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

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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
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Pigment Re
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ingredients. Furth
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Thanks to all my colleagues, friend
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2.10.1 Enzymatic treatments .......
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LIST OF TABLESTable 3. 1 - Factors
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LIST OF FIGURESFigure 2. 1 - Molecu
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Ingredients commonly in</st
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CHAPTER - 2 LITERATURE REVIEW2.1 In
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leached when 5% of soy flour as LOX
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deep and fast glyc
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observed in ra<str
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Chylomicron constituents are remove
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Muscle growth in r
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2.7.2 Corn gluten
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2010). However, due to its imbalanc
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No differences in
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supplemented with 42% of a vegetabl
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Soybean seeds are the richest known
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2.10.1.4 Bleaching
Page 39 and 40: 2.11 ConclusionInclusion of <strong
Page 41 and 42: 4.0%*68%* 5.6%* 12%* 3.5%* 6.5%*Fig
Page 43 and 44: Figure 2. 4 - Schematic representat
Page 45 and 46: 3.1 IntroductionThe characteristic
Page 47 and 48: many factors such as LOX source lev
Page 49 and 50: During the 12-week
Page 51 and 52: 3.2.3 Analytical methodsDry matter
Page 53 and 54: solution was generated by mix<stron
Page 55 and 56: 3.2.4 Statistical analysisData from
Page 57 and 58: Factors defined as
Page 59 and 60: chromatography). Relative amounts o
Page 61 and 62: After carotenoids are absorbed thro
Page 63 and 64: and ended up <stro
Page 65 and 66: Table 3. 2 - Experimental design us
Page 67 and 68: Table 3. 4 - Analysed proximate com
Page 69 and 70: Proximate composition (Analysed; dr
Page 71 and 72: Table 3. 7 - Lipoxygenase activity
Page 73 and 74: 18 1 0 -1 0 120 15 0 10 2519 -1 0 1
Page 75 and 76: Table 3. 10 - HPLC carotenoid profi
Page 77 and 78: Table 3. 12 - Retain</stron
Page 79 and 80: Table 3. 14 - Fillet carotenoid con
Page 81 and 82: Total yellow pigment(mg•kg -1 )25
Page 83 and 84: Figure 3. 4 - Growth curve of ra<st
Page 85 and 86: effects of dietary CGM and<
Page 87 and 88: soybean meal in a
Page 89: were randomly samp
Page 93 and 94: Retained nitrogen
Page 95 and 96: significant (p
Page 97 and 98: Values for all-trans astaxanth<stro
Page 99 and 100: previous reports where colour attri
Page 101 and 102: Proximate composition (analysed), d
Page 103 and 104: Table 4. 2 - Growth performance of
Page 105 and 106: Table 4. 3 - Carcass chemical proxi
Page 107 and 108: Table 4. 4 - Retain</strong
Page 109 and 110: Table 4. 5 - Pigment</stron
Page 111 and 112: Table 4. 6 - Colour attributes <str
Page 113 and 114: FiguresFigure 4. 1 - Growth curves
Page 115 and 116: Figure 4. 3 - Muscle colour attribu
Page 117 and 118: the effects of dietary reduced-caro
Page 119 and 120: astaxanthin deposi
Page 121 and 122: After steeping, ke
Page 123 and 124: 0.9994 and 0.9999,
Page 125 and 126: under acidic and a
Page 127 and 128: oxidation within t
Page 129 and 130: colour difference usin</str
Page 131 and 132: TablesTable 5. 1 - Analyzed proxima
Page 133 and 134: Effects of
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Data are mean (n=2).a Steepwater pH
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CHAPTER - 6 GENERAL DISCUSSIONCurre
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significant reduction in</s
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Reduction of pigme
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though wheat gluten meal conta<stro
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BibliographyAas, G.H., Storebakken,
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Bjerkeng, B., Hatlen, B., Wathne, E
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Choubert, G., Cravedi, J., Laurenti
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Fauconneau, B., Andre, S., Chmaitil
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Johnston, I., Li, X., Vieira, V., N
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Li, M.H., Oberle, D.F., Lucas, P.M.
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Peter, C.M., Han, Y., Bolin
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Skonberg, D.I., Hardy, R.W., Barrow
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Ytrestoyl, T., Struksnæs, G., Kopp
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