strong>g AACC method14-50) were bleached when the slurry was stirred for 120 m<strong>in</strong> at pH 6.5. Aeration through thereaction medium <strong>in</strong>creased pigment bleach<strong>in</strong>g whereas soy flour pre-soak<strong>in</strong>g resulted <strong>in</strong> very lowpigment reduction, nonetheless a synergistic effect was observed when these two factors wereapplied simultaneously reach<strong>in</strong>g a 52% of carotenoids loss. Carotenoid bleach<strong>in</strong>g sped up dur<strong>in</strong>gthe reaction first m<strong>in</strong>utes, when soy flour <strong>in</strong>clusion level was <strong>in</strong>creased to 10 <strong>and</strong> 15%, howeverthis difference was not observed after 30 m<strong>in</strong>. Lipoxygenase activity ranged from 5 to 10 unitsmg of sample, however small differences <strong>in</strong> carotenoid bleach<strong>in</strong>g (1 to 3%) were observed.Spray-dry<strong>in</strong>g (130 °C for 60 m<strong>in</strong>) resulted <strong>in</strong> additional 18% of carotenoid bleach<strong>in</strong>g (Park et al.,1997).Us<strong>in</strong>g the proposed methodology of Park et al., (1997), the effect of different factors (pHvariation, lipoxygenase activity, spray dry<strong>in</strong>g temperature, reaction time, experimental scale <strong>and</strong>stirr<strong>in</strong>g speed) on wet CGM lipoxygenase <strong>in</strong>duce pigment bleach<strong>in</strong>g was studied us<strong>in</strong>g acomplete r<strong>and</strong>omized design. 65% of carotenoids from wet corn gluten meal (520 mg·kg -1 <strong>in</strong>itialcarotenoid content) were bleached at pH 6.5. <strong>Pigment</strong> reduction was 19% <strong>and</strong> 15% lower whenpH was acidified (pH 5.0) <strong>and</strong> alkal<strong>in</strong>ized (pH 8), respectively. No effect of <strong>in</strong>creas<strong>in</strong>g reactionscale up to 2500 g was observed as long as appropriate mechanical stirr<strong>in</strong>g level was applied.Increas<strong>in</strong>g of mechanical stirr<strong>in</strong>g resulted <strong>in</strong> higher pigment reduction (70% pigment bleach<strong>in</strong>g),<strong>and</strong> pigment bleach<strong>in</strong>g maximum rates were reached faster. Spray-dry<strong>in</strong>g <strong>in</strong>duced additionalpigment bleach<strong>in</strong>g <strong>in</strong> wet corn gluten meal from 9% to 26% when temperature was <strong>in</strong>creased26

from 160 to 250 °C. Addition of soy flour to CGM resulted <strong>in</strong> a slight <strong>in</strong>crease <strong>in</strong> prote<strong>in</strong> content<strong>and</strong> improv<strong>in</strong>g wet CGM am<strong>in</strong>o acid balance, ea. lys<strong>in</strong>e (Cha et al., 2000).2.10.2 Other enzymesA total reduction of β-carotene from bread dough (prepared with 100 g of wheat flour)was obta<strong>in</strong>ed with 3,000 <strong>and</strong> 815 U of peroxidase (from soybeans) <strong>and</strong> lipase <strong>in</strong> the presence ofl<strong>in</strong>oleic acid (Gel<strong>in</strong>as et al., 1998).2.10.3 Chemical reagents as pigment bleach<strong>in</strong>g agents2.10.3.1 Benzoyl peroxideBenzoyl peroxide is widely utilized <strong>in</strong> mill<strong>in</strong>g plants from Canada <strong>and</strong> the U.S. as ableach<strong>in</strong>g agent for the yellow pigment content <strong>in</strong> wheat flour (Mercier <strong>and</strong> Gel<strong>in</strong>as, 2001). Thisproduct is a free radical <strong>in</strong>itiator that <strong>in</strong>duces the oxidation of carotenoid molecules by a typicalfree radical reaction. Dur<strong>in</strong>g pigment bleach<strong>in</strong>g, part of the benzoyl peroxide is converted <strong>in</strong>tobenzoic acid (Saiz et al., 2001).2.10.3.2 Hydrogen peroxideHydrogen peroxide (HP) is widely utilized as a food additive. Alkal<strong>in</strong>e HP (pH 11.5)reduced more than half of the colour (expressed as colour difference) <strong>in</strong> wheat distillers’ gra<strong>in</strong>,when 60 g of this commodity was suspended <strong>in</strong> 300 ml of an aqueous HP solution (0.3 M) for 2hours (Abdel-Aal et al., 1996).27

2.11 ConclusionInclusion of <strong>in</strong>creas<strong>in</strong>g levels of cost-effective plant-orig<strong>in</strong> commodities <strong>in</strong>to formulated dietsfor salmonid fish is a common practice widely utilized <strong>in</strong> order to susta<strong>in</strong> <strong>in</strong>creas<strong>in</strong>g productionlevels.Colour <strong>in</strong> fillet from salmonid fish is determ<strong>in</strong>ed by the deposition of carotenoid pigments,ma<strong>in</strong>ly astaxanth<strong>in</strong>, with<strong>in</strong> fiber muscles. This colour attribute plays a key role on consumer’sperception as well as on will<strong>in</strong>gness to pay. Salmonid fish are unable to synthetize carotenoid denovo, hence formulated diets are supplemented with expensive pigment premix.Anecdotal evidence from fish farmers along with a few scientific studies support a reduction<strong>in</strong> astaxanth<strong>in</strong> deposition <strong>and</strong> reduction <strong>in</strong> color attributes <strong>in</strong> fillets from ra<strong>in</strong>bow trout fed dietsconta<strong>in</strong><strong>in</strong>g corn gluten meal. This commodity, a highly digestible high prote<strong>in</strong> (~60% on a drymatter basis) by-product of the corn wet mill<strong>in</strong>g conta<strong>in</strong><strong>in</strong>g substantial levels of yellowxanthophylls (200 – 550 mg kg -1 ), ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>. The effects related to the<strong>in</strong>clusion of substantial levels of corn gluten meal on muscle pigmentation need to be addressed<strong>in</strong> order to avoid potential reduction <strong>in</strong> astaxanth<strong>in</strong> utilization <strong>and</strong> colour expression <strong>in</strong> muscleform ra<strong>in</strong>bow trout.The development of straight-forward/cost-effective process<strong>in</strong>g techniques, aim<strong>in</strong>g to reduceyellow xanthophylls content <strong>in</strong> corn gluten meal <strong>and</strong> to <strong>in</strong>crease this commodity’s nutritionalvalue for salmonid fish are highly necessary. The use of common bleach<strong>in</strong>g agents, eitherapplied directly to corn gluten meal or to corn kernels dur<strong>in</strong>g the corn wet mill<strong>in</strong>g process canbecome a practical tool <strong>in</strong> order to <strong>in</strong>crease the feed<strong>in</strong>g value of corn gluten meal.28

FiguresFigure 2. 1 - Molecular structure of some important carotenoids founds <strong>in</strong> nature.29

4.0%*68%* 5.6%* 12%* 3.5%* 6.5%*Figure 2. 2 - Schematic representation of the wet mill<strong>in</strong>g of corn. * Typical f<strong>in</strong>al product yields.30

**44%* 39%* 3%*Figure 2. 3 - Schematic representation of the dry mill<strong>in</strong>g of corn. * Typical f<strong>in</strong>al product yields.** Approximately 10 L of ethyl alcohol are produced from 1 bushel (35.2 L) of corn kernels.31

Figure 2. 4 - Schematic representation of the mode of action of lipoxygenase dur<strong>in</strong>g lipidoxidation (Taylor <strong>and</strong> Morris 1983). a) Penta-1,4-cis-diene system with<strong>in</strong> l<strong>in</strong>oleic acid, b)Intermediate peroxy radical responsible for carotenoid pigments co-oxidation, c) hydroperoxide,the f<strong>in</strong>al product of the enzyme-catalyzed lipid oxidation.32

CHAPTER - 3 OPTIMIZATION OF THE REDUCTION OF CAROTENOIDS IN CORNGLUTEN MEAL FOR EVALUATION ON GROWTH AND MUSCLE PIGMENTATIONOF RAINBOW TROUT (Oncorhynchus mykiss)Abstract<strong>Corn</strong> gluten meal (CGM) is a common feed <strong>in</strong>gredient that is widely <strong>in</strong>cluded <strong>in</strong> aquaculturediets. CGM has been related to the reduction <strong>in</strong> pigmentation <strong>in</strong> the muscle of salmonid fish dueto <strong>in</strong>terference among carotenoids. Therefore, a bench-scale study was carried out to optimizethe reduction of lute<strong>in</strong>, zeaxanth<strong>in</strong>, β-cryptoxanth<strong>in</strong> <strong>and</strong> β-carotene <strong>in</strong> CGM, us<strong>in</strong>g white soyflake flour lipoxygenase as bleach<strong>in</strong>g agent. A 12-week growth trial was conducted to assess theeffects of regular or treated CGM on growth <strong>and</strong> muscle astaxanth<strong>in</strong> deposition <strong>in</strong> ra<strong>in</strong>bow trout(Oncorhynchus mykiss). Results <strong>in</strong>dicated that lute<strong>in</strong>, zeaxanth<strong>in</strong>, β-cryptoxanth<strong>in</strong> <strong>and</strong> β-carotenewere reduced by 86%, 97%, 100% <strong>and</strong> 100%, respectively <strong>in</strong> the treated CGM. Highly reactiveperoxy radicals produced dur<strong>in</strong>g the reduction of yellow carotenoids <strong>in</strong> the treated CGM <strong>in</strong>ducedlipid rancidity <strong>and</strong> the oxidative destruction of astaxanth<strong>in</strong> <strong>in</strong> the diet supplemented with thiscommodity. A significant (p

3.1 IntroductionThe characteristic p<strong>in</strong>k or red colour <strong>in</strong> fillets of salmonid fish is the result of depositionof dietary carotenoids pigments with<strong>in</strong> muscle cells. This quality trait dramatically affects f<strong>in</strong>alproduct quality <strong>and</strong> <strong>in</strong>fluences costumers’ perception. Fish are unable to synthesise carotenoidsde novo, therefore formulated diets for farmed salmonid fish must be supplemented withsynthetic or natural-orig<strong>in</strong> carotenoids, ma<strong>in</strong>ly astaxanth<strong>in</strong>. These pigments are expensive <strong>and</strong>represent about 10-20% of total feed costs (Bjerkeng, 2000; Choubert et al., 2009).Aquaculture feed formulations have evolved to lessen dependence on expensive<strong>in</strong>gredients derived from mar<strong>in</strong>e resources by <strong>in</strong>creas<strong>in</strong>g <strong>in</strong>clusion level of cost-effective plantorig<strong>in</strong>commodities as alternatives prote<strong>in</strong> sources. <strong>Corn</strong> gluten meal (CGM), a prote<strong>in</strong> rich (60%crude prote<strong>in</strong>), highly palatable <strong>and</strong> digestible by-product of the corn (maize) wet mill<strong>in</strong>g, iscommonly <strong>in</strong>cluded <strong>in</strong> formulated feeds for many farmed fish. <strong>Corn</strong> wet mill<strong>in</strong>g is a process bywhich the ma<strong>in</strong> structural components of corn kernels (i.e. starch, gluten, germ <strong>and</strong> fiber) areseparated <strong>in</strong>to different fractions <strong>and</strong> further ref<strong>in</strong>ed to manufacture high quality products forfood, feed <strong>and</strong> <strong>in</strong>dustrial uses. Dur<strong>in</strong>g corn wet mill<strong>in</strong>g yellow xanthophylls present with<strong>in</strong>endosperm are recovered <strong>in</strong> CGM, consequently this commodity frequently conta<strong>in</strong>s significantamounts of these carotenoids (200-500 mg kg -1 ), ma<strong>in</strong>ly as lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> (Skonberg etal., 1998; Park et al., 1997).Anecdotal evidence from fish farmers along with a small number of scientific studieshave reported lower astaxanth<strong>in</strong> deposition rates as well as higher yellow colour attributes <strong>in</strong>fillets of salmonid fish fed formulated diets conta<strong>in</strong><strong>in</strong>g substantial amounts of CGM (Mundheimet al., 2004; Skonberg et al., 1998). A significant (p

us<strong>in</strong>g SalmoFan colorimetric analysis) was observed <strong>in</strong> fillets of Atlantic salmon (Salmosalar) as graded levels of a vegetable prote<strong>in</strong> blend (CGM <strong>and</strong> full fat soybean meal <strong>in</strong> a 2:1ratio) were <strong>in</strong>cluded <strong>in</strong> diets supplemented with 64 mg kg -1 of astaxanth<strong>in</strong> (Mundheim et al.,2004). Fillets of ra<strong>in</strong>bow trout fed a high CGM (22.5%) diet with no pigment supplementationfor 12 weeks presented yellowish coloration (b* values, tristimulus color analysis) comparedwith those from fish fed a high CGM (22.5%) supplemented with 100 mg kg -1 syntheticcanthaxanth<strong>in</strong> (Skonberg et al., 1998).Before absorption through <strong>in</strong>test<strong>in</strong>al epithelial cells, carotenoids are emulsified <strong>and</strong>further solubilized <strong>in</strong>to lipid mixed micelles, assembled <strong>in</strong>to chilomicrons (very low densitylipoprote<strong>in</strong>s) with<strong>in</strong> enterocytes, released <strong>in</strong>to the blood stream <strong>and</strong> eventually bound to an α-act<strong>in</strong> molecule with<strong>in</strong> muscle fiber (Aas et al., 1999; Furr <strong>and</strong> Clark, 1997; Henmi et al., 1987;Matthews et al., 2006; Salvador et al., 2007). Based on hydrophobic properties <strong>and</strong> molecularsimilarities shared by astaxanth<strong>in</strong> <strong>and</strong> xanthophylls from CGM <strong>and</strong> the reported presence oflute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> with<strong>in</strong> salmonid fish muscle (Kitahara, 1983), a potential<strong>in</strong>teraction/competition among these carotenoids dur<strong>in</strong>g <strong>in</strong>test<strong>in</strong>al absorption, transport with<strong>in</strong>bloodstream <strong>and</strong>/or muscle deposition can be hypothesized when substantial amounts of thiscommodity are used <strong>in</strong> formulated diets for salmonid fish.Lipoxygenases (LOX) are nonheme iron-conta<strong>in</strong><strong>in</strong>g dioxygenases (EC 1.13.11.12) thatcatalyze the oxygenation of polyunsaturated fatty acids conta<strong>in</strong><strong>in</strong>g a cis-cis-1, 4-pentadienesystem. LOX-catalyzed bleach<strong>in</strong>g of carotenoids from CGM has been successfully achieved <strong>in</strong>the past. More than 60% of total carotenoids pigments content was bleached when 5% of soyflour as LOX source was mixed with wet CGM <strong>in</strong> an aqueous medium at pH 6.5 (Cha et al.,2000; Park et al., 1997). <strong>Pigment</strong> bleach<strong>in</strong>g ability of LOX has been reported as dependent upon35

many factors such as LOX source level, LOX source pre-soak<strong>in</strong>g, aeration through the reactionmedium, ascorbic acid addition <strong>and</strong> reaction temperature (Park et al., 1997).Therefore, the objectives of this study were to (1) optimize the reduction of lute<strong>in</strong>, zeaxanth<strong>in</strong>, β-cryptoxanth<strong>in</strong> <strong>and</strong> β-carotene <strong>in</strong> CGM through a practical <strong>and</strong> cost-effective pigment bleach<strong>in</strong>gprocess, us<strong>in</strong>g white soy flake flour (WSFF) LOX as bleach<strong>in</strong>g agent <strong>and</strong> (2) assess the effects ofregular <strong>and</strong> treated CGM on growth <strong>and</strong> muscle pigmentation of ra<strong>in</strong>bow trout.3.2 Materials <strong>and</strong> methods3.2.1 Bleach<strong>in</strong>g of carotenoidsCGM (Casco Canada, London, ON, Canada) was bleached us<strong>in</strong>g a bench-scale proceduredeveloped accord<strong>in</strong>g to Cha et al (2000) with some modifications. CGM (20 g) was suspended <strong>in</strong>distilled water (75 ml) <strong>in</strong> a 250 ml Erlenmeyer flask. Solution pH was adjusted to 6.5 <strong>and</strong> whitesoy flakes flour (WSFF) (Bunge Canada, Hamilton, ON, Canada) was added to the mixture;flasks were then placed on a shak<strong>in</strong>g water bath (120 m<strong>in</strong>, 110 r m<strong>in</strong> -1 ). Potential factorsaffect<strong>in</strong>g bleach<strong>in</strong>g of pigments from CGM were applied to the slurry as described <strong>in</strong> theexperimental design section.Samples of the slurry were taken at 0, 60, <strong>and</strong> 120 m<strong>in</strong> of reaction progress <strong>and</strong>centrifuged at 4,000 x g per 20 m<strong>in</strong> (Eppendorf, centrifuge 5810r). Supernatant was discarded<strong>and</strong> result<strong>in</strong>g sediment was kept frozen (-20 °C) until analysis. Heat-treated (deactivated) WSSF,prepared by heat<strong>in</strong>g soy WSFF <strong>in</strong> an oven at 85 °C for 90 m<strong>in</strong>, was used as a control. Carotenoidbleach<strong>in</strong>g rate was determ<strong>in</strong>ed as the ratio of carotenoid content <strong>in</strong> processed samples (i.e. 60 or120 m<strong>in</strong>) to the pigment concentration <strong>in</strong> the <strong>in</strong>itial sample (0 m<strong>in</strong>). All trials were run <strong>in</strong>triplicate.36

Experimental designs. A Plackett-Burman Screen<strong>in</strong>g Design (PBSD) was used toevaluate significance of seven potential factors affect<strong>in</strong>g bleach<strong>in</strong>g of carotenoids from CGM.Seven variables or factors were tried out <strong>in</strong> this experiment. For each variable a low (-) <strong>and</strong> high(+) level were tested (Table 3.1). The rows <strong>in</strong> Table 3.2 represent different trials or scenarios,<strong>and</strong> each column represents a different variable or factor. A Box-Behnken design (BBD) wasapplied to determ<strong>in</strong>e the quadratic effect of factors screened as active on CGM carotenoidsbleach<strong>in</strong>g. This response surface design led to study the effect of the four factors <strong>in</strong> a s<strong>in</strong>gleblock of 27 sets of test conditions with three central po<strong>in</strong>ts. Experiments order was fullyr<strong>and</strong>omized. Three levels were attributed to each factor (low, medium <strong>and</strong> high) coded as -1, 0,+1 (Table 3.3).3.2.2 Fish muscle pigmentation trialFish husb<strong>and</strong>ry <strong>and</strong> experimental conditions. Ra<strong>in</strong>bow trout (O. mykiss) obta<strong>in</strong>ed fromthe Alma Aquaculture Research Station (Elora, ON, Canada) were adapted to experimentalconditions for two weeks before start<strong>in</strong>g the trial. Dur<strong>in</strong>g that period, fish were fed a commercialdiet (Mart<strong>in</strong> Mills Inc, Elora, ON, Canada) once per day.Groups of twenty five fish (125 g fish -1 , <strong>in</strong>itial body weight) were r<strong>and</strong>omly distributed<strong>in</strong>to 6 (500 L) fibreglass tanks <strong>and</strong> the 3 experimental diets were assigned r<strong>and</strong>omly to tanks.Fish were ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> a fresh water recirculated system, provided with filtered well water at anapproximately 10 L m<strong>in</strong> -1 rate. Water was cont<strong>in</strong>uously aerated <strong>and</strong> temperature was controlledthermostatically at 15 °C. Fish were held under artificial light, with a photoperiod regime of 12 hlight: 12 h dark. The experimental protocol used <strong>in</strong> this trial was developed <strong>in</strong> accordance withboth the guidel<strong>in</strong>e of the Canadian Council on Animal Care (CCAC, 1984) as well as theUniversity of Guelph Animal Care Committee.37

Dur<strong>in</strong>g the 12-week experimental period all fish were h<strong>and</strong>-fed to near-satiety two timesa day on weekdays <strong>and</strong> once daily on weekends. Feed consumption was recorded on a weeklybasis. Bulk weight of the fish <strong>in</strong> each tank was recorded every four weeks. Before start<strong>in</strong>g thetrial, a sample of ten <strong>and</strong> five fish was taken for determ<strong>in</strong>ation of the <strong>in</strong>itial carcass composition<strong>and</strong> for <strong>in</strong>itial muscle pigment content <strong>and</strong> colour determ<strong>in</strong>ation, respectively. At the end of trial,four <strong>and</strong> five fish from each tank were r<strong>and</strong>omly taken for the determ<strong>in</strong>ation of f<strong>in</strong>al carcasscomposition <strong>and</strong> f<strong>in</strong>al muscle pigment content <strong>and</strong> colour, respectively. Fish were killed by anoverdose of trica<strong>in</strong>e methane sulfonate (200 mg L -1 water). Fish sampled for carcass compositiondeterm<strong>in</strong>ation were autoclaved, ground <strong>in</strong>to an homogenous slurry, freeze-dried, ground <strong>in</strong>to apowder <strong>and</strong> stored at -20 °C until analysis. Fish sampled for muscle pigment <strong>and</strong> colourdeterm<strong>in</strong>ation were sk<strong>in</strong>ned <strong>and</strong> colour was determ<strong>in</strong>ed on the right h<strong>and</strong> side fillet right afterslaughter; muscle samples were kept at -80 °C until analysis.Ingredients <strong>and</strong> experimental diets. Bleached CGM (14 batches of 200 g) was preparedas described <strong>in</strong> the bleach<strong>in</strong>g of carotenoids section. Proximate <strong>and</strong> carotenoid composition ofregular <strong>and</strong> pigment bleached CGM is presented <strong>in</strong> Tables 3.4 <strong>and</strong> 3.10 respectively). Threediets were formulated to be isonitrogenous (48 % crude prote<strong>in</strong>) <strong>and</strong> isoenergetic (20 MJ DE kg -1 ) <strong>and</strong> conta<strong>in</strong> no (Diet 1, Control diet), 19% CGM (Diet 2) or 19% bleached CGM (Diet 3) <strong>and</strong>to meet all the nutritional requirements for ra<strong>in</strong>bow trout based on NRC (2011) (Table 3.5). Alldiets were supplemented with 50 mg kg -1 astaxanth<strong>in</strong> (Carophyll®p<strong>in</strong>k, 8% astaxanth<strong>in</strong>, DSMNutritional Products, ON, Canada). All <strong>in</strong>gredients were mixed us<strong>in</strong>g a Hobart mixer (HobartLtd, Don Mills, ON, Canada). The three diets were steam pelleted to appropriate size us<strong>in</strong>g alaboratory pellet mill (California Pellet Mill, San Francisco, CA, USA). Pellets were then driedovernight under forced-air at 60 °C, sieved to appropriate size, <strong>and</strong> stored at 4 °C until used.38

Calculations. Growth rate (thermal unit growth coefficient, TGC), was assessed for eachtank as: (1) TGC = 100×[(FBW 1/3 −IBW 1/3 )×(sum T×D) −1 ], where: FBW = f<strong>in</strong>al body weight (gfish -1 ); IBW = <strong>in</strong>itial body weight (g fish -1 ); sum T×D = sum degrees Celsius×days.Feed efficiency (FE, ga<strong>in</strong>:feed) for each tank was determ<strong>in</strong>ed as: (2) FE = live bodyweight ga<strong>in</strong>/dry feed <strong>in</strong>take, where: feed <strong>in</strong>take=total dry feed/number of fish; live body weightga<strong>in</strong> = (FBW/f<strong>in</strong>al number of fish)−(IBW/<strong>in</strong>itial number of fish); FBW = f<strong>in</strong>al body weight (g);IBW = <strong>in</strong>itial body weight (g). Reta<strong>in</strong>ed nitrogen (RN, g fish -1 ) <strong>and</strong> recovered energy (RE, kJfish -1 ) for each tank were assessed as: (3) RN = (FBW×N content f<strong>in</strong>al )−(IBW×N content <strong>in</strong>itial ) <strong>and</strong>(4) RE = (FBW×GE content f<strong>in</strong>al )−(IBW×GE content <strong>in</strong>itial ), respectively, where: FBW = f<strong>in</strong>al bodyweight (g fish -1 ); IBW = <strong>in</strong>itial body weight (g fish -1 ); N content f<strong>in</strong>al = nitrogen content (%) of thef<strong>in</strong>al carcass sample; N content <strong>in</strong>itial = nitrogen content (%) of the <strong>in</strong>itial carcass sample; GE f<strong>in</strong>al =gross energy (kJ g -1 ) content of the f<strong>in</strong>al carcass sample; GE <strong>in</strong>itial = gross energy (kJ g -1 ) contentof the <strong>in</strong>itial carcass sample.Nitrogen retention efficiency (NRE) <strong>and</strong> energy retention efficiency(ERE) for each tank were calculated as a ratio of <strong>in</strong>gested nitrogen (IN): (5) NRE (% IN) =[[(FBW×N content f<strong>in</strong>al )−(IBW×N content <strong>in</strong>itial )]/IN]×100; <strong>and</strong> as ratio of <strong>in</strong>gested energy (IE): (6)ERE (% IE) = [[(FBW×GE content f<strong>in</strong>al )−(IBW×GE content <strong>in</strong>itial )]/IE]×100, where: FBW = f<strong>in</strong>albody weight (g fish -1 ); IBW=<strong>in</strong>itial body weight (g fish -1 ); N content f<strong>in</strong>al = nitrogen content (%)of the f<strong>in</strong>al carcass sample; N content <strong>in</strong>itial = nitrogen content (%) of the <strong>in</strong>itial carcass sample;GE f<strong>in</strong>al = gross energy (kJ g -1 ) content of the f<strong>in</strong>al carcass sample; GE <strong>in</strong>itial = gross energy (kJ g -1 )content of the <strong>in</strong>itial carcass sample; IN = <strong>in</strong>gested nitrogen (g fish -1 ); IE = <strong>in</strong>gested energy (kJfish -1 ).39

3.2.3 Analytical methodsDry matter <strong>and</strong> ash content of regular <strong>and</strong> bleached CGM, experimental diets,<strong>in</strong>gredients <strong>and</strong> carcass samples were determ<strong>in</strong>ed accord<strong>in</strong>g to AOAC (1995) crude prote<strong>in</strong> (%Nx 6.25) was assessed <strong>in</strong> a LECO analyzer (LECO Corp., St. Joseph, MI, USA), <strong>and</strong> total lipiddeterm<strong>in</strong>ation was conducted with <strong>in</strong> a high pressure extractor (Ankom XT-20 Lipid extractor –ANKOM Technology, Macedon, NY, USA). Gross energy (GE) content was assessed us<strong>in</strong>g a abomb calorimeter (Parr 1271, Parr <strong>in</strong>struments, Mol<strong>in</strong>e, IL, USA). 2-Thiobarbituric acid assay(TBA) number <strong>in</strong> experimental diets was determ<strong>in</strong>ed spectrophometrically accord<strong>in</strong>g to Tironi etal. (2007).Total yellow pigment content of CGM samples was determ<strong>in</strong>ed accord<strong>in</strong>g to Santra et al.(2003) with some modifications. Water-saturated butanol (1.25 mL) was added to 0.25 g ofsample <strong>in</strong>to 1.5 mL micro centrifuge tube. Samples were mixed by vortex<strong>in</strong>g for 30 s <strong>and</strong> thenkept <strong>in</strong> the dark for 16 – 18 h for pigments extraction. Then tubes were centrifuged at 10,000 x gfor 10 m<strong>in</strong> to recover the supernatant. The absorbance of the supernatant was determ<strong>in</strong>ed with aspectrophotometer (Novaspec II, LKB Biochrom) at 450 nm wavelength.Muscle colour determ<strong>in</strong>ation. Instrumental colour determ<strong>in</strong>ation was assessed us<strong>in</strong>g aCHROMA METER tristimulus colorimeter (CR-400, KONICA MINOLTA SENSING, Inc,Japan). Colour sampl<strong>in</strong>g was determ<strong>in</strong>ed: close to the head; below the dorsal f<strong>in</strong> <strong>and</strong> close to thetail; <strong>and</strong> close to the tail, each measurement was performed over <strong>and</strong> under the lateral l<strong>in</strong>e.Colour values are expressed as mean of six read<strong>in</strong>g per sample. Measurements are expressed <strong>in</strong>the colorimetric space were L*, represents lightness (L*=0 for black, L*=100 for white); a*,represents the <strong>in</strong>tensity of the red colour, <strong>and</strong> b* scale, represent<strong>in</strong>g the <strong>in</strong>tensity <strong>in</strong> the yellowcolour. The quantitative hue (H° ab ) <strong>and</strong> chroma (C*) colour attributes were determ<strong>in</strong>ed accord<strong>in</strong>g40

to Christiansen et al. (1995). H° ab is (as the relationship between the yellowness <strong>and</strong> redness) as:(7) H° ab =tan -1 b*/a*. Where 0° (H° ab = 0) represents a red hue <strong>and</strong> 90° (H° ab = 90) represents ayellow hue. The C* expresses saturation <strong>and</strong> clarity of a colour. A high C* value represents ahigh saturation of colour. It was calculated as: (8) C* = (a* 2 + b* 2 ) 1/2 .Lipoxygenase activity determ<strong>in</strong>ation. Lipoxygenase activity <strong>in</strong> WSFF, heat treated WSFF<strong>and</strong> CGM, was determ<strong>in</strong>ed accord<strong>in</strong>g to Gӧkmen et al. (2007) with some modifications. Enzymeextraction was carried out by homogeniz<strong>in</strong>g 5 g of sample with sodium phosphate (25 mL)buffer (pH 6.5) at 4°C us<strong>in</strong>g a magnetic stirrer for 30 m<strong>in</strong>. The homogenate was centrifuged at15,000 x g for 15 m<strong>in</strong> at 4°C. The supernatant conta<strong>in</strong><strong>in</strong>g lipoxygenase was kept as a crudeenzyme extract. The total prote<strong>in</strong> content of the enzyme extract was determ<strong>in</strong>ed us<strong>in</strong>g the Sigmadiagnostics kit for micro prote<strong>in</strong> determ<strong>in</strong>ation (Sigma Oakville, ON, Canada).The substrate solution was obta<strong>in</strong>ed by comb<strong>in</strong><strong>in</strong>g pure l<strong>in</strong>oleic acid (157.2 µL), Tween20 (157.2 µL) <strong>and</strong> nano-pure water (10 mL). 1 N NaOH (1mL) was added <strong>and</strong> the mixture wasdiluted to 200 mL with M/15 sodium phosphate buffer (pH 6.0).Spectrophotometric lipoxygenase activity determ<strong>in</strong>ation was conducted by add<strong>in</strong>g 29.9mL of substrate solution <strong>in</strong> a flask placed <strong>in</strong> a water bath set up at 30 °C. Gently aeration waspassed through the solution for 2 m<strong>in</strong>. The reaction was <strong>in</strong>itiated by mix<strong>in</strong>g the crude enzymeextract (0.1 mL) <strong>and</strong> the substrate solution. Aliquots (1 mL) were transferred <strong>in</strong>to labeled testtubes conta<strong>in</strong><strong>in</strong>g 4 mL of 0.1 Normal NaOH solution at 0.5, 1.0, 1.5, 2.0, 2.5 <strong>and</strong> 3.0 m<strong>in</strong> ofreaction time. NaOH solution stops the enzymatic reaction <strong>and</strong> control optical <strong>in</strong>terference <strong>in</strong> thesolution before absorbance measur<strong>in</strong>g. Hydroperoxide formation was followed us<strong>in</strong>g a VarianInc., spectrophotometer (Palo Alto, CA, USA) as <strong>in</strong>crease <strong>in</strong> absorbance at 234 nm. A blank41

solution was generated by mix<strong>in</strong>g the substrate solution (1 mL) with the 0.1 Normal NaOHsolution (4 mL). One lipoxygenase unit was def<strong>in</strong>ed as the quantity of enzyme that generates 1µmol of hydroperoxide per m<strong>in</strong> per mg of prote<strong>in</strong> under the st<strong>and</strong>ard assay conditions.HPLC analysis. Carotenoids from CGM samples were extracted accord<strong>in</strong>g to Abdel-Aalet al. (2007). An approximately 0.15 g of sample was homogenized <strong>in</strong> 10 mL of water saturatedbutanol for 30 s <strong>in</strong> a PT 18/105 Ultra-turrax homogenizer, kept for 30 m<strong>in</strong> at room temperature,<strong>and</strong> homogenized aga<strong>in</strong> for 30 s. The mixture was centrifuged at 10,000 x g for 5 m<strong>in</strong>, <strong>and</strong> analiquot of the supernatant (0.5 mL) was filtered through a 0.45 µm Nylon Acrodisc syr<strong>in</strong>ge filter.The first two drops of the filtrate were discarded, <strong>and</strong> the rem<strong>in</strong>der was collected for HPLCanalyses.Carotenoids from the diets <strong>and</strong> fish muscle samples were extracted accord<strong>in</strong>g to Bjerkenget al. (1997) with some variations. Feed samples were weight (5.0 g) <strong>and</strong> mixed with 10 mL ofmethanol (conta<strong>in</strong><strong>in</strong>g 500 mg L -1 of 2,6-di-t-butyl-p-cresol) <strong>and</strong> distilled water (5 mL). Sampleswere extracted us<strong>in</strong>g a PT 18/105 Ultra-turrax homogenizer for 30 s. The samples were mixedaga<strong>in</strong> for 30 s with chloroform (15 mL) <strong>and</strong> kept <strong>in</strong> the dark for 10 m<strong>in</strong>, remixed for extra 30 s<strong>and</strong> centrifuged (10 m<strong>in</strong>, 3000 x g, 18°C).Muscle samples (sk<strong>in</strong> <strong>and</strong> bone free) of each fish were chopped <strong>and</strong> ground twice whilefrozen. Samples (7.5 g) were then mixed with 7.5 mL of distilled water <strong>and</strong> 7.5 ml methanol(conta<strong>in</strong><strong>in</strong>g 500 mg L -1 of BHT). The mixture was homogenized us<strong>in</strong>g a PT 18/105 Ultra-turraxhomogenizer for 30 s. Chloroform (21 mL) was then added <strong>and</strong> an additional mixture of 30 s wasapplied. Muscle samples were then centrifuged (15 m<strong>in</strong>, 2500 x g) at 18°C.42

An aliquot (1 mL) of the chloroform phase was transferred <strong>in</strong>to a test tube thenevaporated to dryness on a water bath (ca. 40°C) us<strong>in</strong>g nitrogen gas. Samples were dissolved <strong>in</strong>to4 or 1 mL of water-saturated butanol (feeds <strong>and</strong> muscles, respectively). The solution was filtered(0.45 µm; M<strong>in</strong>isart SRP15, Sartorius), <strong>in</strong>to the sample vials, sealed <strong>and</strong> storage at-80 °C untilanalysis. All carotenoid extractions were conducted under dim light to avoid sample degradationby photo-oxidation.Carotenoids extracts were analysed accord<strong>in</strong>g to Abdel-Aal et al. (2007) by liquidchromatography (1100 series liquid chromatographer, Aligent, Mississauga, ON). <strong>Pigment</strong>s wereseparated us<strong>in</strong>g a short C30 column (YMC Carotenoid, Waters, Mississauga, ON, Canada), setup at 35°C. Composition of the eluted mobile phase was (A) methanol/methyl tert-butilether/nanopure water solution (81:15:4, v/v/v) <strong>and</strong> (B) methyl tert-butyl ether/methanol (90:10,v/v) solution, programed to run as: 0-9 m<strong>in</strong>, 100 - 75% A; 9 - 14 m<strong>in</strong>, 75 - 20% A; 14 - 15 m<strong>in</strong>,25 - 0% A; 15 - 18 m<strong>in</strong>, hold at 0% A; 18 -20 m<strong>in</strong> hold at 100% A. Detection <strong>and</strong> measurementof different pigments were conducted at 450 nm ( for all-trans-lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, alltrans-β-cryptoxanth<strong>in</strong><strong>and</strong> all-trans-β-carotene) <strong>and</strong> 478 nm (for all-trans-astaxanth<strong>in</strong>),respectively.Identification <strong>and</strong> quantification of carotenoids was accomplished us<strong>in</strong>g pure authenticcarotenoids st<strong>and</strong>ards (all-trans-astaxanth<strong>in</strong>, all-trans-lute<strong>in</strong>, all-trans-β-carotene, all-transzeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong>) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Thel<strong>in</strong>earity of the response for each pigment (response area v/s <strong>in</strong>jected amount) was evaluated <strong>and</strong>the coefficient of determ<strong>in</strong>ation (R 2 ) for all-trans-astaxanth<strong>in</strong>, all-trans-lute<strong>in</strong>, all-transzeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong>, <strong>and</strong> all-trans-β-carotene were 0.9997, 0.9996, 0.9996,0.9994 <strong>and</strong> 0.9999, respectively.43

3.2.4 Statistical analysisData from the Plackett-Burman design were analyzed us<strong>in</strong>g a multiple regression analysisperformed by the Analyst application of the SAS/STAT software (SAS <strong>in</strong>stitute, Cary, NC,USA). The design for the Box-Behnken design was generated <strong>and</strong> analyzed us<strong>in</strong>g the ADXapplication of the SAS/STAT software (SAS <strong>in</strong>stitute, Cary, NC, USA).Data from the fish muscle pigmentation trial were analysed as a complete r<strong>and</strong>om designus<strong>in</strong>g the general l<strong>in</strong>ear model of the SAS/STAT software (SAS <strong>in</strong>stitute, Cary, NC, USA). Themeans of dependent variables were compared us<strong>in</strong>g Tukey’s honestly significant difference(HSD) test; significance was considered when p0.05) effects under theexperimental conditions (Table 3.6).44

The antioxidant effects of ascorbic acid on lute<strong>in</strong> degradation have been reported onlyunder alkal<strong>in</strong>e conditions (Shi <strong>and</strong> Chen, 1997). The neutral conditions <strong>in</strong>duced for LOX to reachmaximum activity <strong>in</strong> this study most likely promoted a protective effect <strong>in</strong>stead of bleach<strong>in</strong>gaction of ascorbic acid on carotenoids <strong>in</strong> CGM.Lipoxygenase activity from WSFF used <strong>in</strong> this study was 124.5 units m<strong>in</strong> -1 mg prote<strong>in</strong> -1(Table 3.7) <strong>and</strong> showed the characteristic spectrophotometric response of LOX (Figure 3.1)previously reported <strong>in</strong> the literature (Gӧkmen et al., 2007). The many different purificationmethodologies described for LOX activity determ<strong>in</strong>ation represent a drawback for proper <strong>in</strong>terstudycomparative purposes; however LOX activity, as long as it reaches certa<strong>in</strong> level, is not adeterm<strong>in</strong><strong>in</strong>g factor for bleach<strong>in</strong>g carotenoids <strong>in</strong> CGM (Park et al., 1997). LOX activity reachedby a 5% WSFF <strong>in</strong>clusion was probably large enough to maximize carotenoids oxidation <strong>and</strong>consequently <strong>in</strong>creas<strong>in</strong>g WSFF <strong>in</strong>corporation level showed no significant effects. Heat-treatedWSFF <strong>and</strong> CGM showed no LOX activity.The LOX-catalyzed polyunsaturated fatty acid oxidation promot<strong>in</strong>g the appearance ofhydroperoxydiene molecules (bleach<strong>in</strong>g agent) is oxygen dependent (Cohen et al., 1985). In thisstudy aeration (3.25 L m<strong>in</strong> -1 ) had no significant (p> 0.05) effects on carotenoids reduction. Themechanical energy (generated by shak<strong>in</strong>g at 110 r m<strong>in</strong> -1 ) applied to samples most likelygenerated a suitable oxygen level as well as proper oxygen distribution with<strong>in</strong> the slurry <strong>and</strong>might expla<strong>in</strong> this result.A 4-factor, 27-run Box-Behnken experimental design was run <strong>in</strong> order to determ<strong>in</strong>e thequadratic <strong>and</strong> <strong>in</strong>teractive effect of active factors on the LOX-catalyzed pigment bleach<strong>in</strong>g.45

Factors def<strong>in</strong>ed as no active by the Plackett-Burman analysis were not considered <strong>in</strong> the responsesurface design.Results from the BBD analysis have shown that pigments bleach<strong>in</strong>g ranged from 22% to62% (Table 3.8). L<strong>in</strong>ear term for WSFF pre-soak<strong>in</strong>g, as well as l<strong>in</strong>ear <strong>and</strong> quadratic terms forBP <strong>and</strong> SO were highly significant (p

y lipoxygenase, compared to destruction rates obta<strong>in</strong>ed when pigments were exposed to LOX orl<strong>in</strong>oleic acid separately.Benzoyl peroxide is widely utilized as a free radical <strong>in</strong>itiator <strong>and</strong> bleach<strong>in</strong>g agent <strong>in</strong><strong>in</strong>dustrial food process<strong>in</strong>g (Gel<strong>in</strong>as et al., 1998). Addition of this compound <strong>in</strong>to the reactionmedia most likely <strong>in</strong>creased pigment bleach<strong>in</strong>g by a typical free radical mechanism act<strong>in</strong>g <strong>in</strong> asynergistic fashion with lipoxygenase <strong>in</strong> this study.The effect of different reaction temperatures (when presoak<strong>in</strong>g time is kept constant at 90m<strong>in</strong>) on pigment bleach<strong>in</strong>g is depicted <strong>in</strong> Figures 3.3 A (20°C), B (15°C) <strong>and</strong> C (10°C).Temperature can modify activity of antioxidants such as ascorbyl palmitate, BHA <strong>and</strong> BHT(Frankel, 2005). LOX catalyzed carotenoid co-oxidation is related to the stability of the radical<strong>in</strong>termediates produced. Less stable <strong>in</strong>termediate species promote higher carotenoid bleach<strong>in</strong>gPerez-Galvez <strong>and</strong> M<strong>in</strong>guez-Mosquera, 2002). Whether or not temperature <strong>in</strong>fluences<strong>in</strong>termediate bleach<strong>in</strong>g species stability is yet to be determ<strong>in</strong>ed, <strong>and</strong> may expla<strong>in</strong> the effect of RTon pigment reduction <strong>in</strong> this study.Carotenoids from CGM (expressed as total yellow pigments, assessed byspectrophotometric method) were reduced by 62% when the optimum factor comb<strong>in</strong>ation wasapplied. Additionally, carotenoid profile (i.e. all-trans lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans β-cryptoxanth<strong>in</strong> <strong>and</strong> all-trans β-carotene) obta<strong>in</strong>ed for CGM (determ<strong>in</strong>ed by liquidchromatography) <strong>in</strong> this study (Table 3.10) is <strong>in</strong> the same range of that reported by Moros et al.2002. All-trans lute<strong>in</strong> <strong>and</strong> all-trans zeaxanth<strong>in</strong> were bleached by about 86% <strong>and</strong> 97%respectively whereas β-cryptoxanth<strong>in</strong> <strong>and</strong> β-carotene were 100% reduced <strong>in</strong> bleached CGM,represent<strong>in</strong>g an overall 91% reduction <strong>in</strong> yellow pigments (as assessed by liquid47

chromatography). Relative amounts of pigments such as flavonoids <strong>and</strong> chlorophyll-likecompounds are determ<strong>in</strong>ed at the same wavelength as xanthophylls <strong>and</strong> carotenes <strong>in</strong> cornsamples (Bless<strong>in</strong>, 1962), the presence of these types of compounds may expla<strong>in</strong> the differencesbetween results expressed as total yellow pigment (determ<strong>in</strong>ed us<strong>in</strong>g spectrophotometry) <strong>and</strong><strong>in</strong>dividual carotenoid (obta<strong>in</strong>ed by liquid chromatography) for regular <strong>and</strong> bleached CGMsamples <strong>in</strong> this study.3.3.2 Fish muscle pigmentation trialGrowth <strong>and</strong> feed utilization parameters obta<strong>in</strong>ed <strong>in</strong> this trial (Table 3.11) are comparableto those commonly observed <strong>in</strong> our laboratory for ra<strong>in</strong>bow trout same stra<strong>in</strong> <strong>and</strong> similar size fedhigh nutritional quality formulated diets (Bureau et al., 2006). No significant (p>0.05)differences were obta<strong>in</strong>ed for ga<strong>in</strong> (g fish -1 ), TGC or feed efficiency which is <strong>in</strong> agreement toprevious reports on CGM <strong>in</strong>clusion <strong>in</strong> salmonids formulated diets (Mundheim et al., 2004;Skonberg et al., 1998)Figure 3.4 depicts growth trajectories over time obta<strong>in</strong>ed <strong>in</strong> this study, even though nostatistically significant, all growth parameters for fish fed Diet 3 showed a reduction tendencydur<strong>in</strong>g the last 50 days of the trial. Additionally, a significant (p

Lipid content <strong>and</strong> gross energy levels were slightly, although significantly (p

After carotenoids are absorbed through the <strong>in</strong>test<strong>in</strong>al epithelial cells they are metabolizedaccord<strong>in</strong>g to their molecular structure i.e. cyclization at one or both ends of the molecule,hydrogenation level, presence of oxygen-conta<strong>in</strong><strong>in</strong>g functional groups (Furr <strong>and</strong> Clark, 1997).The major molecular structure similarities shared by astaxanth<strong>in</strong> <strong>and</strong> yellow xanthophylls fromCGM suggest a potential metabolic competition among these pigments before <strong>in</strong>corporation <strong>in</strong>tolymphatic lipoprote<strong>in</strong>s <strong>and</strong> might account for the difference <strong>in</strong> all-trans astaxanth<strong>in</strong> muscledeposition observed <strong>in</strong> this trial.Carotenoids are delivered from the enterocytes <strong>in</strong>to the blood stream where they areexclusively transported by lipoprote<strong>in</strong>s (Salvador et al., 2007). It is hypothesized that polarxanthophylls exist near these structure’s surface (Furr <strong>and</strong> Clark, 1997). Aggregation of differentxanthophyll types with<strong>in</strong> chylomicrons surface might <strong>in</strong>crease astaxanth<strong>in</strong> competition forb<strong>in</strong>d<strong>in</strong>g sites with<strong>in</strong> lipoprote<strong>in</strong> dur<strong>in</strong>g pigment transfer <strong>and</strong> might expla<strong>in</strong> reduction <strong>in</strong> muscleall-trans astaxanth<strong>in</strong> concentration obta<strong>in</strong>ed <strong>in</strong> this trial <strong>in</strong> fish fed Diet 2.The f<strong>in</strong>d<strong>in</strong>gs from this trial are <strong>in</strong> contradiction to those reported by Olsen <strong>and</strong> Baker(2006), who reported no differences (p>0.05) <strong>in</strong> muscle astaxanth<strong>in</strong> concentration of Atlanticsalmon (S. salar) fed diets supplemented with 55 <strong>and</strong> 23 mg kg -1 synthetic astaxanth<strong>in</strong> <strong>and</strong> lute<strong>in</strong>respectively, compared to those from fish fed a diet conta<strong>in</strong><strong>in</strong>g astaxanth<strong>in</strong> (55 mg kg -1 ) as as<strong>in</strong>gle pigment source. The negative effect of dietary CGM on muscle all-trans astaxanth<strong>in</strong>deposition obta<strong>in</strong>ed <strong>in</strong> this trial, as opposed to those observed when us<strong>in</strong>g a synthetic source oflute<strong>in</strong>, suggest a potential <strong>in</strong>teraction of different components from CGM <strong>in</strong>duc<strong>in</strong>g a reduction <strong>in</strong>muscle astaxanth<strong>in</strong> deposition.50

No pigments other than all-trans astaxanth<strong>in</strong> were determ<strong>in</strong>ed <strong>in</strong> muscles from ra<strong>in</strong>bowtrout fed Diet 2 (Table 3.14). Lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> have been previously determ<strong>in</strong>ed <strong>in</strong> musclefrom salmonid fish (Kitahara, 1983). Thus results from this trial suggest that deposition ofyellow xanthophylls from CGM <strong>in</strong> fish muscle might be dietary level dependent. Xanthophyllconcentration <strong>in</strong> commercial CGM varies from 224 to 550 mg kg -1 on a dry matter basis (Park etal., 1997). The relative low pigment concentration <strong>in</strong> CGM used <strong>in</strong> this study (142 mg kg -1 )might expla<strong>in</strong> the lack of yellow carotenoids deposited with<strong>in</strong> muscles from fish feed Diet 2.No significant (p>0.05) differences were determ<strong>in</strong>ed for any colour attribute amongmuscles from fish fed Diet 1<strong>and</strong> 2 <strong>in</strong> spite of the significant (p

<strong>and</strong> ended up <strong>in</strong>duc<strong>in</strong>g lipid rancidity <strong>and</strong> eventually the oxidative destruction of dietaryastaxanth<strong>in</strong>.F<strong>in</strong>d<strong>in</strong>gs from this study suggest that <strong>in</strong>clusion of CGM negatively affect all-transastaxanth<strong>in</strong> deposition <strong>in</strong> muscle from ra<strong>in</strong>bow trout when this commodity is <strong>in</strong>cluded <strong>in</strong> aformulated diet at a level of 19%. More systematic research on this topic is highly necessary <strong>in</strong>order to underst<strong>and</strong> the mechanism(s) <strong>in</strong>volved <strong>in</strong> this phenomenon <strong>and</strong> clarify the exact roleplayed by yellow xanthophylls on astaxanth<strong>in</strong> metabolism <strong>and</strong> deposition. <strong>Corn</strong> gluten meal iswidely utilized <strong>in</strong> diets for many different fish species; accord<strong>in</strong>gly a comprehensiveunderst<strong>and</strong><strong>in</strong>g of the effects of this commodity on important f<strong>in</strong>al product quality parameterssuch as muscle pigmentation is crucial. Additionally, the low capital technology <strong>and</strong>straightforward operational process<strong>in</strong>g methodology developed <strong>in</strong> this study represent anattractive tool for the reduction of natural occurr<strong>in</strong>g yellow carotenoid pigments from CGM.However, potential shortcom<strong>in</strong>g that might negatively affect fish growth performance <strong>and</strong>important flesh quality parameters must be avoid dur<strong>in</strong>g formulation (i.e. proper antioxidantsupplementation).52

TablesTable 3. 1 - Factors <strong>and</strong> levels for screen<strong>in</strong>g us<strong>in</strong>g Plackett-Burman designFactorsLevelsLowHighPS: WSFF pre-soak<strong>in</strong>g (m<strong>in</strong>) 0 30LV: WSFF level (%) 5 15RT: Reaction temperature (°C) 20 30AA: Ascorbic acid (ppm) 0 500BP: Benzoyl peroxide (ppm) 0 300SO: Soy oil (%) 0 5AE: Aeration though the reaction medium (L m<strong>in</strong> -1 ) 0 3.2553

Table 3. 2 - Experimental design us<strong>in</strong>g Plackett-Burman design for screen<strong>in</strong>g of factorsRun # Factors/Levels a Bleach<strong>in</strong>g ofPS LV RT AA BP SO AEcarotenoids (%) b1 + + + - + - - 342 - + + + - + - 373 - - + + + - + 224 + - - + + + - 675 - + - - + + + 496 + - + - - + + 427 + + - + - - + 308 - - - - - - - 19a Low (-) <strong>and</strong> high (+) levels of <strong>in</strong>dependent variables used <strong>in</strong> the trial. b Data are mean (n=3) <strong>and</strong>expressed as total yellow pigments.54

Table 3. 3 - Summary of variables for the Box-Behnken experimental designFactorsLevelsLow Medium HighPS: WSFF pre-soak<strong>in</strong>g (m<strong>in</strong>) 60 90 120RT: Reaction temperature (°C) 10 15 20BP: Benzoyl peroxide (ppm) 0 300 600SO: Soy oil (%) 5 10 1555

Table 3. 4 - Analysed proximate composition of regular <strong>and</strong> bio-bleached CGMNutrients aIngredients bCGMBleached CGMDry matter 91.9 ± 0.0 a 44.7 ±0.3 bCrude prote<strong>in</strong> 72.2 ± 0.1 b 74.8 ± 0.1 aLipids 0.9 ± 0.1 b 5.9 ± 0.0 aNFE 24.7 ± 0.3 a 18.6 ± 0.1 bAsh 2.2 ± 0.1 a 0.8 ± 0.0 bGross energy (MJ kg -1 ) 23.6 ± 0.0 b 28.3 ± 0.7 aa Data are mean (n=2) ± St<strong>and</strong>ard deviation. b As a percentage of dry matter basis, otherwise<strong>in</strong>dicated.56

Table 3. 5 - Formulation, analyzed proximate composition <strong>and</strong> pigment content of experimentaldietsDietIngredients (%) 1 2 3Fish meal 28 16 16<strong>Corn</strong> gluten meal 0 19 0Bleached corn gluten meal 0 0 19Soy flake flour 0 0.5 0.5Soybean oil 0 0.5 0.5Poultry by-product meal (regular) 15 11.5 11.5Feather meal 6 4 4Blood meal, whole, spray-dry 2 2 2Brewer's dried yeast 6 6 6Soybean meal 10 10 10Wheat middl<strong>in</strong>gs 12.4 8.9 8.9Fish oil 12 12 12Vegetable oil 4 4.5 4.5Vitam<strong>in</strong> premix a 1 1 1Lys<strong>in</strong>e (BioLys) 1 1.5 1.5DL-Methion<strong>in</strong>e 0.5 0.5 0.5M<strong>in</strong>eral premix b 0.5 0.5 0.5Ca(H 2 PO 4 ) 2 1.5 1.5 1.5Carophyll®p<strong>in</strong>k (8% Astaxanth<strong>in</strong>) 0.0625 0.0625 0.062557

Proximate composition (Analysed; dry matter basis)Dry matter 94.0 94.0 92.0Crude prote<strong>in</strong> 50.9 50.6 51.3Lipids 23.8 23.1 23.6Ash 9.9 7.5 7.3Energy (MJ kg -1 ) 24.1 24.7 24.7<strong>Pigment</strong> concentration (mg kg -1 , dry matter basis) (Analysed)All-trans astaxanth<strong>in</strong> 48 57 4All-trans lute<strong>in</strong> nd d 28 ndAll-trans zeaxanth<strong>in</strong> nd 15 ndAll-trans β-cryptoxanth<strong>in</strong> nd nd ndAll-trans β-carotene nd nd ndTBA value (mg MDA kg -1 ) c 3.6 b 4.0 b 14.6 aa Vitam<strong>in</strong> premix. Provided per kg of diet = ret<strong>in</strong>yl acetate (vitam<strong>in</strong> A), 75 mg; cholecalciferol(vitam<strong>in</strong> D3), 60 mg; dl-a-tocopherol-acetate (vitam<strong>in</strong> E), 300 mg; menadione Na-bisulfate(vitam<strong>in</strong> K), 1.5 mg; cyanocobalam<strong>in</strong>e (vitam<strong>in</strong> B12), 30 mg; ascorbic acid monophos. (vitam<strong>in</strong>C), 300 mg; D-biot<strong>in</strong>, 210 mg; chol<strong>in</strong>e chloride, 3448 mg; folic acid, 1.5 mg; niac<strong>in</strong>, 15 mg;calcium-d-pantothenate, 33 mg; pyridox<strong>in</strong>e –HCl, 7.5 mg; riboflav<strong>in</strong>, 9 mg; thiam<strong>in</strong>-HCl, 1.5mg. b M<strong>in</strong>eral premix. Provided per kg of diet = sodium chloride (NaCl, 39% Na, 61% Cl), 3077mg; potassium iod<strong>in</strong>e (KI, 24%K, 76%I), 10.5 mg; ferrous sulphate (FeSO 4 , 7H 2 O, 20%Fe), 65mg; manganese sulphate (MnSO 4 , 36%Mn) 88.9 mg; z<strong>in</strong>c sulphate (ZnSO 4 .7H 2 O, 40%Zn), 150mg; copper sulphate (CuSO 4 .5H 2 O, 25%Cu), 28 mg; sodium Selenite (Na 2 SeO 3 , 45.66% Se), 0.7mg. c 2-Thiobarbituric acid assay (TBA). d Not detected.58

Table 3. 6 - Plackett-Burman design resultsEstimate T Value Pr > |t|Intercept 33.96 5.88

Table 3. 7 - Lipoxygenase activity <strong>in</strong> different <strong>in</strong>gredientsIngredient aLipoxygenase activity bWhite soy flake flour 124.5 ± 0.4Heat treated white soy flake flour<strong>Corn</strong> gluten mealnd cnda Data are mean (n=2) ± St<strong>and</strong>ard deviation. b Units m<strong>in</strong> -1 mg prote<strong>in</strong> -1 . c Not detected.60

Table 3. 8 - Box-Behnken experimental designRun #FactorsBleach<strong>in</strong>g ofcarotenoids(%) aCoded valuesActual valuesPS RT BP SO PS RT BP SO1 -1 -1 0 0 60 10 300 10 312 1 -1 0 0 120 10 300 10 223 -1 1 0 0 60 20 300 10 274 1 1 0 0 120 20 300 10 315 0 0 -1 -1 90 15 0 5 326 0 0 1 -1 90 15 600 5 627 0 0 -1 1 90 15 0 15 448 0 0 1 1 90 15 600 15 579 -1 0 0 -1 60 15 300 5 3310 1 0 0 -1 120 15 300 5 2211 -1 0 0 1 60 15 300 15 3512 1 0 0 1 120 15 300 15 3713 0 -1 -1 0 90 10 0 10 2414 0 1 -1 0 90 20 0 10 3415 0 -1 1 0 90 10 600 10 5416 0 1 1 0 90 20 600 10 4717 -1 0 -1 0 60 15 0 10 3861

18 1 0 -1 0 120 15 0 10 2519 -1 0 1 0 60 15 600 10 5720 1 0 1 0 120 15 600 10 3921 0 -1 0 -1 90 10 300 5 3222 0 1 0 -1 90 20 300 5 3423 0 -1 0 1 90 10 300 15 3624 0 1 0 1 90 20 300 15 3825 0 0 0 0 90 15 300 10 2726 0 0 0 0 90 15 300 10 3027 0 0 0 0 90 15 300 10 32a Data are mean (n=3) <strong>and</strong> expressed as total yellow pigments.62

Table 3. 9 - Box-Behnken design l<strong>in</strong>ear, quadratic <strong>and</strong> <strong>in</strong>teraction effectsVariables Estimate t Value Pr > |t|L<strong>in</strong>earPS -3.79 -3.75 0.0004RT 1.06 1.05 0.2972BP 5.68 5.62

Table 3. 10 - HPLC carotenoid profile of regular <strong>and</strong> bleached CGMCarotenoid a Ingredients b Bleach<strong>in</strong>g (%)CGMBleached CGMAll-trans lute<strong>in</strong> 91 ± 0.1 13 ± 0.2 86All-trans zeaxanth<strong>in</strong> 49 ± 0.9 2 ± 1.5 97All-trans β-Cryptoxanth<strong>in</strong> 3 ± 0.2 nd c 100All-trans β -Carotene 15 ± 0.3 nd 100Total 158 14 91a Data are mean (n=2) ± St<strong>and</strong>ard deviation. b On a dry matter basis. c Not detected.64

Table 3. 11 - Growth performance <strong>and</strong> fed efficiency of ra<strong>in</strong>bow trout (IBW=132 g fish -1 ) fedexperimental diets for 12 weeksDietParameter a 1 2 3F<strong>in</strong>al body weight 540 ± 30 531 ± 5 473 ± 21Thermal-unit growthcoefficient0.239 ± 0.01 0.243 ± 0.01 0.220 ± 0.01Feed efficiency (ga<strong>in</strong>:feed) b 0.91 ± 0.05 0.88 ± 0.05 0.89 ± 0.02a Data are mean ± St<strong>and</strong>ard deviation, n=2 tanks. b Feed efficiency (live weight ga<strong>in</strong>:feed DM).65

Table 3. 12 - Reta<strong>in</strong>ed levels <strong>and</strong> retention efficiencies of nitrogen <strong>and</strong> energy by ra<strong>in</strong>bow trout(IBW=132 g fish -1 ) fed experimental diets for 12 weeksDiet1 2 3RN a (g fish -1 ) 11.1 ± 0.6 a 11.2 ± 0.1 a 8.9 ± 0.3 bRE b (kJ fish -1 ) 4815 ± 315 4449 ± 64 4131 ± 162NRE c (% IN e ) 33 ± 2 32 ± 2 31 ± 1ERE f (% IE g ) 48 ± 3 42 ± 2 47 ± 1a RN=reta<strong>in</strong>ed nitrogen (g fish -1 ). b RE=recovered energy (kJ fish -1 ). c NRE=nitrogen retentionefficiency. e IN=<strong>in</strong>gested nitrogen. f ERE=energy retention efficiency. g IE=<strong>in</strong>gested energy.66

Table 3. 13 - Proximate composition of the carcass of ra<strong>in</strong>bow trout a (IBW=132 g fish -1 ) fedexperimental diets for 12 weeksDietNutrient b 1 2 3Moisture 62.7 ± 0.1 c 64.0 ± 0.2 a 63.2 ± 0.0 bCrude prote<strong>in</strong> 16.9 ± 0.2 a 17.0 ± 0.0 a 16.1 ± 0.2 bLipid 18.4 ± 0.2 a 16.7 ± 0.0 b 18.7 ± 0.2 aAsh 2.3 ± 0.1 2.4 ± 0.3 1.9 ± 0.0Gross energy (kJ kg -1 ) 11.2 ± 0.0 a 10.6 ± 0.1 b 11.2 ± 0.1 aa Initial carcass composition: 68% moisture, 15.7% crude prote<strong>in</strong>, 13.7% lipid, 2.3% ash, 9.0kJ·kg -1 gross energy. b As a percentage of dry matter, otherwise <strong>in</strong>dicated.67

Table 3. 14 - Fillet carotenoid concentration <strong>and</strong> colour attributes from ra<strong>in</strong>bow trout a (IBW=132g fish -1 ) fed experimental diets for 12 weeksDiet1 2 3Carotenoid concentration <strong>in</strong> fillets (mg kg -1 , dry matter basis) bAll-trans astaxanth<strong>in</strong> 6 ± 0.5 a 3 ± 0.5 b 0.4 ± 0.4 cAll-trans lute<strong>in</strong> nd c nd ndAll-trans zeaxanth<strong>in</strong> nd nd ndAll-trans β-cryptoxanth<strong>in</strong> nd nd ndAll-trans β-carotene nd nd ndFillet colour attributes dL* 41 ± 1 b 40 ± 1 b 45 ± 2 aa* 9.9 ± 1.3 a 9.6 ± 0.7 a 2.0 ± 0.6 bb* 13.4 ± 0.5 a 12.8 ± 1.0 a 7.8 ± 1.2 bH° ab 54 ± 3 b 53 ± 0 b 76 ± 4 aC* 16.6 ± 1.2 a 16.0 ± 1.2 a 8.1 ± 1.3 ba Initial fillet carotenoid concentration: Astaxanth<strong>in</strong> = nd; Lute<strong>in</strong> = nd; Zeaxanth<strong>in</strong> = nd; β-cryptoxanth<strong>in</strong> = nd; β-carotene = nd. Initial fillet colour attributes: L* = 46; a* = 1.3; b* = 4.8;H° ab = 76; C* = 5.0. b Data are mean ± st<strong>and</strong>ard deviation (n=2 tanks, means of 5 <strong>in</strong>dividuals pertank). c Not detected. d Data are mean ± st<strong>and</strong>ard deviation (n=2 tanks, 5 <strong>in</strong>dividuals per tank,means of six read<strong>in</strong>gs on each fillet). nd = not detected.68

Mol of hydroperoxideFigures14001200100080060040020000 1 2 3 4Time (m<strong>in</strong>)Mol of hydroperoxideFigure 3. 1 - Typical trend for spectrophotometric measurement of lipoxygenase activityobta<strong>in</strong>ed for WSFF <strong>in</strong> the present study.69

Total yellow pigment(mg•kg -1 )2502001501005000 25 50 75 100 125 150Time (m<strong>in</strong>)Heat treated WSFFEnzyme active WSFFFigure 3. 2 - Bleach<strong>in</strong>g pattern of carotenoids observed over time (120 m<strong>in</strong>) for the optimumfactor comb<strong>in</strong>ation, PS = 90 m<strong>in</strong>, RT = 15°C, BP = 600 ppm <strong>and</strong> SO = 5% <strong>in</strong>clusion.70

SO (%)BP (ppm)Figure 3. 3 - Response surface plot show<strong>in</strong>g the effect of SO <strong>and</strong> BP on CGM carotenoidsbleach<strong>in</strong>g, keep<strong>in</strong>g PS fixed at 90 m<strong>in</strong> at three different RT: 20, 15 <strong>and</strong> 10°C for A, B <strong>and</strong> C,respectively.71

Figure 3. 4 - Growth curve of ra<strong>in</strong>bow trout fed the experimental diets for 12 weeks. D1 (Diet 1,Control), D2 (Diet 2, regular CGM), D3 (Diet 3, Bleached CGM.72

CHAPTER - 4 EFFECTS OF FEEDING INCREASING LEVELS OF CORN GLUTENMEAL ON GROWTH AND MUSCLE PIGMENTATION OF RAINBOW TROUT(Oncorhynchus mykiss)Abstract<strong>Corn</strong> gluten meal (CGM) is a highly digestible, cost-effective <strong>in</strong>gredient widely <strong>in</strong>cluded<strong>in</strong> aquaculture feeds. A 24-week growth trial was carried out <strong>in</strong> order to assess the effects of<strong>in</strong>creas<strong>in</strong>g levels of CGM on growth <strong>and</strong> muscle pigment deposition <strong>in</strong> ra<strong>in</strong>bow trout(Oncorhynchus mykiss). Six isonitrogenous <strong>and</strong> isoenergetic (on a digestible energy basis)experimental diets were formulated, us<strong>in</strong>g different <strong>in</strong>gredient comb<strong>in</strong>ations, to conta<strong>in</strong><strong>in</strong>creas<strong>in</strong>g levels of CGM (0 - 18). A commercial feed conta<strong>in</strong><strong>in</strong>g 10% CGM was <strong>in</strong>cluded <strong>in</strong>this trial. All seven diets were supplemented with 50 mg kg -1 astaxanth<strong>in</strong>. Each experimental dietwas fed to apparent satiety to triplicate groups of seventy five fish (<strong>in</strong>itial body weight = 549 gfish -1 ) reared at 8.5 °C. Dietary treatments did not significantly affect f<strong>in</strong>al body weight, thermalgrowth efficiency (TGC) or feed efficiency. Carotenoids determ<strong>in</strong>ation by liquidchromatography showed a significant l<strong>in</strong>ear reduction <strong>in</strong> astaxanth<strong>in</strong> isomer, all-trans astaxanth<strong>in</strong><strong>and</strong> all-trans lute<strong>in</strong> <strong>in</strong> muscle from fish <strong>in</strong> response to <strong>in</strong>creas<strong>in</strong>g levels of CGM. Tristimuluscolour analysis of muscle revealed a significant l<strong>in</strong>ear reduction <strong>in</strong> a* (redness) <strong>and</strong> C*ab(chroma). Additionally, Salmofan TM score showed a significant l<strong>in</strong>ear <strong>and</strong> quadratic reduction to<strong>in</strong>creas<strong>in</strong>g levels of CGM. In conclusion, CGM <strong>in</strong>clusion up to 18% does not significantlyimpact growth parameters. However, all-trans astaxanth<strong>in</strong> muscle concentration as well asimportant colour attributes can be negatively affected at levels exceed<strong>in</strong>g 12% CGM <strong>in</strong> the diet.More systematic research on this topic is needed to discern the mechanism(s) beh<strong>in</strong>d the negative73

effects of dietary CGM <strong>and</strong>/or its <strong>in</strong>tr<strong>in</strong>sic yellow pigment on ra<strong>in</strong>bow trout musclepigmentation.74

4.1 IntroductionColour of muscle from salmonid fish is associated to f<strong>in</strong>al product quality <strong>and</strong> significantly<strong>in</strong>fluences consumers’ perception (Alfnes et al., 2006; Anderson, 2000). This dist<strong>in</strong>ctive fleshquality parameter is achieved by the deposition <strong>and</strong> accumulation of carotenoid pigments, ma<strong>in</strong>lyastaxanth<strong>in</strong>, with<strong>in</strong> muscle fibers. Salmonid fish are unable to synthetize carotenoids de novo(Choubert et al., 2006), therefore formulated diets are supplemented with synthetic or naturallyoccurr<strong>in</strong>g pigments <strong>in</strong> order to satisfy market preferences. Dietary <strong>in</strong>clusion of expensivecarotenoids premixes accounts for up to 10 -20% of feed price (Bjerkeng, 2000; Choubert et al.,2009).Formulation of aquaculture feeds has progressively reduced its dependence upon fish mealas a major prote<strong>in</strong> source. <strong>Corn</strong> gluten meal (CGM), is a highly digestible, prote<strong>in</strong>-rich (60%crude prote<strong>in</strong>) secondary product of the corn wet mill<strong>in</strong>g process that is widely utilized <strong>in</strong> theformulation of diets for a number of fish species. The ma<strong>in</strong> goal of conventional corn wet mill<strong>in</strong>gis to isolate <strong>and</strong> ref<strong>in</strong>e starch, however different structural constituent of the corn kernels (i.e.prote<strong>in</strong>, germ <strong>and</strong> hull) are separated <strong>in</strong>to different fractions along the process. Dur<strong>in</strong>g mill<strong>in</strong>g,naturally occurr<strong>in</strong>g carotenoids from corn kernels, ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, rema<strong>in</strong>embedded with<strong>in</strong> the prote<strong>in</strong> fraction, hence elevated levels (200-500 mg kg -1 ) of yellowxanthophylls are recovered <strong>in</strong> CGM (Park et al., 1997; Skonberg et al., 1998).Results from few scientific studies along with field evidence from fish farmers <strong>in</strong>dicate asignificant impact of dietary CGM on astaxanth<strong>in</strong> deposition <strong>and</strong> colour attributes <strong>in</strong> musclesfrom salmonid fish. Fillets from Atlantic salmon (Salmo salar) (f<strong>in</strong>al body weight=450 g fish -1 )fed diets formulated to conta<strong>in</strong> <strong>in</strong>creas<strong>in</strong>g levels of a plant prote<strong>in</strong> blend (CGM <strong>and</strong> full fat75

soybean meal <strong>in</strong> a 2:1 ratio) <strong>and</strong> 64 mg kg -1 of astaxanth<strong>in</strong>, showed a negative response <strong>in</strong> colourattributes (measured as SalmoFan® scores) after a 11-week feed<strong>in</strong>g trial (Mundheim et al.,2004). A yellowish appearance (based on tristimulus colour analysis) was assessed <strong>in</strong> fillet fromra<strong>in</strong>bow trout (320 g fish -1 of f<strong>in</strong>al body weight) fed a high CGM diet (22.5%) compared to thosefrom fish fed a high CGM diet (22.5%) supplemented with 100 mg kg -1 canthaxanth<strong>in</strong> for 12weeks (Skonberg et al., 1998). More recently, Saez et al. (unpublished results), observed asignificant (p

this trial was to evaluate the effect of <strong>in</strong>creas<strong>in</strong>g levels of CGM (up to 18%) on growth <strong>and</strong>pigment deposition <strong>in</strong> fillets from ra<strong>in</strong>bow trout.4.2 Materials <strong>and</strong> methods4.2.1 Fish husb<strong>and</strong>ry <strong>and</strong> experimental conditionsThis study was conducted at the Alma Aquaculture Research Station (AARS, Alma, ON,Canada). Domestic stra<strong>in</strong> of ra<strong>in</strong>bow trout, (O. mykiss) from the AARS’s stock population wereadapted to experimental conditions for two weeks prior to start the experiment. Dur<strong>in</strong>g thatperiod, fish were fed a commercial (with no pigment supplementation) trout diet (Mart<strong>in</strong> MillsInc, Elora, ON, Canada) once a day.The 7 experimental diets were r<strong>and</strong>omly assigned to each of 21 (1,500 L) fibreglass tanks(n=3). Seventy five fish (549 g fish -1 , <strong>in</strong>itial body weight) were r<strong>and</strong>omly allocated <strong>in</strong>to eachtank <strong>and</strong> kept under a 12 h light: 12 h dark photoperiod regime. Filtered well water was suppliedapproximately at a 30.5 L m<strong>in</strong> -1 rate. Water temperature was kept at 8.5 °C. This experiment wasconducted accord<strong>in</strong>g with the guidel<strong>in</strong>es stated by the Canadian Council on Animal Care(CCAC, 1984) <strong>and</strong> the University of Guelph Animal Care Committee.Fish were h<strong>and</strong>-fed twice a week to near satiation dur<strong>in</strong>g 24 weeks. Dur<strong>in</strong>g the rema<strong>in</strong><strong>in</strong>gfive days, fish were fed us<strong>in</strong>g belt feeders. Daily ration to be dispensed by belt feeders wasdeterm<strong>in</strong>ed by averag<strong>in</strong>g the amount of feed given to that tank dur<strong>in</strong>g the previous two dailyh<strong>and</strong> feed<strong>in</strong>gs.Five fish were sampled at the beg<strong>in</strong>n<strong>in</strong>g of the experiment for the determ<strong>in</strong>ation of <strong>in</strong>itialcarcass composition <strong>and</strong> another sample of five fish was used for <strong>in</strong>itial muscle pigment content<strong>and</strong> colour determ<strong>in</strong>ation. At the end of the experimental period, four <strong>and</strong> ten fish from each tank77

were r<strong>and</strong>omly sampled for f<strong>in</strong>al carcass composition <strong>and</strong> f<strong>in</strong>al muscle pigment content <strong>and</strong>colour determ<strong>in</strong>ation, respectively. Fish were sacrificed by an overdose of MS-222 (300 ppm L -1 ). Fish for carcass composition determ<strong>in</strong>ation were autoclaved, thoroughly ground <strong>in</strong>to a slurry,freeze-dried, ground <strong>in</strong>to a f<strong>in</strong>e powder <strong>and</strong> kept at -20 °C until analysis. Fish to be analysed formuscle pigment content <strong>and</strong> colour determ<strong>in</strong>ation were manually sk<strong>in</strong>ned <strong>and</strong> right h<strong>and</strong> sidefilleted right after slaughter; after muscle colour determ<strong>in</strong>ation muscle samples were stored at -20 °C until pigments determ<strong>in</strong>ation analysis.4.2.2 Experimental dietsSix (6) extruded experimental diets were formulated to be isonitrogenous (48 % crudeprote<strong>in</strong>) <strong>and</strong> isoenergetic (20 MJ DE/kg) <strong>and</strong> to conta<strong>in</strong> different <strong>in</strong>gredient comb<strong>in</strong>ations (i.e.fish meal, soy prote<strong>in</strong> concentrate, feather meal, blood meal, wheat meal, soybean meal <strong>and</strong> fishoil) <strong>and</strong> either no CGM (Diet 1), 4% CGM (Diet 2), 9% CGM (Diet 3), 12% CGM (Diet 4), 14%CGM (Diet 5) <strong>and</strong> 18% CGM (Diet 6), <strong>and</strong> meet all the nutritional requirements for ra<strong>in</strong>bowtrout based on NRC (2011) (Table 4.1). A commercial feed conta<strong>in</strong><strong>in</strong>g 10% CGM (Diet 7) wasused. All diets were supplemented with 50 mg kg -1 astaxanth<strong>in</strong> (Carophyll® p<strong>in</strong>k, 8%astaxanth<strong>in</strong>, DSM Nutritional Products, ON, Canada).Noteworthy is that <strong>in</strong>clusion of graded levels of CGM <strong>in</strong> Diet 1, 3 <strong>and</strong> 6 (0, 9 <strong>and</strong> 18%CGM, respectively) was achieved at expense of soy prote<strong>in</strong> concentrate only (Table 4.1).Conversely, <strong>in</strong> the formulation for Diets 2, 4 <strong>and</strong> 5 (4, 12 <strong>and</strong> 14% CGM, respectively), <strong>in</strong>clusionof several <strong>in</strong>gredients (i.e. fish meal, feather meal, blood meal, wheat meal <strong>and</strong> fish oil) wasmodified <strong>in</strong> order to accomplish desired CGM <strong>in</strong>clusion levels.78

4.2.3 Chemical analysisDry matter <strong>and</strong> ash <strong>in</strong> diets <strong>and</strong> carcass samples were determ<strong>in</strong>ed accord<strong>in</strong>g to AOAC(1995), crude prote<strong>in</strong> (%N x 6.25) determ<strong>in</strong>ation was run us<strong>in</strong>g a LECO analyzer (LECO Corp.,St. Joseph, MI, USA), <strong>and</strong> total lipid analysis was performed us<strong>in</strong>g a petroleum ether extractor(Ankom XT-20 Lipid extractor – ANKOM Technology, Macedon, NY, USA). Gross energy(GE) content of carcass <strong>and</strong> diet samples was assessed us<strong>in</strong>g an automated bomb calorimeter(Parr 1271, Parr <strong>in</strong>struments, Mol<strong>in</strong>e, IL, USA).Carotenoid extraction from feed <strong>and</strong> muscle. Carotenoids conta<strong>in</strong>ed <strong>in</strong> the experimentaldiets <strong>and</strong> fish muscle samples were extracted accord<strong>in</strong>g to Bjerkeng et al. (1997), with somemodification.Diets (10 g) were mixed with methanol (10 mL), conta<strong>in</strong><strong>in</strong>g 2,6-di-t-butyl-p-cresol at 500mg L -1 of (BHT) as antioxidant agent, <strong>and</strong> distilled water (5 ml). Samples mixed us<strong>in</strong>g ah<strong>and</strong>held mixer for 30 s before addition of chloroform (15 ml). Samples were then mixed aga<strong>in</strong>for 30 s <strong>and</strong> kept <strong>in</strong> the dark for 10 m<strong>in</strong>, mixed for 30 s <strong>and</strong> centrifuged (10 m<strong>in</strong>, 3000 x g,18°C). Further mix<strong>in</strong>g with chloroform was performed until no colour was observed.Sk<strong>in</strong>ned muscle samples (deboned) of each fish were ground while frozen, freeze-dried,ground <strong>in</strong>to a f<strong>in</strong>e powder <strong>and</strong> stored at -20 °C until pigment determ<strong>in</strong>ation. Samples (2 g) werethen mixed with distilled water (7.5 ml) <strong>and</strong> methanol (7.5 ml) conta<strong>in</strong><strong>in</strong>g 500 mg L -1 of BHT.The mixture was homogenized with a h<strong>and</strong>held mixer (Ultra-turrax T25, Janke <strong>and</strong> Kunkel, IKALaborteknik, Staufen, Germany) for 30 s. Chloroform (30 ml) was added <strong>and</strong> the samples werehomogenized for an additional 30 s. The muscle samples were centrifuged (15 m<strong>in</strong>, 2500 x g) at18°C.79

An aliquot (1 ml) of chloroform lower phase was pipetted <strong>in</strong>to a test tube <strong>and</strong> evaporatedus<strong>in</strong>g water bath (ca. 40°C) <strong>and</strong> a flow of nitrogen gas. Once dried, samples were dissolved <strong>in</strong>to4 or 1 mL (for feed <strong>and</strong> muscle, respectively) of water-saturated butanol. Carotenoids extractswere filtered (0.45 µm; M<strong>in</strong>isart SRP15, Sartorius), <strong>in</strong>to amber sample vials, air was removedus<strong>in</strong>g a flow of nitrogen gas <strong>and</strong> sealed. Samples were kept frozen (-80 °C) until analysis. Allpigment extractions were performed under dim light <strong>in</strong> order to m<strong>in</strong>imize destructive effect oflight on carotenoids.Carotenoid determ<strong>in</strong>ation. Carotenoids from diets <strong>and</strong> muscle samples were separated<strong>and</strong> quantified accord<strong>in</strong>g to Abdel-Aal et al. (2007) us<strong>in</strong>g a 1100 series liquid chromatographer(Aligent, Mississauga, ON). <strong>Pigment</strong>s separation was performed on a short (10 cm x 4.6 mm,pack<strong>in</strong>g 3 µm) C30 column, YMC Carotenoid (Waters, Mississauga, ON, Canada), operated at35°C. The mobile system gradient used for elution was conducted us<strong>in</strong>g solutions of (A)methanol/methyl tert-butyl ether/nanopure water (81:15:4, v/v/v) <strong>and</strong> (B) methyl tert-butylether/methanol (90:10, v/v), <strong>and</strong> programed as follows: 0-9 m<strong>in</strong>, 100 - 75% A; 9 - 14 m<strong>in</strong>, 75 -20% A; 14 - 15 m<strong>in</strong>, 25 - 0% A; 17 - 18 m<strong>in</strong>, hold at 0% A; 17 - 18 m<strong>in</strong> 0 - 100% A; 18 -20 m<strong>in</strong>hold at 100% A. After separation, detection <strong>and</strong> measurement of carotenoids was conducted at450 nm (all-trans-lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong> <strong>and</strong> all-trans-β-carotene,)<strong>and</strong> 478 nm (all-trans-astaxanth<strong>in</strong>), respectively. The correspondence of the retention times <strong>and</strong>UV/Vis spectra of analysed samples <strong>and</strong> those showed by pure authentic st<strong>and</strong>ards was utilizedfor identification carotenoids.For identification <strong>and</strong> quantification purposes pure authentic st<strong>and</strong>ards of all-transastaxanth<strong>in</strong>,all-trans-lute<strong>in</strong>, all-trans-β-carotene (Sigma-Aldrich Canada Ltda., Oakville, ON),<strong>and</strong> all-trans zeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong> (ChromaDex Inc., Santa Ana, CA) were80

utilized. Five concentrations for each carotenoid were prepared <strong>in</strong> butanol <strong>in</strong> order to assessl<strong>in</strong>earity of the response. The regression analysis (response area versus <strong>in</strong>jected amount) showeda l<strong>in</strong>ear relationship for all-trans-astaxanth<strong>in</strong>, all-trans-lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans-βcryptoxanth<strong>in</strong>,<strong>and</strong> all-trans-β-carotene with the coefficient of determ<strong>in</strong>ation (R 2 ) of 0.9997,0.9996, 0.9996, 0.9994 <strong>and</strong> 0.9999, respectively. Purity of each compound <strong>in</strong> the diets <strong>and</strong>muscle extracts was verified us<strong>in</strong>g isoabsorbance plot or 3D graphic <strong>and</strong> peak purity analyses(ChemStation software).4.2.4 Muscle colour determ<strong>in</strong>ationMuscle colour determ<strong>in</strong>ation was assessed at three po<strong>in</strong>ts over <strong>and</strong> three po<strong>in</strong>t below thelateral l<strong>in</strong>e: close to the head; midway between the head <strong>and</strong> the tail; <strong>and</strong> close to the tail us<strong>in</strong>g aCHROMA METER tristimulus colorimeter (CR-400, KONICA MINOLTA SENSING, Inc,Japan). Muscle colour was expressed as mean of the six read<strong>in</strong>g per fish. All measurementswere performed <strong>in</strong> the colorimetric space L* (lightness, L*=0 for black, L*=100 for white); a*scale represents the <strong>in</strong>tensity <strong>in</strong> red, <strong>and</strong> b* scale represents the <strong>in</strong>tensity <strong>in</strong> yellow.4.2.5 CalculationsThermal unit growth coefficient (TGC), for each tank was calculated as: TGC=100×[(FBW 1/3 −IBW 1/3 )×(sum T×D) -1 ], where: FBW= f<strong>in</strong>al body weight (g fish -1 ); IBW= <strong>in</strong>itialbody weight (g fish -1 ); sum T×D= sum degrees Celsius×days.Feed efficiency (FE, ga<strong>in</strong>:feed) for each tank was assessed as: FE= live body weightga<strong>in</strong>/dry feed <strong>in</strong>take, where: feed <strong>in</strong>take= total dry feed/number of fish; live body weight ga<strong>in</strong>=(FBW f<strong>in</strong>al number of fish -1 )−(IBW <strong>in</strong>itial number of fish -1 ); FBW= f<strong>in</strong>al body weight (g);IBW= <strong>in</strong>itial body weight (g).81

Reta<strong>in</strong>ed nitrogen (RN, g fish -1 ) <strong>and</strong> recovered energy (RE, kJ fish -1 ) for each tank weredeterm<strong>in</strong>ed us<strong>in</strong>g the follow<strong>in</strong>g formulas as: RN= (FBW×N content f<strong>in</strong>al )−(IBW×N content <strong>in</strong>itial )<strong>and</strong> RE= (FBW×GE content f<strong>in</strong>al )−(IBW×GE content <strong>in</strong>itial ), respectively, where: FBW= f<strong>in</strong>al bodyweight (g fish -1 ); IBW= <strong>in</strong>itial body weight (g fish -1 ); N content f<strong>in</strong>al = nitrogen content (%) of thef<strong>in</strong>al carcass sample; N content <strong>in</strong>itial = nitrogen content (%) of the <strong>in</strong>itial carcass sample; GE f<strong>in</strong>al =gross energy (kJ g -1 ) content of the f<strong>in</strong>al carcass sample; GE <strong>in</strong>itial = gross energy (kJ g -1 ) content ofthe <strong>in</strong>itial carcass sample.Nitrogen retention efficiency (NRE) <strong>and</strong> energy retention efficiency (ERE) for each tankdeterm<strong>in</strong>ed based on the rate of <strong>in</strong>gested nitrogen (IN): NRE (% IN)= [[(FBW×Ncontent f<strong>in</strong>al )−(IBW×N content <strong>in</strong>itial )]/IN]×100; <strong>and</strong> based on the rate of <strong>in</strong>gested energy (IE): ERE(% IE)= [[(FBW×GE content f<strong>in</strong>al )−(IBW×GE content <strong>in</strong>itial )]/IE]×100, where: FBW= f<strong>in</strong>al bodyweight (g fish -1 ); IBW= <strong>in</strong>itial body weight (g fish -1 ); N content f<strong>in</strong>al = nitrogen content (%) of thef<strong>in</strong>al carcass sample; N content <strong>in</strong>itial = nitrogen content (%) of the <strong>in</strong>itial carcass sample; GE f<strong>in</strong>al =gross energy (kJ g -1 ) content of the f<strong>in</strong>al carcass sample; GE <strong>in</strong>itial = gross energy (kJ g -1 ) content ofthe <strong>in</strong>itial carcass sample; IN=<strong>in</strong>gested nitrogen (g fish -1 ); IE= <strong>in</strong>gested energy (kJ fish -1 ).The quantitative hue (H°ab) <strong>and</strong> chroma (C*) were assessed accord<strong>in</strong>g to Christiansen etal. (1995). H°ab (yellowness <strong>and</strong> redness relation of the fillet) as: H°ab= tan-1 b*/a*. The hue isan angular dimension where 0° (H°ab= 0) represents the red hue <strong>and</strong> 90° (H°ab= 90) representsthe yellow hue.C* was assessed as: C*= (a* 2 + b* 2 ) 1/2 . C* represents the <strong>in</strong>tensity <strong>and</strong> clarity of thecolour <strong>and</strong> is expressed as the relationship between a* <strong>and</strong> b* values. A more <strong>in</strong>tense the colour,represents a higher the C* values.82

4.2.6 Statistical analysisData were analysed as a complete r<strong>and</strong>om design us<strong>in</strong>g the general l<strong>in</strong>ear model of theSAS/STAT software (SAS version 9.1.3, SAS <strong>in</strong>stitute, Cary, NC, USA). Orthogonal polynomialcontrast analyses were utilized <strong>in</strong> order to determ<strong>in</strong>e the significance of l<strong>in</strong>ear <strong>and</strong> quadraticresponses of growth parameters, HPLC-determ<strong>in</strong>ed carotenoid muscle concentration <strong>and</strong> musclecolor attributes to <strong>in</strong>creas<strong>in</strong>g levels of CGM.In order to address the confound<strong>in</strong>g effects of us<strong>in</strong>g different <strong>in</strong>gredient comb<strong>in</strong>ations <strong>and</strong>to highlight the effects of CGM <strong>in</strong>clusion only, the significance of l<strong>in</strong>ear <strong>and</strong> quadratic responsesare presented separately for Diets 1, 3 <strong>and</strong> 6 (<strong>in</strong>clusion of CGM at expense of soy prote<strong>in</strong>concentrate only) <strong>and</strong> for all dietary treatments.4.3 ResultsTable 4.2 shows the results for growth performance obta<strong>in</strong>ed <strong>in</strong> this trial. F<strong>in</strong>al bodyweight, TGC (thermal growth coefficient) <strong>and</strong> feed efficiency were not significantly (p>0.05)affected by dietary treatments. On average fish grew from 549 to 1275 g fish -1 giv<strong>in</strong>g averageTGC <strong>and</strong> feed efficiency of 0.76 <strong>and</strong> 0.186, respectively.Results for carcass chemical proximate composition are depicted <strong>in</strong> Table 4.3. Nosignificant effects of dietary CGM on carcass composition were determ<strong>in</strong>ed for moisture, crudeprote<strong>in</strong>, lipids, ash or gross energy. When significance for all dietary treatment was taken <strong>in</strong>toaccount, a significant (p

significant (p

address the confound<strong>in</strong>g effect of the <strong>in</strong>clusion of different <strong>in</strong>gredient comb<strong>in</strong>ation <strong>and</strong> highlightthe effects of CGM <strong>in</strong>clusion only, the significance of the l<strong>in</strong>ear <strong>and</strong> quadratic responses wasevaluated separately.Growth performance <strong>and</strong> feed efficiency values achieved <strong>in</strong> this trial were with<strong>in</strong> the rangeof those typically obta<strong>in</strong>ed at Alma Research Station for fish same size <strong>and</strong> stra<strong>in</strong> fed to nearsatiation with nutritionally complete commercial or experimental diets (Dumas et al., 2007).Figure 4.1 depicts growth trajectory performed by ra<strong>in</strong>bow trout fed the experimental diets for24 weeks. CGM <strong>in</strong>clusion level did not have a significant effect on f<strong>in</strong>al body weight, TGC orfeed efficiency which is <strong>in</strong> agreement with previous studies on CGM <strong>in</strong>clusion <strong>in</strong> formulateddiets for fish (Mundheim et al., 2004; Skonberg et al., 1998).The significant reduction <strong>in</strong> carcass crude prote<strong>in</strong> (Table 4.3) <strong>and</strong> energy retentionefficiency (ERE) (Table 4.4), obta<strong>in</strong>ed <strong>in</strong> this study when all dietary treatments were taken <strong>in</strong>toaccount can be attributed to the confound<strong>in</strong>g effect of <strong>in</strong>clusion of different comb<strong>in</strong>ations.<strong>Reduction</strong> of the nutritive value <strong>in</strong> Diets 2, 4 <strong>and</strong> 5 (4, 12 <strong>and</strong> 14% CGM, respectively) due tohigher fish meal substitution might expla<strong>in</strong> these results (Table 4.1).Noteworthy is the occurrence of all-trans lute<strong>in</strong> <strong>and</strong> all-trans zeaxanth<strong>in</strong> (2 <strong>and</strong> 9 mg kg -1 ,respectively) <strong>in</strong> the non-CGM supplemented diet (Diet 1, Table 4.1). The presence of yellowxanthophylls <strong>in</strong> this diet is most probably due to the <strong>in</strong>clusion of soy prote<strong>in</strong> concentrate <strong>and</strong>soybean meal <strong>and</strong> wheat (18%, 10% <strong>and</strong> 10.7%, respectively). Substantial amounts of thesecarotenoids have been previously reported for soybean seeds <strong>and</strong> oil as well as for wheat (Abdel-Aal et al., 2007; Kanamaru et al., 2006; Lee et al., 2009; Slav<strong>in</strong> et al., 2009).85

Values for all-trans astaxanth<strong>in</strong> muscle deposition (16 - 24 mg kg -1 ) obta<strong>in</strong>ed <strong>in</strong> this trial(Table 4.5) are <strong>in</strong> the same range of those previously reported for ra<strong>in</strong>bow trout fed dietssupplemented with 71 to 200 mg kg -1 astaxanth<strong>in</strong> concentrations (Choubert et al., 2006; 2009).Differences <strong>in</strong> fish size <strong>and</strong> trial duration might expla<strong>in</strong> similar outputs achieved with differentastaxanth<strong>in</strong> dietary concentration.One astaxanth<strong>in</strong> isomer was detected <strong>in</strong> samples of diets <strong>and</strong> muscles <strong>in</strong> this trial.Occurrence of cis-astaxanth<strong>in</strong> isomers have been previously reported for both dietssupplemented with all-trans astaxanth<strong>in</strong>, <strong>and</strong> muscle from ra<strong>in</strong>bow trout, respectively (Bjerkenget al. 1997; Bowen et al. 2002). Occurrence of all-trans lute<strong>in</strong> <strong>and</strong> all-trans zeaxanth<strong>in</strong> <strong>in</strong> musclefrom ra<strong>in</strong>bow trout fed all diets used <strong>in</strong> this trial is <strong>in</strong> accordance with previous studies report<strong>in</strong>gdeposition of yellow xanthophylls <strong>in</strong> muscle from salmonid fish (Bowen et al. 2002; Kitahara,1983; Welker et al. 2001).The significant l<strong>in</strong>ear reduction <strong>in</strong> astaxanth<strong>in</strong> isomer <strong>and</strong> all-trans astaxanth<strong>in</strong>concentration <strong>in</strong> muscle from ra<strong>in</strong>bow trout obta<strong>in</strong>ed <strong>in</strong> this trial <strong>in</strong> response to <strong>in</strong>creas<strong>in</strong>g levelsof CGM confirms the hypothesized negative effects of this commodity on ra<strong>in</strong>bow trout musclepigmentation. The concomitant reduction of all-trans lute<strong>in</strong> suggests an overall decrease ofpigment deposition <strong>in</strong> muscle from ra<strong>in</strong>bow trout to <strong>in</strong>creas<strong>in</strong>g levels of dietary CGM (Figure4.2).Astaxanth<strong>in</strong> b<strong>in</strong>ds to an actomyos<strong>in</strong> prote<strong>in</strong> complex (specifically to an α-act<strong>in</strong> prote<strong>in</strong>)with<strong>in</strong> muscle fibers from salmonid fish (Henmi et al., 1987; Matthews et al., 2006). However,before deposition with<strong>in</strong> the muscle, astaxanth<strong>in</strong> undergoes a series of complex metabolicprocesses (Aas et al., 1999; Furr <strong>and</strong> Clark, 1997; Hemni et al., 1987; Matthews et al., 2006;86

Salvador et al., 2007). With<strong>in</strong> the digestive tract, astaxanth<strong>in</strong> is solubilized <strong>in</strong>to lipid emulsions,further transferred <strong>in</strong>to bile salt micelles <strong>and</strong> f<strong>in</strong>ally absorbed through the <strong>in</strong>test<strong>in</strong>al epithelialcells (Borel et al., 1996, Ishida <strong>and</strong> Bartley, 2005). With<strong>in</strong> the <strong>in</strong>test<strong>in</strong>al lumen, astaxanth<strong>in</strong> isassembled <strong>in</strong>to nascent chylomicrons, released <strong>in</strong>to the bloodstream <strong>and</strong> carried by lipoprote<strong>in</strong>stoward peripheral tissue <strong>and</strong> the liver (Salvador et al., 2007).Similar metabolic pathways have been proposed for different types of carotenoidsmolecules. Additionally, <strong>in</strong>teractions for uptake <strong>and</strong>/or blood-carry<strong>in</strong>g capacity betweencanthaxanth<strong>in</strong> <strong>and</strong> astaxanth<strong>in</strong> <strong>in</strong> Atlantic salmon have been reported (Kiessl<strong>in</strong>g et al., 2003).Whether the occurrence of substantial amounts of yellow xanthophylls from CGM <strong>in</strong> the dietresults <strong>in</strong> an <strong>in</strong>teraction/competition with astaxanth<strong>in</strong> molecules dur<strong>in</strong>g solubilisation,absorption, transport <strong>and</strong>/or deposition rema<strong>in</strong>s unclear <strong>and</strong> might expla<strong>in</strong> results from this study.Results from this trial differ from those reported by Olsen <strong>and</strong> Baker (2006) who assessedsimilar astaxanth<strong>in</strong> levels <strong>in</strong> muscle among fillets form Atlantic salmon fed a diet supplementedwith synthetic astaxanth<strong>in</strong> <strong>and</strong> lute<strong>in</strong> at levels of 55 <strong>and</strong> 23 mg kg -1 , respectively, compared tothose obta<strong>in</strong>ed from fish fed a control diet supplemented with astaxanth<strong>in</strong> (55 mg kg -1 ) as s<strong>in</strong>glesource of pigment). <strong>Reduction</strong> of astaxanth<strong>in</strong> muscle deposition to dietary CGM obta<strong>in</strong>ed <strong>in</strong> thisstudy, as opposed to that obta<strong>in</strong>ed <strong>in</strong> response to <strong>in</strong>clusion of a synthetic purified source oflute<strong>in</strong>, suggests the possible <strong>in</strong>teraction effect of other components with<strong>in</strong> CGM contribut<strong>in</strong>g tothe reduction <strong>in</strong> astaxanth<strong>in</strong> muscle deposition.Muscle a* (redness), b* (Yellowness), Cab (Chroma) <strong>and</strong> Salmofan TM colour attributeswere significantly affected by <strong>in</strong>creas<strong>in</strong>g levels of CGM <strong>in</strong> the diet (Figure 4.3) <strong>and</strong> by theconcomitant reduction <strong>in</strong> all-trans astaxanth<strong>in</strong> muscle concentration. This is <strong>in</strong> agreement with87

previous reports where colour attributes <strong>in</strong> muscle from ra<strong>in</strong>bow trout were dependent uponastaxanth<strong>in</strong> concentration <strong>in</strong> muscle (Choubert et al., 2006; Christiansen et al., 1995).Significance of the l<strong>in</strong>ear or/<strong>and</strong> quadratic responses for muscle all-trans astaxanth<strong>in</strong>, alltranslute<strong>in</strong> <strong>and</strong> all-trans zeaxanth<strong>in</strong> (Table 4.5) as well as for muscle redness (a*), chroma(C*ab) <strong>and</strong> salmoFan TM score, (Table 4.6) was affected by the <strong>in</strong>clusion of different <strong>in</strong>gredientcomb<strong>in</strong>ations used <strong>in</strong> this trial, highlight<strong>in</strong>g the artifact effect of diets formulation. Interest<strong>in</strong>gly,results obta<strong>in</strong>ed by Diets 2, 4 <strong>and</strong> 5 (4, 12 <strong>and</strong> 14% CGM, respectively) were with<strong>in</strong> the sametrend (or slightly off) of those obta<strong>in</strong>ed with Diets 1, 3 <strong>and</strong> 9 (0, 9 <strong>and</strong> 18% CGM, respectively)(Figures 4.2 <strong>and</strong> 4.3), where <strong>in</strong>clusion of CGM was done at expense of soy prote<strong>in</strong> concentrateonly, imply<strong>in</strong>g a consistent adverse effect of dietary CGM on pigment deposition <strong>and</strong> colourattributes <strong>in</strong> muscle from ra<strong>in</strong>bow trout.Results from this study <strong>in</strong>dicate that growth performance of ra<strong>in</strong>bow trout was notsignificantly affected by CGM dietary <strong>in</strong>clusion up to 18%. However astaxanth<strong>in</strong> muscledeposition as well as muscle colour attributes can be reduced by CGM <strong>in</strong>clusion levels over12%. The mechanism(s) controll<strong>in</strong>g this phenomenon as well as the specific role played by<strong>in</strong>tr<strong>in</strong>sic yellow xanthophylls of this commodity on astaxanth<strong>in</strong> utilization by ra<strong>in</strong>bow troutrema<strong>in</strong> to be clarified. F<strong>in</strong>ally, CGM is widely utilized <strong>in</strong> formulation for salmonid fish, hencemore studies on the effect of this commodity on astaxanth<strong>in</strong> deposition <strong>and</strong> key muscle qualityparameters are necessary.88

TablesTable 4. 1 - Formulation, analyzed proximate composition <strong>and</strong> pigment content of experimentaldietsIngredients Diet 10%CGMDiet 24%CGMDiet 39%CGMDiet 412%CGMDiet 514%CGMDiet 618%CGMDiet 7 aCommercial10% CGMFish meal, herr<strong>in</strong>g 16.0 12.0 16.0 12.0 8.0 16.0 -<strong>Corn</strong> gluten meal 0.0 4.0 9.0 11.5 13.7 18.0 -Soy prote<strong>in</strong> concentrate 18.0 0.0 9.0 11.5 13.8 0.0 -Poultry by-product meal 18.0 18.0 18.0 18.0 18.0 18.0 -Feather meal 6.0 15.0 6.0 6.0 6.0 6.0 -Blood meal, spray-dried 0.0 4.0 0.0 0.0 0.0 0.0 -Wheat, gra<strong>in</strong> 10.7 15.7 10.7 9.3 8.4 10.7 -Soybean meal, 52%CP 10.0 10.0 10.0 10.0 10.0 10.0 -Fish oil 11.0 11.0 11.0 11.4 11.8 11.0 -Canola oil 7.0 7.0 7.0 7.0 7.0 7.0 -Vitam<strong>in</strong> premix b 1.1 1.1 1.1 1.1 1.1 1.1 -Bio-Lys® (52% lys<strong>in</strong>e) 1.0 1.0 1.0 1.0 1.0 1.0 -DL-Methion<strong>in</strong>e 0.2 0.2 0.2 0.2 0.2 0.2 -Chol<strong>in</strong>e chloride 0.3 0.3 0.3 0.3 0.3 0.3 -M<strong>in</strong>eral premix c 0.2 0.2 0.2 0.2 0.2 0.2 -NaCl 0.3 0.3 0.3 0.3 0.3 0.3 -Rovimix stay-C (25%) 0.09 0.09 0.09 0.09 0.09 0.09 -Carophyll® p<strong>in</strong>k0.063 0.063 0.063 0.063 0.063 0.063 -(8% astaxanth<strong>in</strong>)89

Proximate composition (analysed), dry matter basisDry matter 89.9 90.6 90.7 90.7 90.2 90.4 90.4Crude prote<strong>in</strong> 52.2 51.3 51.6 51.4 51.7 50.7 51.5Lipids 18.0 18.9 17.5 18.4 18.2 18.2 18.8Ash 8.3 7.2 7.5 7.2 6.8 7.3 8.5Energy (MJ kg -1 ) 19.0 19.3 19.0 19.2 19.3 19.2 19.1<strong>Pigment</strong> concentration (mg kg -1 ) (analysed), dry matter basisAll-E-astaxanth<strong>in</strong> 49 43 41 53 54 46 43Astaxanth<strong>in</strong> isomers d 4 6 4 6 6 5 6All-E-lute<strong>in</strong> 2 3 7 9 11 15 9Lute<strong>in</strong> isomers e ND h N 3 3 4 6 3All-E-zeaxanth<strong>in</strong> 9 10 11 13 16 17 13Zeaxanth<strong>in</strong> isomers f ND 11 1 13 15 3 14All-E-β-cryptoxanth<strong>in</strong> ND ND 1 1 1 2 NDAll-E- β-carotene ND ND 1 1 1 2 1β-Carotene isomers g ND ND 2 2 3 3 2aCommercial feed conta<strong>in</strong><strong>in</strong>g 10% CGM.bVitam<strong>in</strong> premix. Provided per kg of diet = ret<strong>in</strong>yl acetate (vitam<strong>in</strong> A), 75 mg; cholecalciferol(vitam<strong>in</strong> D3), 60 mg; dl-a-tocopherol-acetate (vitam<strong>in</strong> E), 300 mg; menadione Na-bisulfate(vitam<strong>in</strong> K), 1.5 mg; cyanocobalam<strong>in</strong>e (vitam<strong>in</strong> B12), 30 mg; ascorbic acid monophos. (vitam<strong>in</strong>C), 300 mg; D-biot<strong>in</strong>, 210 mg; chol<strong>in</strong>e chloride, 3448 mg; folic acid, 1.5 mg; niac<strong>in</strong>, 15 mg;calcium-d-pantothenate, 33 mg; pyridox<strong>in</strong>e –HCl, 7.5 mg; riboflav<strong>in</strong>, 9 mg; thiam<strong>in</strong>-HCl, 1.5mg.90

cM<strong>in</strong>eral premix. Provided per kg of diet = sodium chloride (NaCl, 39% Na, 61% Cl), 3077 mg;potassium iod<strong>in</strong>e (KI, 24%K, 76%I), 10.5 mg; ferrous sulphate (FeSO 4 , 7H 2 O, 20%Fe), 65 mg;manganese sulphate (MnSO 4 , 36%Mn) 88.9 mg; z<strong>in</strong>c sulphate (ZnSO 4 .7H 2 O, 40%Zn), 150 mg;copper sulphate (CuSO 4 .5H 2 O, 25%Cu), 28 mg; sodium Selenite (Na 2 SeO 3 , 45.66% Se), 0.7 mg.dAstaxanth<strong>in</strong> isomers = 1 Astaxanth<strong>in</strong> isomer; e Lute<strong>in</strong> isomers = Summation of 3 lute<strong>in</strong> isomers;fZeaxanth<strong>in</strong> isomers = 1 Zeaxanth<strong>in</strong> isomer; g β-Carotene isomers = Summation of 3 β-Caroteneisomers.hND = Not detected.91

Table 4. 2 - Growth performance of ra<strong>in</strong>bow trout (IBW= 549 g fish -1 ) fed experimental diets for24 weeksCGMFBW a TGC b FE c Ga<strong>in</strong>:feedlevel (%)Diet 1 0 1302 0.190 0.77Diet 2 4 1241 0.180 0.75Diet 3 9 1297 0.189 0.77Diet 7 (Commercial) 10 1300 0.192 0.75Diet 4 12 1286 0.188 0.79Diet 5 14 1252 0.182 0.75Diet 6 18 1245 0.180 0.75Significance * for Diets 1, 3 <strong>and</strong> 6 dL<strong>in</strong>ear NS f NS NSQuadratic NS NS NSSignificance * for all dietary treatments eL<strong>in</strong>ear NS f NS NS92

Quadratic NS NS NSData are mean, n=3 tanks.aFBW= f<strong>in</strong>al body weight.bTGC = Thermal growth coefficient.c FE = Feed efficiency (live weight ga<strong>in</strong>:feed DM).* Significance= significance of the l<strong>in</strong>ear <strong>and</strong> quadratic contrasts of dependent variables for d dietswhere CGM <strong>in</strong>clusion was done at expense of soy prote<strong>in</strong> concentrate only, <strong>and</strong> for e all dietarytreatments used <strong>in</strong> this trial (as described <strong>in</strong> the material <strong>and</strong> methods section); f NS = nostatistically significant (p>0.05).93

Table 4. 3 - Carcass chemical proximate composition a of ra<strong>in</strong>bow trout b (IBW= 549 g fish -1 ) fedexperimental diets for 24 weeksCGMMoistureCrudeLipidsAshGross Energylevel(%)Prote<strong>in</strong>(%)(%)(kJ/kg)(%)(%)Diet 1 0 64.5 17.7 16.0 2.5 8.9Diet 2 4 64.5 17.5 16.9 2.5 9.2Diet 3 9 63.0 17.5 17.7 2.5 9.5Diet 7 (Commercial) 10 64.4 16.4 17.5 2.4 9.3Diet 4 12 65.2 17.5 15.2 2.6 8.6Diet 5 14 64.1 17.9 16.2 2.6 9.0Diet 6 18 64.4 16.9 16.4 2.5 8.6Significance * for Diets 1, 3 <strong>and</strong> 6 cL<strong>in</strong>ear NS e NS NS NS NSQuadratic NS NS NS NS NSSignificance * for all dietary treatments d94

L<strong>in</strong>ear NS e p0.05).95

Table 4. 4 - Reta<strong>in</strong>ed levels <strong>and</strong> retention efficiencies of nitrogen <strong>and</strong> energy by ra<strong>in</strong>bow trout(IBW= 549 g fish -1 ) fed experimental diets for 24 weeksCGMRN a (g/fish) RE b (kJ/fish) NRE c (% IN d ) ERE e (% IE f )level(%)Diet 1 0 21 7259 26 48Diet 2 4 19 7113 25 49Diet 3 9 20 7969 25 54Diet 7 (Commercial) 10 21 8610 25 55Diet 4 12 20 6713 26 46Diet 5 14 20 6975 26 48Diet 6 18 20 6412 27 45Significance * for Diets 1, 3 <strong>and</strong> 6 gL<strong>in</strong>ear NS i NS NS NSQuadratic NS NS NS NS96

Significance * for all dietary treatments hL<strong>in</strong>ear NS i NS NS NSQuadratic NS NS NS p0.05).97

Table 4. 5 - <strong>Pigment</strong> content <strong>in</strong> muscle of ra<strong>in</strong>bow trout a (IBW= 549 g fish -1 ) fed experimentaldiets for 24 weeksCGM levelAstaxanth<strong>in</strong>All-transAll-transAll-trans(%)isomers (mgastaxanth<strong>in</strong>lute<strong>in</strong>zeaxanth<strong>in</strong>kg -1 )(mg kg -1 )(mg kg -1 )(mg kg -1 )Diet 1 0 2 24 3 4Diet 2 4 2 18 3 3Diet 3 9 2 19 2 3Diet 7 (Commercial) 10 2 15 3 4Diet 4 12 2 19 3 4Diet 5 14 2 19 3 4Diet 6 18 2 16 2 3Significance * for Diets 1, 3 <strong>and</strong> 6 bL<strong>in</strong>ear p

L<strong>in</strong>ear NS d NS p

Table 4. 6 - Colour attributes <strong>in</strong> muscle from ra<strong>in</strong>bow trout a (IBW= 549 g fish -1 ) fedexperimental diets for 24 weeksCGML b a* c b* d H°ab e C*ab f SF glevel(%)Diet 1 0 40.9 15.5 14.4 42.6 21.1 26Diet 2 4 41.6 14.6 14.0 43.4 20.4 26Diet 3 9 41.9 14.7 14.5 44.2 20.7 26Diet 7 (Commercial) 10 42.9 12.6 12.2 43.3 18.0 25Diet 4 12 41.5 14.1 14.2 44.8 20.1 26Diet 5 14 42.7 14.9 15.6 46.0 21.7 25Diet 6 18 41.8 11.7 11.2 43.1 16.3 24Significance * for Diets 1, 3 <strong>and</strong> 6 hL<strong>in</strong>ear NS j p

L<strong>in</strong>ear NS j NS NS NS NS NSQuadratic NS p

FiguresFigure 4. 1 - Growth curves of ra<strong>in</strong>bow trout fed the experimental diets for 24 weeks.102

Figure 4. 2 - Muscle pigment concentration to graded levels of corn gluten meal <strong>in</strong> ra<strong>in</strong>bow troutfed the experimental diets for 24 weeks. Larger markers on each trend l<strong>in</strong>e represent resultsobta<strong>in</strong>ed by diets where <strong>in</strong>clusion of corn gluten meal was at expense of soy prote<strong>in</strong> concentrateonly (0, 9 <strong>and</strong> 18% CGM <strong>in</strong>clusion).103

Figure 4. 3 - Muscle colour attributes to graded levels of corn gluten meal <strong>in</strong> ra<strong>in</strong>bow trout fedthe experimental diets for 24 weeks. Larger markers on each trend l<strong>in</strong>e represent results obta<strong>in</strong>edby diets where <strong>in</strong>clusion of corn gluten meal was at expense of soy prote<strong>in</strong> concentrate only (0, 9<strong>and</strong> 18% CGM <strong>in</strong>clusion).104

CHAPTER - 5 BLEACHING OF CAROTENOIDS FROM CORN GLUTEN MEALTHROUGH A MODIFIED STEEPING PROCESS TO IMPROVE ITS VALUE AS ASALMONID FISH INGREDIENT: EFFECTS OF KERNEL VARIETY, HYDROGENPEROXIDE AND STEEPWATER PHAbstract<strong>Corn</strong> gluten meal (CGM) is a highly digestible <strong>in</strong>gredient commonly used <strong>in</strong> diets forsalmonids. However the use of significant (>12%) levels of this <strong>in</strong>gredient has been related toreduction <strong>in</strong> pigment deposition <strong>in</strong> muscle of ra<strong>in</strong>bow trout (Oncorhynchus mykiss), possibly dueto negative effects of yellow xanthophylls on astaxanth<strong>in</strong> deposition <strong>in</strong> the muscle. Cost effectivemethods for decreas<strong>in</strong>g the level of yellow xanthophylls <strong>in</strong> corn products may improve theirvalue as a fish feed <strong>in</strong>gredient. A laboratory-scale (10 g) corn wet mill<strong>in</strong>g procedure was used toassess the bleach<strong>in</strong>g of carotenoids from CGM dur<strong>in</strong>g steep<strong>in</strong>g. Kernels from two maize (Zeamays L) varieties (HiC7 <strong>and</strong> HiC23 rich <strong>in</strong> lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, respectively) were used <strong>in</strong> thistrial. Hydrogen peroxide was added <strong>in</strong>to steepwater at 0, 0.15 or 0.30 M under either acidic (pH~2) or alkal<strong>in</strong>e (pH ~8) conditions. <strong>Corn</strong> kernels were steeped for 48 h at 52 °C. About 50% oftotal determ<strong>in</strong>ed carotenoids were bleached dur<strong>in</strong>g steep<strong>in</strong>g for the two kernel varieties at 0.30 Mof hydrogen peroxide concentration under acidic conditions (pH~2). <strong>Corn</strong> variety significantlyaffected bleach<strong>in</strong>g of all pigments determ<strong>in</strong>ed except for all-trans β-carotene. Steepwater pH hada significant effect on all-trans zeaxanth<strong>in</strong> <strong>and</strong> all-trans β-cryptoxanth<strong>in</strong>. Hydrogen peroxideaddition <strong>in</strong>to steepwater significantly affected all yellow carotenoids monitored. Mass balancefor total carotenoid determ<strong>in</strong>ed <strong>in</strong> corn gluten meal showed no significant effect of corn variety,suggest<strong>in</strong>g that bleach<strong>in</strong>g rates were proportional to the <strong>in</strong>itial pigment content <strong>in</strong> the kernels.Scal<strong>in</strong>g-up of this procedure (us<strong>in</strong>g commercial-scale equipment) is necessary <strong>in</strong> order to assess105

the effects of dietary reduced-carotenoid CGM on astaxanth<strong>in</strong> deposition <strong>in</strong> muscle of salmonidfish.106

5.1 IntroductionDeposition of carotenoids pigments, ma<strong>in</strong>ly astaxanth<strong>in</strong>, with<strong>in</strong> muscle fibers results <strong>in</strong> thedist<strong>in</strong>ctive red/p<strong>in</strong>k colour of fillet from salmonid fish. This important flesh quality attributeplays a crucial role on f<strong>in</strong>al product quality <strong>and</strong> significantly affects consumers’ perception(Alfnes et al., 2006; Anderson, 2000). Salmonid fish are not able to synthesize astaxanth<strong>in</strong> denovo,therefore synthetic or natural pigments are regularly <strong>in</strong>cluded <strong>in</strong> formulated diets. Thesefeed additives represent up to 10 - 20% of total feed costs (Bjerkeng, 2000; Choubert et al.,2009).Increas<strong>in</strong>g levels of plant-prote<strong>in</strong> <strong>in</strong>gredients have been progressively <strong>in</strong>cluded <strong>in</strong>aquaculture feed formulations. <strong>Corn</strong> gluten meal (CGM), is a low phosphorus, prote<strong>in</strong>-rich(~60% crude prote<strong>in</strong>) by-product of the corn wet mill<strong>in</strong>g, which has found wide use <strong>in</strong> feeds formany fish species. <strong>Corn</strong> wet mill<strong>in</strong>g is an <strong>in</strong>dustrial process aim<strong>in</strong>g to separate <strong>and</strong> ref<strong>in</strong>e themajor structural components <strong>in</strong> corn gra<strong>in</strong>s (i.e. starch, gluten, germ <strong>and</strong> fiber) <strong>in</strong> order toproduce valuable products for <strong>in</strong>dustrial, food <strong>and</strong> feed applications. Dur<strong>in</strong>g mill<strong>in</strong>g, carotenoidsfrom corn gra<strong>in</strong>s, ma<strong>in</strong>ly lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, rema<strong>in</strong> embedded with<strong>in</strong> the gluten fraction <strong>and</strong>are recovered <strong>in</strong> CGM, accord<strong>in</strong>gly high levels (200 - 550 mg kg -1 ) of carotenoids are conta<strong>in</strong>ed<strong>in</strong> this commodity (Park et al., 1997; Skonberg et al., 1998).Significant reduction <strong>in</strong> colour attributes of muscle from salmonid fish fed dietssupplemented with CGM has been reported. Low (p

astaxanth<strong>in</strong> deposition, redness <strong>and</strong> choma (a* <strong>and</strong> C*ab values, tristimulus color analysis), <strong>and</strong>fillet colour (assessed by SalmoFan TMcolorimetric analysis) was assessed <strong>in</strong> muscle fromra<strong>in</strong>bow trout fed diets formulated us<strong>in</strong>g different <strong>in</strong>gredient comb<strong>in</strong>ations, <strong>and</strong> to conta<strong>in</strong><strong>in</strong>creas<strong>in</strong>g levels of CGM (up to 18% <strong>in</strong>clusion level) <strong>and</strong> 50 mg kg -1of astaxanth<strong>in</strong>(unpublished data).The reduction of fish muscle pigmentation <strong>in</strong> response to dietary CGM is possibly relatedto a negative effect of yellow carotenoids on astaxanth<strong>in</strong> deposition <strong>in</strong> the muscle. Accord<strong>in</strong>gly,reduc<strong>in</strong>g the concentration of xanthophyll pigments <strong>in</strong> CGM might improve its value as a fishfeed <strong>in</strong>gredient.Bleach<strong>in</strong>g of yellow pigments from CGM us<strong>in</strong>g different process<strong>in</strong>g techniques (i.e.natural maturation, enzymatic oxidation <strong>and</strong> solvent extraction) has been attempted withpromis<strong>in</strong>g results (Cha et al., 2000; Gél<strong>in</strong>as et al., 1998; Park et al., 1997; Saez et al.,unpublished results). However, reduction of pigment content from corn kernels dur<strong>in</strong>g the courseof wet mill<strong>in</strong>g might represent a more practical approach from a commercial po<strong>in</strong>t of view.Conventional wet mill<strong>in</strong>g <strong>in</strong>volves the <strong>in</strong>cubation of corn kernels <strong>in</strong> steepwater (solutionconta<strong>in</strong><strong>in</strong>g 1- 2% lactic acid <strong>and</strong> 0.1 - 0.2 % sulfur dioxide) for 30 - 48 h under acidic conditions(pH ~ 2). Dur<strong>in</strong>g steep<strong>in</strong>g, kernels absorb steepwater (promot<strong>in</strong>g soften<strong>in</strong>g of kernels for furthergr<strong>in</strong>d<strong>in</strong>g) <strong>and</strong> sulfur dioxide breakes down prote<strong>in</strong> disulfide bonds <strong>in</strong>duc<strong>in</strong>g the release of<strong>in</strong>soluble starch (Dailey, 2002). Addition of a bleach<strong>in</strong>g agent <strong>in</strong>to steepwater would <strong>in</strong>duce theabsorption of oxidative species <strong>in</strong>side the kernel, allow<strong>in</strong>g the bleach<strong>in</strong>g of carotenoids beforecorn is separated <strong>in</strong>to it component parts <strong>and</strong> consequently reduc<strong>in</strong>g the recovery of yellowpigments <strong>in</strong> the gluten fraction.108

The aim of this study was to assess the bleach<strong>in</strong>g of carotenoid pigments from CGMdur<strong>in</strong>g steep<strong>in</strong>g us<strong>in</strong>g a laboratory bench-scale (10 g) corn wet mill<strong>in</strong>g process us<strong>in</strong>g twodifferent high-carotenoid varieties of corn gra<strong>in</strong>s. Steepwater composition was modified by theaddition of hydrogen peroxide (0.15 <strong>and</strong> 0.3 M under either acid or alkal<strong>in</strong>e conditions).Additionally, yields of different fractions, mass balance of total starch <strong>and</strong> crude prote<strong>in</strong> as wellas the mass balance for carotenoids with<strong>in</strong> the gluten meal fraction were assessed.5.2 Materials <strong>and</strong> methods<strong>Corn</strong> gra<strong>in</strong>s from two maize (Zea mays L.) l<strong>in</strong>es were produced <strong>and</strong> k<strong>in</strong>dly donated byProfessor Elizabeth A. Lee of the Maize Breed<strong>in</strong>g & Genetics Laboratory, University of Guelph.(Guelph, ON, Canada). Variety HiC-7 (Pedigree notation URO01089 x CG102-2(G)-1-2-1) <strong>and</strong>variety HiC-23 (Pedigree notation AR13026 x CG60/CG62-5(R)-1-11-1) were selected for thistrial based on their high content of lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, respectively (Burt et al., 2011). Table5.1 depicts proximate composition <strong>and</strong> carotenoid profile from the two different corn kernelvarieties used <strong>in</strong> this study.5.2.1 Bench-scale maize wet mill<strong>in</strong>g<strong>Corn</strong> gra<strong>in</strong>s were processed us<strong>in</strong>g a bench-scale mill<strong>in</strong>g process as proposed by Perez etal., (2001) with some modifications. Before steep<strong>in</strong>g, gra<strong>in</strong>s were manually cleaned <strong>in</strong> order toremove any foreign material or broken kernels. Seventy (70) mL of steepwater were mixed withdry corn kernels (~10 g) <strong>in</strong> a 250 mL beaker <strong>and</strong> steeped for 48 h <strong>in</strong> a shak<strong>in</strong>g water bath at 52°C <strong>and</strong> 100 r m<strong>in</strong> -1 .Hydrogen peroxide (H 2 O 2 ) was added <strong>in</strong>to steepwater at 0, 0.15 or 0.30 ppm under eitheracidic (pH ~2) or alkal<strong>in</strong>e (pH ~8) conditions (addition of 4M NaOH solution).109

After steep<strong>in</strong>g, kernels <strong>and</strong> steepwater were separated <strong>and</strong> gra<strong>in</strong> outer layers (hull) <strong>and</strong>germ fractions were h<strong>and</strong> dissected from the gra<strong>in</strong>s. Hull-free degermed endosperms were thenmixed with 100 ml of distilled water <strong>and</strong> further ground for 3 m<strong>in</strong> <strong>in</strong> a Dupont gr<strong>in</strong>der (Omnimixer 17105, Kennesaw, GA, USA) at approximately 10,000 r m<strong>in</strong> -1 . The resultant slurry wasmanually sieved us<strong>in</strong>g sta<strong>in</strong>less steel sieves (0.131 <strong>and</strong> 0.074 mm open<strong>in</strong>g size). <strong>Gluten</strong> fractionwas recovered from the sieves <strong>and</strong> the slurry pass<strong>in</strong>g through was further centrifuged at 1200 gfor 10 m<strong>in</strong> so higher density starch fraction can be isolated.Different corn wet mill<strong>in</strong>g fractions (i.e. fiber, germ, gluten <strong>and</strong> starch) were dried <strong>in</strong> anoven at 35 °C until dried (overnight), ground <strong>and</strong> stored at -20°C until analysis. Steepwater wasdried at 105 °C <strong>and</strong> solids <strong>in</strong> steepwater were determ<strong>in</strong>ed by mass difference. Each corn wetmill<strong>in</strong>g fraction yield was determ<strong>in</strong>ed as the ratio of the totally dried isolated fraction to the<strong>in</strong>itial amount of dried corn kernels.5.2.2 General analysis methodsSamples were analysed for dry matter <strong>and</strong> ash accord<strong>in</strong>g to AOAC (1995), crude prote<strong>in</strong>(%N x 6.25) us<strong>in</strong>g a LECO analyzer (LECO Corp., St. Joseph, MI, USA), <strong>and</strong> total lipid us<strong>in</strong>gpetroleum ether <strong>in</strong> a high pressure extractor (Ankom XT-20 Lipid extractor – ANKOMTechnology, Macedon, NY, USA). Total starch was determ<strong>in</strong>ed us<strong>in</strong>g a Megazyme® total starchdeterm<strong>in</strong>ation Kit (Megazyme International Irel<strong>and</strong>, Bray, Co. Wicklow, Irel<strong>and</strong>).Carotenoids extraction <strong>and</strong> determ<strong>in</strong>ation. Carotenoids from CGM samples wereextracted accord<strong>in</strong>g to Abdel-Aal et al. (2007). Samples (0.15 g) were mixed with 10 mL ofwater saturated butanol for 30 s <strong>in</strong> a PT 18/105 Ultra-turrax homogenizer (Stratford, CT, USA),110

kept for 30 m<strong>in</strong> at room temperature, <strong>and</strong> homogenized aga<strong>in</strong> for 30 s. The mixture wascentrifuged at 10,000 x g for 5 m<strong>in</strong>, <strong>and</strong> an aliquot of the supernatant (0.5 mL) was filteredthrough a 0.45 µm Nylon Acrodisc syr<strong>in</strong>ge filter (Cole-Parmer Canada Inc., Montreal, QC). Thefirst two drops of the filtrate were discarded, <strong>and</strong> the rem<strong>in</strong>der was collected for HPLC analyses.Carotenoids from corn gluten meal samples were separated <strong>and</strong> quantified accord<strong>in</strong>g toAbdel-Aal et al. (2007) us<strong>in</strong>g a 1100 series liquid chromatographer (Aligent, Mississauga, ON).<strong>Pigment</strong>s separation was performed on a short (10 cm x 4.6 mm, pack<strong>in</strong>g 3 µm) C30 column,YMC Carotenoid (Waters, Mississauga, ON, Canada), operated at 35°C. The mobile systemgradient used for elution was conducted us<strong>in</strong>g solutions of (A) methanol/methyl tert-butilether/nanopure water (81:15:4, v/v/v) <strong>and</strong> (B) methyl tert-butyl ether/methanol (90:10, v/v), <strong>and</strong>programed as follows: 0-9 m<strong>in</strong>, 100 - 75% A; 9 - 14 m<strong>in</strong>, 75 - 20% A; 14 - 15 m<strong>in</strong>, 25 - 0% A;17 - 18 m<strong>in</strong>, hold at 0% A; 17 - 18 m<strong>in</strong> 0 - 100% A; 18 -20 m<strong>in</strong> hold at 100% A. Afterseparation, detection <strong>and</strong> measurement of carotenoids was conducted at 450 nm (all-trans-lute<strong>in</strong>,all-trans zeaxanth<strong>in</strong>, all-trans-β-cryptoxanth<strong>in</strong> <strong>and</strong> all-trans-β-carotene,) <strong>and</strong> 478 nm (all-transastaxanth<strong>in</strong>),respectively. The correspondence of the retention times <strong>and</strong> UV/vis spectra ofanalysed samples <strong>and</strong> those showed by pure authentic st<strong>and</strong>ards was utilized for identification ofcarotenoids.For identification <strong>and</strong> quantification purposes five pure authentic st<strong>and</strong>ards carotenoids(all-trans-astaxanth<strong>in</strong>, all-trans-lute<strong>in</strong>, all-trans-β-carotene, all-trans zeaxanth<strong>in</strong>, all-trans-βcryptoxanth<strong>in</strong>)were utilized. Five concentrations for each carotenoid were prepared <strong>in</strong> butanol <strong>in</strong>order to assess l<strong>in</strong>earity of the response. The regression analysis (response area v/s <strong>in</strong>jectedamount) showed a l<strong>in</strong>ear relationship for all-trans-lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans-βcryptoxanth<strong>in</strong>,<strong>and</strong> all-trans-β-carotene with the coefficient of determ<strong>in</strong>ation (R 2 ) 0.9996, 0.9996,111

0.9994 <strong>and</strong> 0.9999, respectively. Purity of each compound <strong>in</strong> the diets <strong>and</strong> muscle extracts wasverified us<strong>in</strong>g isoabsorbance plot or 3D graphic <strong>and</strong> peak purity analyses (ChemStationsoftware).Nutrient mass balance. Mass balance of dry matter, total starch <strong>in</strong> the starch fraction, <strong>and</strong>mass balance of crude prote<strong>in</strong> <strong>and</strong> yellow xanthophylls <strong>in</strong> the corn gluten meal fraction weredeterm<strong>in</strong>ed based on the nutrient content <strong>in</strong> the <strong>in</strong>itial kernel samples <strong>and</strong> the percentage ofnutrient recovered on each fraction.5.2.3 Statistical analysisFactorial ANOVA analysis was carried out <strong>in</strong> order to determ<strong>in</strong>e the significance of eachs<strong>in</strong>gle factor as well as factors <strong>in</strong>teraction on the yield from the different fractions, HPLCanalysed yellow carotenoids <strong>in</strong> corn gluten meal <strong>and</strong> nutrient mass balance <strong>in</strong> starch <strong>and</strong> corngluten meal fractions. All data were analysed us<strong>in</strong>g the SAS/STAT software (SAS version 9.1.3,SAS <strong>in</strong>stitute, Cary, NC, USA).5.3 ResultsTable 5.2 shows the results for the mass balance of dry matter <strong>and</strong> yields of the differentcorn fractions obta<strong>in</strong>ed <strong>in</strong> this trial. Yield of gluten fraction decreased for the two kernel varieties<strong>and</strong> steepwater pH <strong>in</strong> response to <strong>in</strong>creas<strong>in</strong>g concentrations of hydrogen peroxide. Yield of starch<strong>in</strong>creased for HiC7 variety under acidic steep<strong>in</strong>g conditions <strong>and</strong> for HiC23 under both acid <strong>and</strong>alkal<strong>in</strong>e conditions <strong>in</strong> response to hydrogen peroxide <strong>in</strong>clusion. On the other h<strong>and</strong>, no clear trendwas observed for HiC7 <strong>in</strong> alkal<strong>in</strong>e conditions. The yield of germ fraction <strong>in</strong>creased for both typesof corn kernel <strong>and</strong> steepwater pH when hydrogen peroxide concentration was <strong>in</strong>creased. The112

yield of fiber fraction did not show a clear trend, however higher values were obta<strong>in</strong>ed with theHiC7 kernel variety. F<strong>in</strong>ally, the yield of solids <strong>in</strong> steepwater showed no clear trend.Total starch <strong>and</strong> crude prote<strong>in</strong> content <strong>in</strong> starch <strong>and</strong> gluten fraction are presented <strong>in</strong> Table5.3. Total starch content <strong>in</strong> starch fraction ranged from 81% to 93% <strong>and</strong> did not show any cleartrend. Crude prote<strong>in</strong> content <strong>in</strong> starch fraction was m<strong>in</strong>imal (ranged from 0.16% to 0.29%). Totalstarch content <strong>in</strong> the gluten fraction showed no clear trend <strong>and</strong> varied from 24% to 26%. Crudeprote<strong>in</strong> content <strong>in</strong> gluten fraction did not show a clear trend <strong>in</strong> this study <strong>and</strong> varied from 29% to43%.The carotenoid profile of corn gluten fractions produced <strong>in</strong> this trial is presented <strong>in</strong> Table5.4. Concentrations of all-trans lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong>, all-trans β-cryptoxanth<strong>in</strong> <strong>and</strong> all-transβ-carotene <strong>in</strong> gluten fractions obta<strong>in</strong>ed from variety HiC7 were reduced to <strong>in</strong>creas<strong>in</strong>g levels ofhydrogen peroxide under either acidic <strong>and</strong> alkal<strong>in</strong>e conditions. All carotenoids from the glutenfractions produced from variety HiC23 were reduced under acidic condition as hydrogenperoxide concentration was <strong>in</strong>creased. On the other h<strong>and</strong>, under alkal<strong>in</strong>e conditions, pigmentreduction <strong>in</strong> gluten fraction obta<strong>in</strong>ed from variety HiC23 was m<strong>in</strong>imal.A reduction was observed <strong>in</strong> the concentration of all different type of pigments fromvariety HiC7 under both acidic <strong>and</strong> alkal<strong>in</strong>e conditions as well as from variety HiC23 underacidic conditions to <strong>in</strong>creas<strong>in</strong>g levels of hydrogen peroxide. Conversely, carotenoid level <strong>in</strong> corngluten meal obta<strong>in</strong>ed from kernel variety HiC23 under alkal<strong>in</strong>e tended to be at a relativelyconstant level.Results for mass balance of total starch <strong>and</strong> crude prote<strong>in</strong> are shown <strong>in</strong> Table 5.5. Massbalance of total starch <strong>in</strong>creased with hydrogen peroxide <strong>in</strong>clusion for the two corn varieties113

under acidic <strong>and</strong> alkal<strong>in</strong>e conditions. Conversely, mass balance of crude prote<strong>in</strong> was reduced <strong>in</strong>variety HiC7 <strong>and</strong> HiC23 under acidic <strong>and</strong> alkal<strong>in</strong>e conditions, respectively.Tables 5.6 <strong>and</strong> 5.7 present mass balance of carotenoid pigments <strong>and</strong> the significance offactors applied, respectively. Mass balance of pigments <strong>in</strong> corn gluten meal produced from bothkernel varieties showed a general reduction trend to hydrogen peroxide level under both acidic<strong>and</strong> alkal<strong>in</strong>e conditions.5.4 DiscussionBleach<strong>in</strong>g of yellow carotenoids from corn gluten meal was assessed us<strong>in</strong>g a laboratoryscalewet mill<strong>in</strong>g process. Hydrogen peroxide, a bleach<strong>in</strong>g agent widely utilized for colourreduction <strong>in</strong> cellulosic <strong>and</strong> dairy products (Zeronian <strong>and</strong> Inglesby, 1995; Abdel-Aal et al., 1996;Kang et al., 2010) was <strong>in</strong>cluded <strong>in</strong> steepwater <strong>and</strong> applied under either acidic (pH~2) or alkal<strong>in</strong>e(pH~11) conditions. Two different corn kernel varieties were chosen based on their highcarotenoid content <strong>and</strong> composition (Table 5.1).The wet mill<strong>in</strong>g process used <strong>in</strong> this trial allowed the separation of corn kernels <strong>in</strong>to fivedifferent fractions (i.e. starch, gluten, germ, fiber <strong>and</strong> solids <strong>in</strong> steepwater). Yield of solids <strong>in</strong>steepwater obta<strong>in</strong>ed <strong>in</strong> this trial did not show a clear trend <strong>and</strong> values were high (rang<strong>in</strong>g from6% to 17%) compared to those previously reported for this fraction (Zehr et al., 1995). Thisresult suggests that large amounts of soluble compounds from corn kernels were leached <strong>in</strong>tosteep water dur<strong>in</strong>g the course of steep<strong>in</strong>g.Yields of germ <strong>and</strong> fiber fractions obta<strong>in</strong>ed <strong>in</strong> this trial (Table 5.2) were similar to thoseregularly achieved dur<strong>in</strong>g <strong>in</strong>dustrial operations. H<strong>and</strong>-dissection for hull <strong>and</strong> germ removalapplied <strong>in</strong> this study most probably led to an efficient isolation of these fractions. Yields of114

gluten meal fraction achieved <strong>in</strong> this study (Table 5.2) were about 1.6 to 4.1 folds higher (<strong>and</strong>yields of starch were 0.8 to 2.0 folds lower) than those previously reported for experimental cornwet mill<strong>in</strong>g procedures (Zehr et al., 1995; Eckhoff et al., 1996; S<strong>in</strong>gh et al., 1997; Dowd, 2003).These results suggest an <strong>in</strong>complete separation of the starch <strong>and</strong> prote<strong>in</strong> fractions.With<strong>in</strong> kernel endosperm, starch particles are embedded <strong>in</strong> prote<strong>in</strong> matrixes; sulfur dioxide<strong>and</strong> lactic acid dissolved <strong>in</strong> steepwater promotes the breakdown of disulfide bonds <strong>in</strong> prote<strong>in</strong>s,thus promot<strong>in</strong>g the release of starch (Krochta et al., 1981; Eckhoff et al., 1999). Low starchyields obta<strong>in</strong>ed <strong>in</strong> a laboratory-scale (10 g) wet mill<strong>in</strong>g process were related to non-uniformgr<strong>in</strong>d<strong>in</strong>g (Vignaux et al., 2006).Yields of starch <strong>and</strong> gluten fractions typically obta<strong>in</strong>ed under<strong>in</strong>dustrial conditions are difficult to replicate under bench-scale laboratory conditions ma<strong>in</strong>ly dueto equipment limitations. Moreover, high-prote<strong>in</strong> corn kernels have been correlated with highernumbers of prote<strong>in</strong> matrixes embedd<strong>in</strong>g starch (Vignaux et al., 2006), thus limit<strong>in</strong>g the release ofstarch granules. The high crude prote<strong>in</strong> level conta<strong>in</strong>ed <strong>in</strong> kernel varieties utilized <strong>in</strong> this trial(Table 5.1) might partially expla<strong>in</strong> the results from this trial.Incomplete separation of starch <strong>and</strong> prote<strong>in</strong> fractions obta<strong>in</strong>ed <strong>in</strong> this study can beattributed to mill<strong>in</strong>g shortcom<strong>in</strong>gs associated with equipment limitations. These shortcom<strong>in</strong>gsprobably did not have an effect on pigment reduction <strong>in</strong> the gluten fraction which is the ma<strong>in</strong>objective of this study.Increase of hydrogen peroxide concentration under acidic conditions led to lower gluten,<strong>and</strong> higher starch yields (Table 5.2). The effect of hydrogen peroxide on prote<strong>in</strong> oxidation <strong>in</strong>biological systems is very well documented (Stadtman <strong>and</strong> Lev<strong>in</strong>e, 2000). The highconcentration of hydrogen peroxide (0.03 M) used <strong>in</strong> this trial might have <strong>in</strong>duced prote<strong>in</strong>115

oxidation with<strong>in</strong> the endosperm of kernels <strong>and</strong> consequently improved millability of the prote<strong>in</strong>matrix.Total starch <strong>and</strong> crude prote<strong>in</strong> content <strong>in</strong> the starch fraction ranged from 81% to 93% <strong>and</strong>from 0.2% <strong>and</strong> 0.3%, respectively. On the other h<strong>and</strong>, total starch <strong>and</strong> crude prote<strong>in</strong> <strong>in</strong> the glutenfraction varied from 24% to 26% for all the treatments <strong>in</strong> this trial (Table 5.3). Substantialamounts of starch <strong>and</strong> low crude prote<strong>in</strong> level <strong>in</strong> the gluten fraction are consistent with the<strong>in</strong>complete separation of this fraction obta<strong>in</strong>ed <strong>in</strong> this trial. However, commercial corn glutenmeals with higher total starch content have been previously reported <strong>in</strong> the literature (Ji et al.,2012).Carotenoid profiles determ<strong>in</strong>ed for the high carotenoid corn varieties utilized <strong>in</strong> this studywere <strong>in</strong> the same range of those previously reported for these types of kernels (Burt et al., 2011)<strong>and</strong> higher than those reported for regular corn gra<strong>in</strong>s (Moros et al., 2002). HiC7 varietyconta<strong>in</strong>ed high levels of all-trans lute<strong>in</strong> content (58 mg kg -1 ) <strong>and</strong> a total pigment concentration of102 mg kg -1 , whereas HiC7 variety conta<strong>in</strong>ed high levels of all-trans zeaxanth<strong>in</strong> (56 mg kg -1 ) <strong>and</strong>a total carotenoid content of 84 mg kg -1 (Table 5.1).Carotenoid profiles of the corn gluten meal fraction produced us<strong>in</strong>g steepwater with nohydrogen peroxide <strong>in</strong>clusion under acidic conditions (Table 5.4) were <strong>in</strong> the range of thosereported for commercial corn gluten meal (Moros et al., 2002). Differences <strong>in</strong> carotenoidcomposition among corn gluten meals obta<strong>in</strong>ed from different corn kernel varieties reflectedpigment composition of corn gra<strong>in</strong>s utilized <strong>in</strong> this study (Table 5.1).Concentration of hydrogen peroxide <strong>in</strong> steepwater significantly affected carotenoidpigment reduction <strong>in</strong> corn gluten meal (Table 5.4). The effectiveness of hydrogen peroxide on116

pigment bleach<strong>in</strong>g has been previously reported. Hydrogen peroxide (used as part of oxidationsystem based on the Fenton’s system at a concentration of 0.03%) reduced more than 30% <strong>and</strong>60% (measured as loss of absorbance) of total lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong>, respectively <strong>in</strong> a fivem<strong>in</strong>utereaction at 25°C (Woodall et al., 1997). Through the course of corn steep<strong>in</strong>g progression,steepwater penetrates (through the tip cap) <strong>and</strong> disperses with<strong>in</strong> different structural compartmentof the gra<strong>in</strong> (i.e. germ <strong>and</strong> endosperm) by the action of capillary forces (Krochta et al., 1981).Hydrogen peroxide <strong>in</strong>cluded <strong>in</strong> steepwater <strong>in</strong> this trial, probably <strong>in</strong>duced an oxidation processwith<strong>in</strong> corn endosperm, thus bleach<strong>in</strong>g yellow carotenoids from the gluten fraction <strong>and</strong>consequently reduc<strong>in</strong>g the carotenoid recovery <strong>in</strong> corn gluten meal.<strong>Corn</strong> variety significantly affected the bleach<strong>in</strong>g of all-trans lute<strong>in</strong>, all-trans zeaxanth<strong>in</strong> <strong>and</strong>all-trans β-cryptoxanth<strong>in</strong> (Table 5.4). Dur<strong>in</strong>g steep<strong>in</strong>g, the rate of steepwater uptake is dependenton the permeability of the tip cap <strong>and</strong> aleurone layer (prote<strong>in</strong> layer cover<strong>in</strong>g endosperm <strong>in</strong> corngra<strong>in</strong>s) (Krochta et al., 1981). Structural differences attributed to lower hydrogen concentrations<strong>in</strong> response to fungal degradation have been determ<strong>in</strong>ed for different varieties of dried corn gra<strong>in</strong>(assessed us<strong>in</strong>g neutron tomography analysis) (Clevel<strong>and</strong> et al., 2008). Differences <strong>in</strong> <strong>in</strong>ternalstructure might change the degree of permeability <strong>in</strong> different varieties of corn kernels thusreduc<strong>in</strong>g contact between hydrogen peroxide <strong>and</strong> carotenoid molecules. Whether the two kernelsvarieties used <strong>in</strong> this trial presented different <strong>in</strong>ternal structure rema<strong>in</strong>s unknown <strong>and</strong> mightexpla<strong>in</strong> this result.Steepwater pH showed a significant effect on the bleach<strong>in</strong>g of all-trans zeaxanth<strong>in</strong> <strong>and</strong> alltransβ-cryptoxanth<strong>in</strong> (Table 5.4). Lower bleach<strong>in</strong>g rations of yellow carotenoids were observedunder alkal<strong>in</strong>e conditions for both types of kernels. Alkal<strong>in</strong>e hydrogen peroxide has beenreported as an effective bleach<strong>in</strong>g agent for the reduction of dark colour (expressed as total117

colour difference us<strong>in</strong>g a colorimeter) <strong>in</strong> wheat stillage (Abdel-Aal et al., 1996). However,sodium hydroxide utilized to modify steepwater alkal<strong>in</strong>ity <strong>in</strong> this trial most probably <strong>in</strong>creasedthe density of steep<strong>in</strong>g solution (Perry <strong>and</strong> Green, 1984) thus limit<strong>in</strong>g absorption of steepwaterby the kernel.Mass balance of starch obta<strong>in</strong>ed <strong>in</strong> this trial <strong>in</strong>dicated that 35% to 48% of the starchcontent <strong>in</strong> the <strong>in</strong>itial corn sample was not recovered (Table 5.5). These values are high comparedto those previously reported (6% to17% of starch loss) (Zehr et al., 1995; S<strong>in</strong>gh et al., 1997;Dowd, 2003). The occurrence of fermentation dur<strong>in</strong>g steep<strong>in</strong>g has been reported (Gawienowskiet al., 1999). Whether fermentation of starch occurred dur<strong>in</strong>g steep<strong>in</strong>g rema<strong>in</strong>s unknown <strong>and</strong>might expla<strong>in</strong> the high levels of unrecovered starch obta<strong>in</strong>ed <strong>in</strong> this study.On the other h<strong>and</strong>, values for prote<strong>in</strong> recovery were <strong>in</strong> general high, from 74% to 112%.This type of results has been previously associated to a low millability of corn kernels <strong>and</strong> tohigh recovery of starch <strong>in</strong> the gluten fraction (S<strong>in</strong>gh et al., 1997). Additionally, overestimationof prote<strong>in</strong> mass balance observed <strong>in</strong> this trial (e.i. values exceed<strong>in</strong>g 100%) can be attributed tosampl<strong>in</strong>g errors associated to the small quantity of sample used for the quantitative determ<strong>in</strong>ationof nitrogen (LECO, Dumas method).Mass balance for carotenoids obta<strong>in</strong>ed <strong>in</strong> this trial <strong>in</strong>dicated that a maximum of 65% <strong>and</strong>63% of total determ<strong>in</strong>ed yellow pigments conta<strong>in</strong>ed <strong>in</strong> the <strong>in</strong>itial sample were recovered <strong>in</strong> corngluten meals produced from varieties HiC7 <strong>and</strong> HiC23, respectively. This result <strong>in</strong>dicates thatabout 25% of yellow carotenoids are lost dur<strong>in</strong>g the wet mill<strong>in</strong>g process used <strong>in</strong> this trial evenunder acidic conditions (pH~2) <strong>and</strong> no hydrogen peroxide <strong>in</strong>clusion. No significant effect wasdeterm<strong>in</strong>ed for corn variety on mass balance of carotenoids <strong>in</strong> this study, suggest<strong>in</strong>g that118

leach<strong>in</strong>g rates obta<strong>in</strong>ed <strong>in</strong> this study were proportional to the <strong>in</strong>itial pigment content on eachcorn variety.The laboratory-scale wet mill<strong>in</strong>g used <strong>in</strong> this trial (steep<strong>in</strong>g corn kernels <strong>in</strong> a hydrogenperoxide conta<strong>in</strong><strong>in</strong>g solution at either acid or alkal<strong>in</strong>e conditions) produced high yields of corngluten meal (with high total starch content) <strong>and</strong> low yields of starch, which were attributed to an<strong>in</strong>complete separation of the gluten <strong>and</strong> starch fractions.High yield of gluten fraction but low yields of starch fraction obta<strong>in</strong>ed <strong>in</strong> this study wereattributed to gr<strong>in</strong>d<strong>in</strong>g equipment limitation. <strong>Reduction</strong> of yellow carotenoids content <strong>in</strong> corngluten meal was proportional to the pigment content <strong>in</strong> corn gra<strong>in</strong>s. Recovery of crude prote<strong>in</strong>did not show a clear trend, however up to 40% reduction was assessed as hydrogen peroxidelevel was <strong>in</strong>creased for the HiC7 variety under acidic conditions.Scale-up <strong>and</strong> use of commercial scale mill<strong>in</strong>g equipment as well as <strong>in</strong>-vivo pigmentationtrial are highly necessary <strong>in</strong> order to assess the effects of low-pigment corn gluten meal <strong>in</strong>clusion<strong>in</strong> formulated diets on muscle pigment deposition <strong>in</strong> muscle from salmonid fish.119

TablesTable 5. 1 - Analyzed proximate composition <strong>and</strong> pigment content of the two corn kernelvarieties used <strong>in</strong> the study<strong>Corn</strong> kernel varietyHiC7HiC23Analysed proximate composition (%), dry matter basisMoisture 10.5 ± 0.01 9.6 ± 0.03Crude prote<strong>in</strong> 11.7 ± 0.07 12.1 ± 0.05Total starch 69.3 ± 2.0 67.3 ± 0.3Lipids 4.5 ± 0.2 4.2 ± 0.3Ash 1.3 ± 0.02 1.5 ± 0.01Analysed pigment concentration (mg kg -1 ), dry matter basisAll-trans lute<strong>in</strong> 58 ± 4.3 14 ± 0.9All-trans zeaxanth<strong>in</strong> 34 ± 2.5 56 ± 6.1All-trans β-cryptoxanth<strong>in</strong> 7 ± 1.9 10 ± 0.8All-trans-β-carotene 4 ± 1.5 4 ± 0.9Total carotenoids 102 ± 10.2 84 ± 8.6Data are mean (n=2) ± st<strong>and</strong>ard deviation.120

Table 5. 2 - Yields of different fractions <strong>and</strong> mass balance of dry matter obta<strong>in</strong>ed us<strong>in</strong>g the laboratory scale corn wet mill<strong>in</strong>g underdifferent steep<strong>in</strong>g conditions<strong>Corn</strong>SteepwaterH 2 O 2Dry matter contentDry matter content <strong>in</strong> different fractions (%)varietypH(Mol L -1 )<strong>in</strong> <strong>in</strong>itial kernelStarch <strong>Gluten</strong> Germ Fiber Solids <strong>in</strong>Unaccountedsample (g)steepwaterfractionHiC7 2.0 0.00 8.9 44 (3.9) 32 (2.8) 8 (0.7) 9 (0.8) 7 (0.6) 0 (0.0)0.15 9.0 42 (3.8) 29 (2.6) 8 (0.7) 9 (0.8) 9 (0.8) 3 (0.2)0.30 9.1 50 (4.5) 19 (1.8) 8 (0.8) 9 (0.8) 10 (0.9) 4 (0.3)8.0 0.00 9.1 40 (3.6) 33 (3.0) 8 (0.8) 9 (0.8) 9 (0.8) 1 (0.1)0.15 9.1 40 (3.6) 32 (2.9) 11 (1.0) 9 (0.8) 6 (0.5) 3 (0.2)0.30 9.0 39 (3.5) 31 (2.8) 9 (0.8) 10 (0.9) 8 (0.7) 4 (0.3)HiC23 2.0 0.00 9.1 40 (3.6) 31 (2.8) 6 (0.5) 7 (0.7) 12 (1.1) 5 (0.4)0.15 9.1 44 (2.5) 28 (2.5) 7 (0.6) 7 (0.6) 10 (0.9) 5 (0.5)0.30 9.1 43 (4.0) 22 (2.0) 7 (0.6) 8 (0.7) 8 (0.8) 11 (1.0)8.0 0.00 9.2 28 (2.5) 48 (4.4) 8 (0.7) 7 (0.7) 9 (0.8) 1 (0.1)0.15 9.3 39 (3.6) 31 (2.9) 11 (1.0) 9 (0.8) 8 (0.7) 3 (0.3)0.30 9.1 31(2.8) 31 (2.8) 8 (0.7) 8 (0.7) 17 (1.5) 5 (0.5)121

<strong>Effects</strong> of<strong>Corn</strong> variety *** ** * **** NS ****Steepwater pH **** **** ** NS **** ****H 2 0 2 * **** * NS **** ****<strong>Corn</strong> variety * Steepwater pH NS ** NS NS **** ****<strong>Corn</strong> variety * H 2 0 2 * ** NS NS **** ****Steepwater pH * H 2 0 2 * ** * NS **** ****<strong>Corn</strong> variety * Steepwater pH * H 2 0 2 NS *** NS NS **** ****Data are mean (n=2). Values <strong>in</strong> brackets are expressed <strong>in</strong> g.; NS= Not significant; *=p

Table 5. 3 - Total starch <strong>and</strong> crude prote<strong>in</strong> content <strong>in</strong> starch <strong>and</strong> gluten fractions<strong>Corn</strong> variety Steepwater pH H 2 O 2 (Mol L -1 ) Starch fraction <strong>Gluten</strong> fractionTotalCrudeTotalCrudestarch (%)prote<strong>in</strong> (%)starch (%)Prote<strong>in</strong> (%)HiC7 2.0 0.00 84.9 0.26 24.8 41.10.15 83.1 0.16 24.3 37.10.30 82.6 0.27 24.9 41.08.0 0.00 84.5 0.20 25.1 29.20.15 81.0 0.27 24.2 43.70.30 82.7 0.23 23.2 41.9HiC23 2.0 0.00 87.0 0.29 25.1 37.60.15 92.7 0.25 24.5 39.90.30 90.3 0.25 24.7 41.98.0 0.00 85.8 0.25 26.1 27.60.15 89.8 0.23 23.9 39.10.30 88.9 0.25 25.1 30.5123

<strong>Effects</strong> of<strong>Corn</strong> variety NS NS NS ****Steepwater pH **** NS NS ****H 2 0 2 NS NS * NS<strong>Corn</strong> variety * Steepwater pH NS NS NS ****<strong>Corn</strong> variety * H 2 0 2 NS NS NS *Steepwater pH * H 2 0 2 * NS NS *<strong>Corn</strong> variety * Steepwater pH * H 2 0 2 NS NS NS NSData are mean (n=2); NS= Not significant; *=p

Table 5. 4 - Carotenoids profile <strong>in</strong> corn gluten meal fraction produced under experimental conditions<strong>Corn</strong> variety Steepwater pH H 2 O 2All-transAll-transAll-transAll-transTotal carotenoids(Mol L -1 )Lute<strong>in</strong>Zeaxanth<strong>in</strong>β-Cryptoxanth<strong>in</strong>β-CaroteneHiC7 2.0 0.00 121 75 9 6 2110.15 65 40 8 3 1160.30 61 14 7 1 1078.0 0.00 92 55 8 4 1600.15 86 55 8 3 1530.30 67 40 8 2 118HiC23 2.0 0.00 18 84 11 5 1160.15 17 55 9 6 870.30 14 37 6 0 588.0 0.00 18 78 12 5 1130.15 18 69 12 6 1040.30 18 84 11 5 116125

<strong>Effects</strong> of<strong>Corn</strong> variety **** *** **** NS ****Steepwater pH NS * * NS NSH 2 0 2 **** **** *** * ****<strong>Corn</strong> variety * Steepwater pH NS * ** NS NS<strong>Corn</strong> variety * H 2 0 2 **** NS NS NS NSSteepwater pH * H 2 0 2 * ** NS NS ***<strong>Corn</strong> variety * Steepwater pH * H 2 0 2 * NS NS NS NSData are mean (n=2); NS= Not significant; *=p

Table 5. 5 - Mass balance of total starch <strong>and</strong> crude prote<strong>in</strong> obta<strong>in</strong>ed us<strong>in</strong>g the laboratory-scale corn wet mill<strong>in</strong>g<strong>Corn</strong>SteepwaterH 2 O 2Total starchCrude prote<strong>in</strong>varietypH(Mol L -1 )Initial aStarch bCGM cUnaccountedInitial aStarch bCGM cUnaccounted(g)(%)(%)fraction (%)(g)(%)(%)fraction (%)HiC7 2.0 0.00 6.2 54 (3.3) 11 (0.7) 35 (2.2) 1.0 1 (0.01) 112 (1.2) -13 (-0.1)0.15 6.2 50 (3.1) 10 (0.6) 40 (2.5) 1.1 1 (0.01) 93 (1.0) 7 (0.1)0.30 6.3 59 (3.7) 7 (0.4) 34 (2.1) 1.1 1 (0.01) 68 (0.7) 31 (0.3)8.0 0.00 6.3 50 (3.1) 12 (0.8) 38 (2.4) 1.1 1 (0.01) 106 (1.1) -7 (-0.1)0.15 6.3 53 (3.3) 11 (0.7) 35 (2.2) 1.1 1 (0.01) 109 (1.2) -10 (-0.1)0.30 6.2 51 (3.1) 11 (0.7) 38 (2.4) 1.0 1 (0.01) 108 (1.1) -8 (-0.1)HiC23 2.0 0.00 6.1 50 (3.0) 11 (0.7) 39 (2.4) 1.1 1 (0.01) 74 (0.8) 25 (0.3)0.15 6.1 53 (3.2) 10 (0.6) 38 (2.3) 1.1 1 (0.01) 100 (1.1) 0 (0.0)0.30 6.1 53 (3.3) 8 (0.5) 39 (2.4) 1.1 1 (0.01) 77 (0.8) 23(-0.2)8.0 0.00 6.2 35 (2.2) 19 (1.1) 46 (2.9) 1.1 1 (0.01) 109 (1.2) -10 (-0.1)0.15 6.3 52 (3.2) 11 (0.7) 37 (2.3) 1.1 1 (0.01) 101 (1.1) -1 (0.0)0.30 6.1 41 (2.5) 12 (0.7) 48 (2.9) 1.1 1 (0.01) 78 (0.9) 21 (0.2)127

<strong>Effects</strong> of<strong>Corn</strong> variety **** **** **** * **** ****Steepwater pH **** **** **** NS **** ****H 2 0 2 **** **** * NS **** ****<strong>Corn</strong> variety * Steepwater pH **** **** ** NS NS ND<strong>Corn</strong> variety * H 2 0 2 **** **** **** * *** ***Steepwater pH * H 2 0 2 **** **** **** NS * *<strong>Corn</strong> variety * Steepwater pH * H 2 0 2 NS **** NS NS **** ****Data are mean (n=2); Values <strong>in</strong> brackets are expressed <strong>in</strong> g.; NS= Not significant; *=p

Table 5. 6 - Mass balance of carotenoids obta<strong>in</strong>ed <strong>in</strong> the laboratory-scale corn wet mill<strong>in</strong>gAll-transAll-transAll-transAll-transTotal carotenoidslute<strong>in</strong>zeaxanth<strong>in</strong>β-cryptoxanth<strong>in</strong>β-carotene<strong>Corn</strong>pHH 2 O 2bIn cGF dUn eInGFUnInGFUnInGFUnInGFUnvarietya(μg)(%)(%)(μg)(%)(%)(μg)(%)(%)(μg)(%)(%)(μg)(%)(%)HiC7 2.0 0.00 515 66 34 303 70 30 64 41 59 32 49 51 914 65 350.15 518 33 67 305 34 66 64 34 66 32 22 78 919 33 670.30 524 21 79 309 22 78 65 20 80 33 6 94 931 20 808.0 0.00 523 53 47 308 54 46 65 38 62 33 40 60 929 52 480.15 522 48 52 308 52 48 65 38 62 33 31 69 927 48 520.30 516 37 63 304 37 63 64 35 65 32 21 79 916 36 64HiC23 2.0 0.00 130 37 63 505 46 54 86 35 65 36 35 65 757 43 570.15 131 32 68 508 27 73 87 27 73 36 44 56 762 29 710.30 131 22 78 507 15 85 86 15 85 36 0 100 760 15 858.0 0.00 132 60 40 511 67 33 87 63 37 36 54 46 766 64 360.15 134 39 61 519 38 62 88 39 61 37 46 54 779 39 61129

Data are mean (n=2).a Steepwater pH.0.30 131 38 62 506 47 53 86 35 65 36 36 64 759 43 57b H 2 O 2 (Mol L -1 ).c Carotenoid content <strong>in</strong> <strong>in</strong>itial kernel sample (dry matter basis).d Carotenoid content <strong>in</strong> gluten fraction (dry matter basis).130

Table 5. 7 - Significance of s<strong>in</strong>gle effect as well as of two-way <strong>and</strong> three-way <strong>in</strong>teraction of factors on carotenoids mass balanceAll-transAll-transAll-transAll-transTotal carotenoidslute<strong>in</strong>Zeaxanth<strong>in</strong>β-Cryptoxanth<strong>in</strong>β-Carotene<strong>Effects</strong> of<strong>Corn</strong> variety NS NS NS NS NSSteepwater pH **** **** **** NS ****H 2 0 2 **** **** **** * ****<strong>Corn</strong> variety * Steepwater pH NS * *** NS *<strong>Corn</strong> variety * H 2 0 2 * NS * NS NSSteepwater pH * H 2 0 2 NS * NS NS *<strong>Corn</strong> variety * Steepwater pH * H 2 0 2 *** * * NS *NS= Not significant; *=p

CHAPTER - 6 GENERAL DISCUSSIONCurrent formulation of aquaculture feeds is based on the use of comb<strong>in</strong>ation of numerous<strong>in</strong>gredients <strong>in</strong>clud<strong>in</strong>g fish meal, rendered prote<strong>in</strong> <strong>in</strong>gredient <strong>and</strong> plant-orig<strong>in</strong> meals (Alexis et al.,1985; Cha et al., 2000; de Francesco et al., 2004). The <strong>in</strong>clusion levels of fish meal have beenreduced by <strong>in</strong>creas<strong>in</strong>g the utilization of cost-effective alternative prote<strong>in</strong>s. A large quantity ofresearch describ<strong>in</strong>g the effect of new <strong>in</strong>gredient on growth <strong>and</strong> nutrient digestibility <strong>and</strong>deposition has been conducted; however m<strong>in</strong>or efforts have been given to assess<strong>in</strong>g the effectson these <strong>in</strong>gredients on flesh quality such as texture <strong>and</strong> colour. A major goal of fish farm<strong>in</strong>g isthat colour of their products meets the high st<strong>and</strong>ards that consumers dem<strong>and</strong> (Anderson, 2000).Colour is a quality trait that dramatically <strong>in</strong>fluences the economic value of f<strong>in</strong>al products,consequently fillets present<strong>in</strong>g poor muscle pigmentation will be downgraded (i.e. reduction ofthe economic value of the product) at process<strong>in</strong>g plants (Anderson, 2000; Johnston et al., 2006).Colour is the result of pigment, ma<strong>in</strong>ly astaxanth<strong>in</strong>, deposition <strong>and</strong> accumulation with<strong>in</strong>muscle fibers. Formulated diets for farmed salmonid fish must be supplemented with premixesconta<strong>in</strong><strong>in</strong>g synthetic or natural pigments <strong>in</strong> order to reach market expectations. Dietsupplementation with expensive pigments represents up to 15% of f<strong>in</strong>al cost (Bjerkeng, 2000;Choubert et al., 2009). Given the high price of commercial carotenoid premixes <strong>and</strong> the higheconomic impact of colour attributes on fillet’s economic value, accurate <strong>in</strong>formation on theeffects of feed <strong>in</strong>gredients on astaxanth<strong>in</strong> muscle deposition is important for the properdevelopment of aquaculture feed formulations.<strong>Corn</strong> gluten meal (CGM) is a by-product of the corn wet mill<strong>in</strong>g process with highdigestibility <strong>and</strong> high prote<strong>in</strong> content that has become <strong>in</strong>creas<strong>in</strong>gly used <strong>in</strong> diets for many fish132

species. However, anecdotal evidence from fish formulators along with results from fewscientific studies suggest that high <strong>in</strong>clusion levels of this community results <strong>in</strong> the reduction ofcolour attributes <strong>in</strong> muscle from salmonid fish (Mundheim et al., 2004; Skonberg et al., 1998).The manufacture of corn gluten meal from white varieties of corn might represent a solution forthis problem. However the production levels of white corn are significantly lower compare toproduction levels of yellow corn. Therefore the development of a practical <strong>and</strong> cost-effectiveprocess<strong>in</strong>g method for the reduction of yellow pigments <strong>in</strong> corn gluten meal, us<strong>in</strong>g soybeanlipoxygenase, was assessed <strong>in</strong> Chapter 3.<strong>Reduction</strong> of the pigment level <strong>in</strong> corn gluten meal, us<strong>in</strong>g soybean lipoxygenase, has beenattempted by some research groups (Cha et al., 2000; Park et al., 1997) with promis<strong>in</strong>g results.However the experimental designs utilized <strong>in</strong> those studies did not consider many of the potentialfactors affect<strong>in</strong>g the activity of lipoxygenase <strong>and</strong> thus the degree of pigment bleach<strong>in</strong>g.Moreover, even though these studies explored the scale up of the developed pigment bleach<strong>in</strong>gmethodology, they did not assess the effect of pigment-reduced corn gluten meal <strong>in</strong> <strong>in</strong>-vivomuscle pigmentation trials.Screen<strong>in</strong>g <strong>and</strong> optimization of factors affect<strong>in</strong>g the bleach<strong>in</strong>g of corn gluten meal wereassessed us<strong>in</strong>g Plackett-Burman <strong>and</strong> Box-Behnken designs, respectively. Up to 91% of totalpigments were destroyed, from a CGM sample with an <strong>in</strong>itial content of 158 mg kg -1 , when anoptimal factor comb<strong>in</strong>ation was applied.The pigment bleach<strong>in</strong>g process developed <strong>in</strong> Chapter 3 was successfully scaled up <strong>and</strong> thusthe evaluation of pigment-bleached <strong>and</strong> regular corn gluten meal on muscle pigmentation <strong>in</strong>ra<strong>in</strong>bow trout was evaluated (Chapter 3). Growth parameters were not affected, however a133

significant reduction <strong>in</strong> astaxanth<strong>in</strong> concentration <strong>in</strong> the fillets of ra<strong>in</strong>bow trout was observed <strong>in</strong>response to the <strong>in</strong>clusion of 19% of corn gluten meal <strong>in</strong> the diet. Nonetheless, the significantreduction <strong>in</strong> muscle astaxanth<strong>in</strong> concentration did not affect any of the colour attributes assessed<strong>in</strong> this study. Similar f<strong>in</strong>d<strong>in</strong>gs have been previously reported <strong>and</strong> it is hypothesized thatvariations <strong>in</strong> light scatter<strong>in</strong>g properties <strong>and</strong> lipid content <strong>in</strong> the muscle might affect colourexpression (Johnston et al., 2000).M<strong>in</strong>imal amounts of astaxanth<strong>in</strong> were determ<strong>in</strong>ed <strong>in</strong> muscle from fish fed the dietsupplemented with bleached corn gluten meal. This result suggests that highly reactive freeradicals produced dur<strong>in</strong>g bleach<strong>in</strong>g of pigments from corn gluten meal were not effectivelyscavenged. These reactive oxygen species <strong>in</strong>duced lipid oxidation <strong>and</strong> the eventual destruction ofdietary astaxanth<strong>in</strong>. Sufficient antioxidant supplementation must be considered when <strong>in</strong>gredientprocessed us<strong>in</strong>g oxidative treatment are <strong>in</strong>cluded <strong>in</strong> the formulation of compounds feed <strong>in</strong> orderto avoid reduction of nutritional quality of f<strong>in</strong>al feed.Based on the significant reduction of astaxanth<strong>in</strong> deposition <strong>in</strong> fillets from ra<strong>in</strong>bow troutobserved <strong>in</strong> Chapter 3, <strong>and</strong> <strong>in</strong> order to underst<strong>and</strong> at what levels of <strong>in</strong>clusion corn gluten mealcan potentially affect astaxanth<strong>in</strong> muscle concentration <strong>and</strong>/or colour attributes, the effects of<strong>in</strong>creas<strong>in</strong>g levels of dietary corn gluten meal on growth <strong>and</strong> muscle pigmentation of ra<strong>in</strong>bowtrout was explored (Chapter 4).Neither growth parameters nor feed efficiency was affected by corn gluten meal <strong>in</strong>clusion.However, consistently with f<strong>in</strong>d<strong>in</strong>g of Chapter 3, a significant reduction <strong>in</strong> astaxanth<strong>in</strong> muscledeposition was observed <strong>in</strong> response to <strong>in</strong>creas<strong>in</strong>g levels of corn gluten meal. Inclusion levels of134

corn gluten meal exceed<strong>in</strong>g 12% reduced astaxanth<strong>in</strong> muscle concentration as well as importantcolour attributes <strong>in</strong> the flesh.Low concentrations of all-trans lute<strong>in</strong> <strong>and</strong> all-trans zeaxanth<strong>in</strong> were determ<strong>in</strong>ed <strong>in</strong> musclefrom ra<strong>in</strong>bow trout fed all diets used <strong>in</strong> this trial. This result is <strong>in</strong> accordance with previousstudies report<strong>in</strong>g deposition of yellow xanthophylls <strong>in</strong> muscle from salmonid fish (Bowen et al.2002; Kitahara, 1983; Welker et al. 2001).Consider<strong>in</strong>g the significant reduction of carotenoids from corn gluten meal obta<strong>in</strong>ed <strong>in</strong>Chapter 3, but aim<strong>in</strong>g to develop a more practical process<strong>in</strong>g methodology from a commercialpo<strong>in</strong>t of view, the bleach<strong>in</strong>g of carotenoid pigments from corn gluten meal dur<strong>in</strong>g steep<strong>in</strong>g(dur<strong>in</strong>g conventional corn wet mill<strong>in</strong>g process) was addressed <strong>in</strong> Chapter 5.Two varieties of corn kernels, with high carotenoid content <strong>and</strong> different pigment profile,were utilized <strong>in</strong> order to simulate variation <strong>in</strong> pigment content of different batches of cornkernels used <strong>in</strong> commercial production. Hydrogen peroxide, an antioxidant widely utilized forcolour reduction of cellulosic <strong>and</strong> dairy products, was added <strong>in</strong>to steepwater as a bleach<strong>in</strong>gagent. Based on the effective reduction <strong>in</strong> colour of wheat stillage us<strong>in</strong>g alkal<strong>in</strong>e hydrogenperoxide (Abdel-Aal et al., 1996), pH of steepwater was modified <strong>in</strong> order to assess thebleach<strong>in</strong>g of carotenoids under acidic <strong>and</strong> alkal<strong>in</strong>e conditions.High gluten yields but low starch yields were obta<strong>in</strong>ed <strong>and</strong> attributed to the <strong>in</strong>completeseparation of these two fractions due to limitation related to gr<strong>in</strong>d<strong>in</strong>g equipment utilized <strong>in</strong> thistrial thus not affect<strong>in</strong>g bleach<strong>in</strong>g of pigments. About 50% of the <strong>in</strong>itial carotenoid content wasbleached us<strong>in</strong>g this methodology. <strong>Corn</strong> variety, hydrogen peroxide <strong>and</strong> steepwater pH had asignificant effect on carotenoid bleach<strong>in</strong>g.135

<strong>Reduction</strong> of pigments from corn gluten meal dur<strong>in</strong>g steep<strong>in</strong>g of corn kernels dur<strong>in</strong>g cornwet mill<strong>in</strong>g represent a straight forward, cost-effective approach that might f<strong>in</strong>d a wide use <strong>in</strong>commercial production of low carotenoid corn gluten meal for specific applications such asaquaculture feed manufactur<strong>in</strong>g.6.1 Metabolism of carotenoids <strong>in</strong> fishMetabolism of carotenoid pigments <strong>in</strong> fish is assumed to be similar to that of dietary fat <strong>in</strong>mammals (Olsen <strong>and</strong> Baker, 2006).After <strong>in</strong>gestion carotenoids are emulsified <strong>in</strong>to lipidemulsions. Polar xanthophylls distribute with<strong>in</strong> the emulsions’ surface from where they arespontaneously transferred <strong>in</strong>to bile salt micelles before be<strong>in</strong>g absorbed through the <strong>in</strong>test<strong>in</strong>alepithelial cells (Borel et al., 1996). Whether occurrence of different types of xanthophylls with<strong>in</strong>lipid emulsions affects astaxanth<strong>in</strong> transfer <strong>and</strong>/or allocation <strong>in</strong>to mixed micelles rema<strong>in</strong>s to beclarified.Mixed lipid micelles reach the <strong>in</strong>test<strong>in</strong>al bush border membrane where carotenoids areabsorbed through passive diffusion. <strong>Pigment</strong>s can compete for absorption or facilitate theabsorption of another (Ishida <strong>and</strong> Bartley, 2005). Whether the presence of a mixture of polarxanthophyll with<strong>in</strong> the surface of mixed micelles can reduce astaxanth<strong>in</strong> absorption <strong>in</strong> ra<strong>in</strong>bowtrout needs to be addresses.The mechanisms govern<strong>in</strong>g carotenoids translocation <strong>and</strong> <strong>in</strong>corporation <strong>in</strong>to lipoprote<strong>in</strong>swith<strong>in</strong> the enterocyte are unknown. As no specific <strong>in</strong>tracellular transport for carotenoids has beendescribed, a non-discrim<strong>in</strong>atory transport has been proposed (Parker, 1996; Dem<strong>in</strong>g <strong>and</strong> Erdman,1999), this suggests that when mixtures of different xanthophylls are absorbed, comb<strong>in</strong>ations of136

different pigments would be carried <strong>in</strong>to the enterocyte’s Golgi apparatus <strong>and</strong> assembled <strong>in</strong>tonascent chylomicrons, result<strong>in</strong>g <strong>in</strong> lower astaxanth<strong>in</strong> concentrations be<strong>in</strong>g aggregated <strong>in</strong>tochylomicrons.Carotenoids are transported by lipoprote<strong>in</strong>s with<strong>in</strong> the blood stream from the <strong>in</strong>test<strong>in</strong>al cells<strong>and</strong> to peripheral tissue. A potential <strong>in</strong>teraction or competition between canthaxanth<strong>in</strong> <strong>and</strong>astaxanth<strong>in</strong> for uptake <strong>and</strong>/or blood-carry<strong>in</strong>g capacity <strong>in</strong> Atlantic salmon has been reported(Kiessl<strong>in</strong>g et al., 2003). Whether lute<strong>in</strong> <strong>and</strong>/or zeaxanth<strong>in</strong> may <strong>in</strong>teract <strong>in</strong> the same fashion withastaxanth<strong>in</strong> for transport with<strong>in</strong> blood stream rema<strong>in</strong>s to be explored.The aff<strong>in</strong>ity of astaxanth<strong>in</strong> for b<strong>in</strong>d<strong>in</strong>g sites with<strong>in</strong> muscle fibers has been previously studied.Whether lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> show aff<strong>in</strong>ity similar to astaxanth<strong>in</strong> for prote<strong>in</strong> complex with<strong>in</strong>muscle or if there are selective accumulation mechanisms for different types pigment <strong>in</strong> muscleof salmonid fish rema<strong>in</strong>s to be explored.6.2 Factors other than carotenoids <strong>in</strong> corn gluten meal reduc<strong>in</strong>g astaxanth<strong>in</strong> muscledepositionA consistent significant reduction <strong>in</strong> muscle astaxanth<strong>in</strong> deposition <strong>in</strong> response to dietarycorn gluten meal was obta<strong>in</strong>ed <strong>in</strong> Chapters 3 <strong>and</strong> 4. This f<strong>in</strong>d<strong>in</strong>g contradicts those previouslyreported by Olsen <strong>and</strong> Baker (2006), who observed no reduction <strong>in</strong> astaxanth<strong>in</strong> deposition <strong>in</strong>muscle from Atlantic salmon (Salmo salar) fed diets conta<strong>in</strong><strong>in</strong>g a purified synthetic source oflute<strong>in</strong> <strong>and</strong> synthetic astaxanth<strong>in</strong>, compared to those fed a diet supplemented astaxanth<strong>in</strong> as as<strong>in</strong>gle source of carotenoid. Anecdotal evidence from feed producers suggests that dietary<strong>in</strong>clusion of wheat gluten meal results <strong>in</strong> the reduction of muscle astaxanth<strong>in</strong> deposition, even137

though wheat gluten meal conta<strong>in</strong>s only about 6% - 35% of the carotenoid concentration present<strong>in</strong> corn gluten meal.Consider<strong>in</strong>g this evidence, it can be hypothesized that the observed reduction <strong>in</strong> muscleastaxanth<strong>in</strong> deposition might be attributed to the <strong>in</strong>teraction of component(s), other than yellowpigments, present <strong>in</strong> both corn <strong>and</strong> wheat gluten meal such am<strong>in</strong>o acid composition for <strong>in</strong>stance6.3 Future researchThe results obta<strong>in</strong>ed <strong>in</strong> this thesis suggest that future research on the evaluation of plantorig<strong>in</strong><strong>in</strong>gredients on flesh quality is necessary. In-vitro studies focus<strong>in</strong>g on the behaviour ofdifferent pigments <strong>and</strong> comb<strong>in</strong>ations of pigments dur<strong>in</strong>g emulsification, transference <strong>and</strong>transport with<strong>in</strong> bile mixed micelles will shed light on the potential <strong>in</strong>teraction or competitionamong carotenoids before absorption with<strong>in</strong> the <strong>in</strong>test<strong>in</strong>al wall.Evaluation of the digestibility <strong>and</strong> blood appearance of pigments <strong>in</strong> fish fed dietssupplemented with plant-prote<strong>in</strong> <strong>in</strong>gredients might be helpful to underst<strong>and</strong> how these nutrientsare absorbed <strong>and</strong> transported with<strong>in</strong> the blood stream. This can be achieved by the developmentof <strong>in</strong>-vitro digestibility trials <strong>and</strong> the determ<strong>in</strong>ation of specific apo-lipoprote<strong>in</strong>s <strong>in</strong> the bloodstream for example.Assessment of the aff<strong>in</strong>ity of lute<strong>in</strong> <strong>and</strong> zeaxanth<strong>in</strong> for b<strong>in</strong>d<strong>in</strong>g sites with<strong>in</strong> muscle fibermight help underst<strong>and</strong><strong>in</strong>g whether reduction <strong>in</strong> astaxanth<strong>in</strong> concentration <strong>in</strong> fillet from salmonidfish is due to competition among different pigments for deposition with<strong>in</strong> the muscle.138

Whether reduction <strong>in</strong> muscle astaxanth<strong>in</strong> deposition is the result of the <strong>in</strong>teraction offactors different than carotenoid level might be also of <strong>in</strong>terest for future research. The potentialeffects of factors such as the high level of glutamic acid <strong>and</strong>/or the low lys<strong>in</strong>e content <strong>in</strong> bothcorn <strong>and</strong> wheat gluten meal rema<strong>in</strong>s to be explored.139

BibliographyAas, G.H., Storebakken, T., Ruyter, B., 1999. Blood appearance, metabolic transformation <strong>and</strong>plasma transport prote<strong>in</strong>s of 14C-astaxanth<strong>in</strong> <strong>in</strong> Atlantic salmon (Salmo salar L.). FishPhysio. Biochem. 21, 325-334.Abdel-Aal, E., Sosulski, F.W., Sokhansanj, S., 1996. Bleach<strong>in</strong>g of wheat distillers' gra<strong>in</strong>s <strong>and</strong> itsfibre <strong>and</strong> prote<strong>in</strong> fractions with alkal<strong>in</strong>e hydrogen peroxide. Lebensm. Wiss. U, Technol. 29,210-216.Abdel-Aal, E.M., Young, J.C., Rabalski, I., Hucl, P., Fregeau-Reid, J., 2007. Identification <strong>and</strong>quantification of seed carotenoids <strong>in</strong> selected wheat species. J. Agric. Food Chem. 55, 787-794.Alexis, M., Papaparaskeva-Papoutsoglou, E., Theochari, V., 1985. Formulation of practical dietsfor ra<strong>in</strong>bow trout (Salmo gardneri) made by partial or complete substitution of fish meal bypoultry by-products <strong>and</strong> certa<strong>in</strong> plant by-products. Aquaculture 50, 61-73.Alfnes, F., Guttormsen, A.G., Ste<strong>in</strong>e, G., Kolstad, K., 2006. Consumers' will<strong>in</strong>gness to pay forthe color of salmon: A choice experiment with real economic <strong>in</strong>centives. Am. J. Agric.Econ. 88, 1050-1061.Anderson, S., 2000. Salmon color <strong>and</strong> the consumer. In: Johnston, R.S. <strong>and</strong> Shriver, A.L. (Eds.),Proceed<strong>in</strong>gs of the Tenth Biennial Conference of the International Institute of FisheriesEconomics <strong>and</strong> Trade Presentations, 2000, 1-3.140

Ando, S. <strong>and</strong> Hatano, M., 1988. Isolation of apolipoprote<strong>in</strong>s from carotenoid-carry<strong>in</strong>glipoprote<strong>in</strong> <strong>in</strong> the serum of chum salmon, Oncorhynchus keta. J. Lipid Res. 29, 1264-1271.AOAC, 1995. Official Methods of Analysis of OAC International, Sixteenth Edition. NationalAcademy Press, Wash<strong>in</strong>gton, DC., 1018 pp.Axelrod, B., Cheesbrough, T.M., Laakso, S., 1981. Lipoxygenase from soybeans: EC 1.13.11.12l<strong>in</strong>oleate:Oxygen oxidoreductase. In: John M. Lowenste<strong>in</strong> (Ed.), Methods <strong>in</strong> Enzymology.Academic Press, pp. 441-451.Baker, R.T.M., Pfeiffer, A.M., Schöner, F.J., Smith-Lemmon, L., 2002. <strong>Pigment</strong><strong>in</strong>g efficacy ofastaxanth<strong>in</strong> <strong>and</strong> canthaxanth<strong>in</strong> <strong>in</strong> fresh-water reared Atlantic salmon, Salmo salar. Anim.Feed Sci. Technol. 99, 97-106.Barone, R., Briante, R., D'Auria, S., Febbraio, F., Vaccaro, C., Del Giudice, L., Borrelli, G., DiFonzo, N., Nucci, R., 1999. Purification <strong>and</strong> characterization of a lipoxygenase enzymefrom durum wheat semol<strong>in</strong>a. J. Agric. Food Chem. 47, 1924-1931.Bjerkeng, B., 2000. Carotenoid pigmentation of salmonid fishes-recent progress. In: Cruz-Suarez, L.E., Ricque-Marie, D., Tapia-Salazar, M. (Eds.), Avances En Nutricion AcuıcolaV. Memorias Del V Simposium Internacional De Nutricion Acuıcola. 2000, 19-22.Bjerkeng, B. <strong>and</strong> Berge, G.M., 2000. Apparent digestibility coefficients <strong>and</strong> accumulation ofastaxanth<strong>in</strong> E/Z isomers <strong>in</strong> Atlantic salmon (Salmo salar L.) <strong>and</strong> Atlantic halibut(Hippoglossus hippoglossus L.). Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 127,423-432.141

Bjerkeng, B., Hatlen, B., Wathne, E., 1999. Deposition of astaxanth<strong>in</strong> <strong>in</strong> fillets of Atlanticsalmon (Salmo salar) fed diets with herr<strong>in</strong>g, capel<strong>in</strong>, s<strong>and</strong>eel, or Peruvian high PUFA oils.Aquaculture 180, 307-319.Bjerkeng, B., Storebakken, T., Liaaen-Jensen, S., 1992. <strong>Pigment</strong>ation of ra<strong>in</strong>bow trout from startfeed<strong>in</strong>g to sexual maturation. Aquaculture 108, 333-346.Bjerkeng, B., 2008. Carotenoids <strong>in</strong> aquaculture: Fish <strong>and</strong> crustaceans. In: Britton, G., Liaaen-Jensen, h.c.S., Pf<strong>and</strong>er, H. (Eds.), Carotenoids, Natural Functions. Basel, Switzerlnd.Bjerkeng, B., Føll<strong>in</strong>g, M., Lagocki, S., Storebakken, T., Olli, J.J., Alsted, N., 1997.Bioavailability of all-E-astaxanth<strong>in</strong> <strong>and</strong> Z-isomers of astaxanth<strong>in</strong> <strong>in</strong> ra<strong>in</strong>bow trout(Oncorhynchus mykiss). Aquaculture 157, 63-82.Bless<strong>in</strong>, C.W. 1962. Carotenoids of corn <strong>and</strong> sorghum. I. Analytical procedure. Cereal Chem 39,236-242.Borel, P., Grolier, P., Arm<strong>and</strong>, M., Partier, A., Lafont, H., Lairon, D., Azais-Braesco, V., 1996.Carotenoids <strong>in</strong> biological emulsions: solubility, surface-to-core distribution, <strong>and</strong> releasefrom lipid droplets. J. Lipid Res. 37, 250-261.Bowen, J., Soutar, C., Serwata, R.D., Lagocki, S., White, D.A., Davies, S.J., Young, A.J., 2002.Utilization of (3S,3'S)-astaxanth<strong>in</strong> acyl esters <strong>in</strong> pigmentation of ra<strong>in</strong>bow trout(Oncorhynchus mykiss). Aquacult. Nutr. 8, 59-68.142

Brenna, O.V. <strong>and</strong> Berardo, N., 2004. Application of near-<strong>in</strong>frared reflectance spectroscopy(NIRS) to the evaluation of carotenoids content <strong>in</strong> maize. J. Agric. Food Chem. 52, 5577-5582.Bureau, D., Hua, K., Cho, C.Y., 2006. Effect of feed<strong>in</strong>g level on growth <strong>and</strong> nutrient deposition<strong>in</strong> ra<strong>in</strong>bow trout (Oncorhynchus mykiss Walbaum) grow<strong>in</strong>g from 150 to 600g. Aquac. Res.37, 1090-1098.Burt, A.J., Gra<strong>in</strong>ger, C.M., Smid, M.P., Shelp, B.J., Lee, E.A., 2011. Allele m<strong>in</strong><strong>in</strong>g of exoticmaize germplasm to enhance macular carotenoids. Crop Sci. 51, 991-1004.Canadian Council on Animal Care. 1984. Guide to the Care <strong>and</strong> use of Experimental Animals.Ottawa, Ontario, Canada.Castenmiller, J. <strong>and</strong> West, C.E., 1998. Bioavailability <strong>and</strong> bioconversion of carotenoids. Annu.Rev. Nutr. 18, 19-38.Cha, J.Y., Flores, R.A., Park, H., 2000. <strong>Reduction</strong> of carotenoids <strong>in</strong> corn gluten meal with soyflour. Transcription of the ASAE 43, 1169-1174.Chikere, A.C., Galunsky, B., Overmeyer, A., Brunner, G., Kasche, V., 2000. Activity <strong>and</strong>stability of immobilised soybean lipoxygenase-1 <strong>in</strong> aqueous <strong>and</strong> supercritical carbon dioxidemedia. Biotechnol. Lett. 22, 1815-1821.Choubert, G., Milicua, J., Gomez, R., Sancé, S., Petit, H., Nègre-Sadargues, G., Castillo, R.,Trilles, J., 1995. Utilization of carotenoids from various sources by ra<strong>in</strong>bow trout: musclecolour, carotenoid digestibility <strong>and</strong> retention. Aquacult. Int. 3, 205-216.143

Choubert, G., Cravedi, J., Laurentie, M., 2009. Effect of alternate distribution of astaxanth<strong>in</strong> onra<strong>in</strong>bow trout (Oncorhynchus mykiss) muscle pigmentation. Aquaculture 286, 100-104.Choubert, G., Mendes-P<strong>in</strong>to, M., Morais, R., 2006. <strong>Pigment</strong><strong>in</strong>g efficacy of astaxanth<strong>in</strong> fed tora<strong>in</strong>bow trout Oncorhynchus mykiss: Effect of dietary astaxanth<strong>in</strong> <strong>and</strong> lipid sources.Aquaculture 257, 429-436.Christiansen, R., Struksnaes, G., Estermann, R., Torrissen, O., 1995. Assessment of flesh colour<strong>in</strong> Atlantic salmon, Salmo salar L. Aquac. Res. 26, 311-321.Clevel<strong>and</strong> I.V., T.E., Hussey, D.S., Chen, Z.-., Jacobson, D.L., Brown, R.L., Carter-Wientjes, C.,Clevel<strong>and</strong>, T.E., Arif, M., 2008. The use of neutron tomography for the structural analysisof corn kernels. J. Cereal Sci. 48, 517-525.Cohen, B., Grossman, S., Kle<strong>in</strong>, B.P., P<strong>in</strong>sky, A., 1985. <strong>Pigment</strong> bleach<strong>in</strong>g by soybeanlipoxygenase type-2 <strong>and</strong> the effect of specific chemical modifications. BBA - Lipid Met.837, 279-287.<strong>Corn</strong> Ref<strong>in</strong>ers Association, 2010. Annual report. Tell<strong>in</strong>g the positive story of corn. Wash<strong>in</strong>gton,DC, USA.Dailey, O., 2002. Effect of lactic acid on prote<strong>in</strong> solubilization <strong>and</strong> starch yield <strong>in</strong> corn wet-millsteep<strong>in</strong>g: a study of hybrid effects. Cereal Chemistry. 79, 257-260.Davis, K., 2001. <strong>Corn</strong> mill<strong>in</strong>g, process<strong>in</strong>g <strong>and</strong> generation of co-products. M<strong>in</strong>nesota NutritionConference, M<strong>in</strong>nesota <strong>Corn</strong> Growers Association, Technical Symposium, 2001,M<strong>in</strong>nesota.144

de Francesco, M., Parisi, G., Medale, F., Lupi, P., Kaushik, S.J., Poli, B.M., 2004. Effect of longtermfeed<strong>in</strong>g with a plant prote<strong>in</strong> mixture based diet on growth <strong>and</strong> body/fillet quality traitsof large ra<strong>in</strong>bow trout (Oncorhynchus mykiss). Aquaculture 236, 413-429.De Gracia, M., Owen, F., Lowry, S., 1989. <strong>Corn</strong> gluten meal <strong>and</strong> blood meal mixture for dairycows <strong>in</strong> midlactation. J Dairy Sci 72, 3064-3069.Dem<strong>in</strong>g, D.N. <strong>and</strong> Erdman, W., 1999. Mammalian carotenoid absorption <strong>and</strong> metabolism. PureAppl. Chem. 71, 2213-2223.Dowd, M.K., 2003. Improvements to laboratory-scale maize wet-mill<strong>in</strong>g procedures. IndustrialCrops <strong>and</strong> Products 18, 67-76.Dumas, A., France, J., Bureau, D.P., 2007. Evidence of three growth stanzas <strong>in</strong> ra<strong>in</strong>bow trout(Oncorhynchus mykiss) across life stages <strong>and</strong> adaptation of the thermal-unit growthcoefficient. Aquaculture 267, 139-146.Eckhoff, S.R., Du, L., Yang, P., Rausch, K.D., Wang, D.L., Li, B.H., Tumbleson, M.E., 1999.Comparison between alkali <strong>and</strong> conventional corn wet-mill<strong>in</strong>g: 100-g procedures. CerealChem. 76, 96-99.Eckhoff, S., S<strong>in</strong>gh, S., Zehr, B., Rausch, K., Fox, E., Mistry, A., Haken, A., Niu, Y., Zou, S.,Buriak, P., 1996. A 100-g laboratory corn wet-mill<strong>in</strong>g procedure. Cereal Chem. 73, 54-57.FAO (Food <strong>and</strong> Agriculture Organization of the United Nations). 2012. The state of worldfisheries <strong>and</strong> aquaculture. Rome, Italy.145

Fauconneau, B., Andre, S., Chmaitilly, J., Le Bail, P.Y., Krieg, F., Kaushik, S.J., 1997. Controlof skeletal muscle fibres <strong>and</strong> adipose cells size <strong>in</strong> the flesh of ra<strong>in</strong>bow trout. Journal of FishBiology 50, 296-314.Frankel, E.N., 2005. Lipid Oxidation. The Oily Press, Bridgwater, Engl<strong>and</strong>. 470 pp.Funaba, M., Matsumoto, C., Matsuki, K., Gotoh, K., Kaneko, M., Iriki, T., Hatano, Y., Abe, M.,2002. Comparison of corn gluten meal <strong>and</strong> meat meal as a prote<strong>in</strong> source <strong>in</strong> dry foodsformulated for cats. Am J Vet Res 69, 1247-1251.Furr, H.C. <strong>and</strong> Clark, R.M., 1997. Intest<strong>in</strong>al absorption <strong>and</strong> tissue distribution of carotenoids. J.Nutr. Biochem. 8, 364-377.Galitsky, C., Worrell, E., Ruth, M., 2003. Energy efficiency improvement <strong>and</strong> cost sav<strong>in</strong>gopportunities for the corn wet mill<strong>in</strong>g <strong>in</strong>dustry. An energy star guide for energy <strong>and</strong> plantmanagers. University of California, Berkeley. USA.Gawienowski, M., Eckhoff, S., Yang, P., Rayapati, P., B<strong>in</strong>der, T., Brisk<strong>in</strong>, D., 1999. Fate ofmaize DNA dur<strong>in</strong>g steep<strong>in</strong>g, wet-mill<strong>in</strong>g, <strong>and</strong> process<strong>in</strong>g. Cereal Chem. 76, 371-374.Gél<strong>in</strong>as, P., Poitras, E., McK<strong>in</strong>non, C.M., Mor<strong>in</strong>, A., 1998. Oxido-reductases <strong>and</strong> lipases asdough-bleach<strong>in</strong>g agents. Cereal Chem. 75, 810-814.Gökmen, V., Bahceci, S., Acar, J., 2002. Characterization of crude lipoxygenase extract fromgreen pea us<strong>in</strong>g a modified spectrophotometric method. Eur. Food Res. Technol. 215, 42-45.146

Gökmen, V., Serpen, A., Atli, A., Köksel, H., 2007. A practical spectrophotometric approach forthe determ<strong>in</strong>ation of lipoxygenase activity of durum wheat. Cereal Chem. 84, 290.Hardy, R.W., Torrissen, O.J., Scott, T.M., 1990. Absorption <strong>and</strong> distribution of 14C-labeledcanthaxanth<strong>in</strong> <strong>in</strong> ra<strong>in</strong>bow trout (Oncorhynchus mykiss). Aquaculture 87, 331-340.Henmi, H., Hata, M., Hata, M., 1989. Astaxanth<strong>in</strong> <strong>and</strong>/or canthaxanth<strong>in</strong>-actomyos<strong>in</strong> complex <strong>in</strong>salmon muscle. Bull. Chem. Soc. Jpn. 55, 1583-1589.Henmi, H., Iwata, T., Hata, M., Hata, M., 1987. Studies on the carotenoids <strong>in</strong> the muscle ofsalmons, I. Intracellular distribution of carotenoids <strong>in</strong> the muscle. Tohoku J. Agr. Res. 37,101-111.Ishida, B.K. <strong>and</strong> Bartley, G.E., 2005. Carotenoids: Chemistry, sources, <strong>and</strong> physiology. In:Caballero, B., Allen, L., Prentice, A. (Eds.), Encyclopedia of Human Nutrition, 2nd Ed.Elsevier, Oxford, U.K., pp. 330-338.Jackson, H., Braun, C., Braun, C., Ernst, H., 2008. The chemistry of novel xanthophyllcarotenoids. Am J Cardiol. 101, 50D-57D.Jaren-Galan, M. <strong>and</strong> M<strong>in</strong>guez-Mosquera, M., 1999. Effect of pepper lipoxygenase activity <strong>and</strong>its l<strong>in</strong>ked reactions on pigments of the pepper fruit. J. Agric. Food Chem. 47, 4532-4536.Ji, Y.F., Zuo, L.F., Wang, F.F., Li, D.F., Lai, C., 2012. Nutritional value of 15 corn gluten mealsfor grow<strong>in</strong>g pigs: chemical composition, energy content <strong>and</strong> am<strong>in</strong>o acid digestibility. ArchTierernahr 66, 283-312.147

Johnston, I., Li, X., Vieira, V., Nickell, D., D<strong>in</strong>gwall, A., Alderson, R., Campbell, P., Bickerdike,R., 2006. Muscle <strong>and</strong> flesh quality traits <strong>in</strong> wild <strong>and</strong> farmed Atlantic salmon. Aquaculture256, 323-336.Johnston, I.A., Alderson, R., S<strong>and</strong>ham, C., D<strong>in</strong>gwall, A., Mitchell, D., Selkirk, C., Nickell, D.,Baker, R., Robertson, B., Whyte, D., Spr<strong>in</strong>gate, J., 2000. Muscle fibre density <strong>in</strong> relation tothe colour <strong>and</strong> texture of smoked Atlantic salmon (Salmo salar L.). Aquaculture 189, 335-349.Junqueira, R., Rocha, F., Moreira, M., Castro, I., 2007. Effect of proof<strong>in</strong>g time <strong>and</strong> wheat flourstrength on bleach<strong>in</strong>g, sensory characteristics, <strong>and</strong> volume of French breads with addedsoybean lipoxygenase. Cereal Chem. 84, 443-449.Kang, E., Campbell, R., Bastian, E., Drake, M., 2010. Invited review: Annatto usage <strong>and</strong>bleach<strong>in</strong>g <strong>in</strong> dairy foods. J. Dairy Sci. 93, 3891-3901.Kanamaru, K., Wang, S., Abe, J., Yamada, T., Kitamura, K., 2006. Identification <strong>and</strong>characterization of wild soybean (Glyc<strong>in</strong>e soja Sieb. et Zecc.) stra<strong>in</strong>s with high lute<strong>in</strong>content. Breed. Sci. 56, 231-234.Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B., Laroche, M., 1995. Partialor total replacement of fish meal by soybean prote<strong>in</strong> on growth, prote<strong>in</strong> utilization, potentialestrogenic or antigenic effects, cholesterolemia <strong>and</strong> flesh quality <strong>in</strong> ra<strong>in</strong>bow trout,Oncorhynchus mykiss. Aquaculture 133, 257-274.148

Kiessl<strong>in</strong>g, A., Olsen, R., Buttle, L. 2003. Given the same dietary carotenoid <strong>in</strong>clusion, Atlanticsalmon, Salmo salar (L.) displays higher blood levels of canthaxanth<strong>in</strong> than astaxanth<strong>in</strong>.Aquacult. Nutrition 9, 253-261.Kitahara, T. 1983. Behavior of carotenoids <strong>in</strong> the chum salmon (Oncorhynchus keta) dur<strong>in</strong>ganadromous migration. Comp. Biochem. Physiol. B. 76, 97-101.Kitahara, T. 1984. Carotenoids <strong>in</strong> the Pacific salmon dur<strong>in</strong>g the mar<strong>in</strong>e period. Comp. Biochem.Physiol. B. 78, 859-862.Krochta, J., Look, K., Wong, L., 1981. Modification of corn wet-mill<strong>in</strong>g steep<strong>in</strong>g conditions toreduce energy consumption. J. Food Process Pres. 5, 39-47.Leclercq, E., Dick, J.R., Taylor, J.F., Bell, J.G., Hunter, D., Migaud, H., 2010. Seasonalvariations <strong>in</strong> sk<strong>in</strong> pigmentation <strong>and</strong> flesh quality of Atlantic salmon (Salmo salar L.):implications for quality management. J. Agric. Food Chem. 58, 7036-7045.Lee, J.D., Shannon, J.G., So, Y.S., Sleper, D.A., Nelson, R.L., Lee, J.H., Choung, M.G., 2009.Environmental effects on lute<strong>in</strong> content <strong>and</strong> relationship of lute<strong>in</strong> <strong>and</strong> other seedcomponents <strong>in</strong> soybean. Plant Breed<strong>in</strong>g 128, 97-100.Leenhardt, F., Lyan, B., Rock, E., Boussard, A., Potus, J., Chanliaud, E., Remesy, C., 2006.Wheat lipoxygenase activity <strong>in</strong>duces greater loss of carotenoids than vitam<strong>in</strong> E dur<strong>in</strong>gbreadmak<strong>in</strong>g. J. Agric. Food Chem. 54, 1710-1715.149

Li, M.H., Oberle, D.F., Lucas, P.M., 2011. Evaluation of corn distillers dried gra<strong>in</strong>s withsolubles <strong>and</strong> brewers yeast <strong>in</strong> diets for channel catfish Ictalurus punctatus (Raf<strong>in</strong>esque).Aquacult. Res. 42, 1424-1430.Matthews, S.J., Ross, N.W., Lall, S.P., Gill, T.A., 2006. Astaxanth<strong>in</strong> b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> <strong>in</strong> Atlanticsalmon. Comp. Biochem. Phys. B. 144, 206-214.Mente, E., Deguara, S., Santos, M.B., Houlihan, D., 2003. White muscle free am<strong>in</strong>o acidconcentrations follow<strong>in</strong>g feed<strong>in</strong>g a maize gluten dietary prote<strong>in</strong> <strong>in</strong> Atlantic salmon (Salmosalar L.). Aquaculture 225, 133-147.Mercier, M., Gel<strong>in</strong>as, P. 2001. Effect of lipid oxidation on dough bleach<strong>in</strong>g. Cereal Chem. 78,36-38.Moros, E.E., Darnoko, D., Cheryan, M., Perk<strong>in</strong>s, E.G., Jerrell, J., 2002. Analysis of xanthophylls<strong>in</strong> corn by HPLC. J. Agric. Food Chem. 50, 5787-5790.Moyano, F.J., Cardenete, G., De la Higuera, M., 1991. Nutritive <strong>and</strong> metabolic utilization ofprote<strong>in</strong>s with high glutamic acid content by the ra<strong>in</strong>bow trout (Oncorhynchus mykiss).Comp. Biochem. Phys. A 100, 759-762.Mundheim, H., Aksnes, A., Hope, B., 2004. Growth, feed efficiency <strong>and</strong> digestibility <strong>in</strong> salmon(Salmo salar L.) fed different dietary proportions of vegetable prote<strong>in</strong> sources <strong>in</strong>comb<strong>in</strong>ation with two fish meal qualities. Aquaculture 237, 315-331.National Research Council (NRC)., 2011. Nutrient Requirements of Fish <strong>and</strong> Shrimp. TheNational Academies Press, Wash<strong>in</strong>gton, DC., 376 pp.150

No, H.K. <strong>and</strong> Storebakken, T., 1991. <strong>Pigment</strong>ation of ra<strong>in</strong>bow trout with astaxanth<strong>in</strong> at differentwater temperatures. Aquaculture 97, 203-216.O’brien, 2009. Recent trends <strong>in</strong> U.S. wet <strong>and</strong> dry corn mill<strong>in</strong>g production. AgMRC. RenewableEnergy Newsletter. USA.Olsen, R.E. <strong>and</strong> Baker, R.T.M., 2006. Lute<strong>in</strong> does not <strong>in</strong>fluence flesh astaxanth<strong>in</strong> pigmentation<strong>in</strong> the Atlantic salmon (Salmo salar L.). Aquaculture 258, 558-564.Park, H., Flores, R.A., Johnson, L.A., 1997. Preparation of fish feed <strong>in</strong>gredients: reduction ofcarotenoids <strong>in</strong> corn gluten meal. J. Agric. Food Chem. 45, 2088-2092.Parker, R.S., 1996. Absorption, metabolism, <strong>and</strong> transport of carotenoids. The FASEB Journal10, 542-551.Pereira, T.; Oliva-Teles, A. Evaluation of corn gluten meal as a prote<strong>in</strong> source <strong>in</strong> diets forgilthead seabream (Sparus aurataL.). Aquac. Res. 2003, 34, 1111-1117.Perez, O.E., Haros, M., Suarez, C., 2001. <strong>Corn</strong> steep<strong>in</strong>g: <strong>in</strong>fluence of time <strong>and</strong> lactic acid onisolation <strong>and</strong> thermal properties of starch. J. Food Eng. 48, 251-256.Pérez-Gálvez, A. <strong>and</strong> Mínguez-Mosquera, M.I., 2002. Degradation of non-esterified <strong>and</strong>esterified xanthophylls by free radicals. BBA Gen. Subjects 1569, 31-34.Perry, R. <strong>and</strong> Green, D., 1984. Perry's chemical eng<strong>in</strong>eers' h<strong>and</strong>book. McGraw-Hill, New York,NY, USA, 2640 pp.151

Peter, C.M., Han, Y., Bol<strong>in</strong>g-Frankenbach, S.D., Parsons, C.M., Baker, D.H., 2000. Limit<strong>in</strong>gorder of am<strong>in</strong>o acids <strong>and</strong> the effects of phytase on prote<strong>in</strong> quality <strong>in</strong> corn gluten meal fed toyoung chicks. J. Anim. Sci. 78, 2150-2156.Rajas<strong>in</strong>gh, H., Øyehaug, L., Våge, D., Omholt, S., 2006. Carotenoid dynamics <strong>in</strong> Atlanticsalmon. BMB Biology 4, 1-15.Regost, C., Arzel, J., Kaushik, S., 1999. Partial or total replacement of fish meal by corn glutenmeal <strong>in</strong> diet for turbot (Psetta maxima). Aquaculture 180, 99-117.Roba<strong>in</strong>a, L., Moyano, F., Izquierdo, M., Socorro, J., Vergara, J., Montero, D., 1997. <strong>Corn</strong> gluten<strong>and</strong> meat <strong>and</strong> bone meals as prote<strong>in</strong> sources <strong>in</strong> diets for gilthead sea bream (Sparus aurata):Nutritional <strong>and</strong> histological implications. Aquaculture 157, 347-359.Rock, D.W., Klopfenste<strong>in</strong>, T.J., Ward, J.K., Britton, R.A., McDonnell, M.L., 1983. Evaluation ofslowly degraded prote<strong>in</strong>s: dehydrated alfalfa <strong>and</strong> corn gluten meal. J. Anim. Sci. 56, 476-482.Saiz, A.I., Manrique, G.D., Fritz, R., 2001. Determ<strong>in</strong>ation of benzoyl peroxide <strong>and</strong> benzoic acidlevels by HPLC dur<strong>in</strong>g wheat Fflour bleach<strong>in</strong>g process. J. Agric. Food Chem. 49, 98-102.Salvador, A.M., Alonso-Damian, A., Choubert, G., Milicua, J.C.G., 2007. Effect of soybeanphospholipids on canthaxanth<strong>in</strong> lipoprote<strong>in</strong>s transport, digestibility, <strong>and</strong> deposition <strong>in</strong>ra<strong>in</strong>bow trout (Oncorhynchus mykiss) muscle. J. Agric. Food Chem. 55, 9202-9207.152

Salvador, A.M., Alonso-DamiaÌ•n, A., Choubert, G., Milicua, J.C.G., 2009. Impact of differentdietary phospholipid levels on cholesterol <strong>and</strong> canthaxanth<strong>in</strong> lipoprote<strong>in</strong>’s serum transport<strong>and</strong> muscle deposition <strong>in</strong> ra<strong>in</strong>bow trout. J. Agric. Food Chem. 57, 2016-2021.Santra, M., Rao, V.S., Tamhankar, S.A., 2003. Modification of AACC procedure for measur<strong>in</strong>g(beta)-carotene <strong>in</strong> early generation durum wheat. Cereal Chem. 80, 130.Sasse, C.E. <strong>and</strong> Baker, D.H., 1973. Availability of sulfur am<strong>in</strong>o acids <strong>in</strong> corn <strong>and</strong> corn glutenmeal for grow<strong>in</strong>g chicks. J. Anim. Sci. 37, 1351-1355.Saunders, J.A., Rosentrater , K.A., Krishnan, P.G., 2008. Potential bleach<strong>in</strong>g techniques for corndistillers gra<strong>in</strong>s. J. Food Technol. 6, 242-252.Schiedt, K., Leuenberger, F., Vecchi, M., Gl<strong>in</strong>z, E., 1985. Absorption, retention <strong>and</strong> metabolictransformations of carotenoids <strong>in</strong> ra<strong>in</strong>bow trout, salmon <strong>and</strong> chicken. Pure Appl. Chem. 57,685-692.Shi, X. <strong>and</strong> Chen, F., 1997. Stability of lute<strong>in</strong> under various storage conditions. Nahrung 41, 38-41.Shukla, R. <strong>and</strong> Cheryan, M., 2001. Ze<strong>in</strong>: the <strong>in</strong>dustrial prote<strong>in</strong> from corn. Industrial Crops <strong>and</strong>Products 13, 171-192.S<strong>in</strong>gh, S., Johnson, L., Pollak, L., Fox, S., Bailey, T., 1997. Comparison of laboratory <strong>and</strong> pilotplantcorn wet-mill<strong>in</strong>g procedures. Cereal Chem. 74, 40-44.153

Skonberg, D.I., Hardy, R.W., Barrows, F.T., Dong, F.M., 1998. Color <strong>and</strong> flavor analyses offillets from farm-raised ra<strong>in</strong>bow trout (Oncorhynchus mykiss) fed low-phosphorus feedsconta<strong>in</strong><strong>in</strong>g corn or wheat gluten. Aquaculture 166, 269-277.Slav<strong>in</strong>, M., Cheng, Z., Luther, M., Kenworthy, W., Yu, L., 2009. Antioxidant properties <strong>and</strong>phenolic, isoflavone, tocopherol <strong>and</strong> carotenoid composition of Maryl<strong>and</strong>-grown soybeanl<strong>in</strong>es with altered fatty acid profiles. Food Chem. 114, 20-27.Stadtman, E.R. <strong>and</strong> Lev<strong>in</strong>e, R.L., 2000. Prote<strong>in</strong> oxidation. Ann NY Acad. Sci. 899, 191-2008.Storebakken, T. <strong>and</strong> Choubert, G., 1991. Flesh pigmentation of ra<strong>in</strong>bow trout fed astaxanth<strong>in</strong> orcanthaxanth<strong>in</strong> at different feed<strong>in</strong>g rates <strong>in</strong> freshwater <strong>and</strong> saltwater. Aquaculture 95, 289-295.Surai, P.F., Speake, B.K., Sparks, N.H.C., 2001. Carotenoids <strong>in</strong> avian nutrition <strong>and</strong> embryonicdevelopment. 1. Absorption, availability <strong>and</strong> levels <strong>in</strong> plasma <strong>and</strong> egg yolk. Poultry Sci. 38,1-27.Sutton, J., Balfry, S., Higgs, D., Huang, C.H., Skura, B., 2006. Impact of iron-catalyzed dietarylipid peroxidation on growth performance, general health <strong>and</strong> flesh proximate <strong>and</strong> fatty acidcomposition of Atlantic salmon (Salmo salar L.) reared <strong>in</strong> seawater. Aquaculture 257, 534-557.Tacon, A. <strong>and</strong> Metian, M., 2009. Fish<strong>in</strong>g for feed or fish<strong>in</strong>g for food: <strong>in</strong>creas<strong>in</strong>g globalcompetition for small pelagic forage fish. Ambio 38, 294-302.Taylor, G. <strong>and</strong> Morris, H., 1983. Lipoxygenase pathways. Brit. Med. Bull. 39, 219-222.154

Tironi, V.A., Tomas, M.C., Añon, M.C., 2007. Lipid <strong>and</strong> prote<strong>in</strong> deterioration dur<strong>in</strong>g the chilledstorage of m<strong>in</strong>ced sea salmon (Pseudopercis semifascista). J Sci Food Agric 87, 2239-2246.Torrissen, O.J. <strong>and</strong> Ingebrigtsen, K., 1992. Tissue distribution of 14C-astaxanth<strong>in</strong> <strong>in</strong> the Atlanticsalmon (Salmo salar). Aquaculture 108, 381-385.Trono, D., Pastore, D., Di Fonzo, N., 1999. Carotenoid dependent <strong>in</strong>hibition of durum wheatlipoxygenase. J. Cereal Sci. 29, 99-102.Tucker, C. <strong>and</strong> Hargreaves, J., 2004. Biology <strong>and</strong> culture of channel catfish. Amsterdam, TheNederl<strong>and</strong>s.United States of Department of Agriculture, 2011. Gra<strong>in</strong>: world markets <strong>and</strong> trade. Office ofGlobal Analysis. USA.Vignaux, N., Fox, S.R., Johnson, L.A., 2006. A 10-g laboratory wet-mill<strong>in</strong>g procedure for maize<strong>and</strong> comparison with larger scale laboratory procedures. Cereal Chem. 83, 482-490.Welker, C., De Negro, P., Sarti, M., 2001. Green algal carotenoids <strong>and</strong> yellow pigmentation ofra<strong>in</strong>bow trout fish. Aquacult. Int. 9, 87-93.Woodall, A., Britton, G., Jackson, M., 1997. Carotenoids <strong>and</strong> protection of phospholipids <strong>in</strong>solution or <strong>in</strong> liposomes aga<strong>in</strong>st oxidation by peroxyl radicals: relationship betweencarotenoid structure <strong>and</strong> protective ability. Biochi. Biophys Acta 1336, 575-586.Yonekura, L. <strong>and</strong> Nagao, A., 2007. Intest<strong>in</strong>al absorption of dietary carotenoids. Mol. Nutr. FoodRes. 51, 107-115.155

Ytrestoyl, T., Struksnæs, G., Koppe, W., Bjerkeng, B., 2005. <strong>Effects</strong> of temperature <strong>and</strong> feed<strong>in</strong>take on astaxanth<strong>in</strong> digestibility <strong>and</strong> metabolism <strong>in</strong> Atlantic salmon, Salmo salar. Comp.Biochem. Physiol. Part B: Biochem. Mol. Biol. 142, 445-455.Ytrestoyl, T. <strong>and</strong> Bjerkeng, B., 2007. Dose response <strong>in</strong> uptake <strong>and</strong> deposition of <strong>in</strong>traperitoneallyadm<strong>in</strong>istered astaxanth<strong>in</strong> <strong>in</strong> Atlantic salmon (Salmo salar L.) <strong>and</strong> Atlantic cod (Gadusmorhua L.). Aquaculture 263, 179-191.Zehr, B., Eckhoff, S., S<strong>in</strong>gh, S., Keel<strong>in</strong>g, P., 1995. Comparison of wet-mill<strong>in</strong>g properties amongmaize <strong>in</strong>bred l<strong>in</strong>es <strong>and</strong> their hybrids. Cereal Chem. 72, 491-497.Zeronian, S., <strong>and</strong> Inglesby., M. 1995. Bleach<strong>in</strong>g of cellulose by hydrogen peroxide. Cellulose 2,265-272.156