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Studies on colour of egg yolk Fadl , Essam Bahgat 1971

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STUDIES ON COLOUR OF EGG YOLK b y ESSAM BAHGAT FADL B.Sc,, University of Alexandria, Egypt A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Food Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA M a y , 1971 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q uirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may "be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a llowed w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f P o o d S c i e n c e The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date May 2 0 , 1971 i i ABSTRACT The e f f e c t o f v a r y i n g c o m b i n a t i o n s o f i r r a d i a t i o n t r e a t m e n t , f r e e z i n g p r o c e d u r e s and d u r a t i o n of s t o r a g e on t h e c o l o r o f n a t u r a l l y and a r t i f i c i a l l y pigmented egg y o l k determined by two o b j e c t i v e methods was s t u d i e d . The r e l a t i o n s h i p between t h e two methods o f c o l o r e v a l u a t i o n was a l s o d e t e r m i n e d . N a t u r a l l y pigmented y o l k s were o b t a i n e d f rom eggs l a i d by p u l l e t s o f a s i n g l e s t r a i n o f S i n g l e Comb White L e g h o r n f e d a s t a n d a r d d i e t . A r t i f i c i a l l y pigmented y o l k was p r e p a r e d by a d d i t i o n o f e i t h e r b e t a - c a r o t e n e o r c a n t h a x a n t h i n t o t h e n a t u r a l l y p igmented m a t e r i a l . C o l o r o f y o l k was a s s e s s e d : 1. On t h e b a s i s o f pigment c o n c e n t r a t i o n d e t e r m i n e d by a bsorbance o f a c e t o n e e x t r a c t and e x p r e s s e d as b e t a - c a r o t e n e e q u i v a l e n t (BCE) and 2. On t h e b a s i s o f c h r o m a t i c i t y c o o r d i n a t e s ( x , y ) , l i g h t n e s s (%Y), dominant w a v e l e n g t h (DWL) and e x c i t a t i o n p u r i t y (EP) d e t e r m i n e d by r e f l e c t a n c e s p e c t r o p h o t o m e t r y . B o t h i r r a d i a t i o n dose ( 0 , 0.5, 1.0 and 2.0 Mrad) and t i m e o f i r r a d i a t i o n ( b e f o r e o r a f t e r f r e e z i n g ) had s i g n i f i c a n t e f f e c t on the c h r o m a t i c i t y c o o r d i n a t e s , BCE v a l u e s and e x c i t a t i o n p u r i t y o f n a t u r a l l y and a r t i f i c i a l l y pigmented i i i y o l k samples. H i g h e r r a d i a t i o n d o s e s and i r r a d i a t i o n b e f o r e f r e e z i n g were a s s o c i a t e d w i t h d e c r e a s e d c h r o m a t i c i t y c o o r d i n a t e s , BCE v a l u e s and e x c i t a t i o n p u r i t y . I n a r t i f i c i a l l y p i gmented samples i n c r e a s e s i n i r r a d i a t i o n dose and i r r a d i a t i o n b e f o r e f r e e z i n g r e s u l t e d i n s i g n i f i c a n t d e c r e a s e s i n l i g h t n e s s . Samples f r o z e n and s t o r e d a t -"10 F° had c o n s i s t e n t l y h i g h e r mean c h r o m a t i c i t y v a l u e s and l o w e r e x c i t a t i o n p u r i t y t h a n t h o s e a t -35 F°. The t e m p e r a t u r e e f f e c t on BCE v a l u e s was i n c o n s i s t e n t among e x p e r i m e n t s . A f t e r 30 days s t o r a g e mean x - v a l u e s were l o w e r and mean y - r v a l u e s were h i g h e r t h a n a f t e r 10 days s t o r a g e . These changes were a s s o c i a t e d w i t h a l m o s t no change i n D W L o r E P . N i t r o g e n - p a c k e d samples had c o n s i s t e n t l y l o w e r BCE v a l u e s t h a n a i r - p a c k e d and t h i s d i f f e r e n c e was s i g n i f i c a n t i n a l l b u t E x p e r i m e n t 1. No c o r r e s p o n d i n g d i f f e r e n c e s were f o u n d i n c h r o m a t i c i t y c o o r d i n a t e s , l i g h t n e s s , DWL o r EP. C o r r e l a t i o n a n a l y s e s r e v e a l e d h i g h l y s i g n i f i c a n t (P^O.01) l i n e a r r e l a t i o n s h i p s between BCE and b o t h c h r o m a t i c i t y v a l u e s and l i g h t n e s s r a n g i n g f r o m + 0.09 t o + 0.79. iv TABLE OP CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 4 MATERIALS AND METHODS 12 Experiment 1 12 Experiment 2 14 Experiment 3 16 Experiment 4 16 S t a t i s t i c a l Methods 16 RESULTS 18 R e f l e c t a n c e Measurements 18 C h r o m a t i c i t y c o o r d i n a t e s 18 Li g h t n e s s 19 •Dominant Wavelength (DWL) 21 E x c i t a t i o n P u r i t y (EP) 21 Absorbance Measurements 21 Reading a t 4-50 ran 22 Reading at 460 nm 22 C o r r e l a t i o n M a t r i x 23 DISCUSSION 42 V Page SUMMARY AND CONCLUSION 4-5 LIST OF REFERENCES 4-7 APPENDIX A 53 APPENDIX B ' 50 APPENDIX C 67 APPENDIX D 74-LIST OF TABLES Mean (x) v a l u e f o r f r e s h samples and a l l t r e a t e d samples by experiment Mean (x) v a l u e f o r c o n t r o l samples and t r e a t e d samples by experiment Mean (y) v a l u e f o r f r e s h samples and a l l t r e a t e d samples by experiment Mean (y) v a l u e f o r c o n t r o l samples and t r e a t e d samples by experiment Mean (#Y) v a l u e f o r f r e s h samples and a l l t r e a t e d samples by experiment Mean (%Y) v a l u e f o r c o n t r o l samples and t r e a t e d samples by experiment Mean b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm f o r f r e s h samples and a l l t r e a t e d samples by experiment Mean b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm f o r c o n t r o l samples and t r e a t e d samples by experiment Mean b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm f o r f r e s h samples and a l l t r e a t e d samples by experiment Mean b e t a - c a r o t e n e e q u i v a l e n t at 460 nm f o r c o n t r o l samples and t r e a t e d samples by I v i i T a b l e Page e x p e r i m e n t 28 11 Mean v a l u e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . E x p e r i m e n t s 1, 2 , 3 and 4 . 29 12 Mean v a l u e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . E x p e r i m e n t s 1 , 2 , 3 and 4 . 30 13 Mean v a l u e f o r (#Y). E x p e r i m e n t s 1, 2 , 3 and 4 . 31 14 Mean v a l u e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm. E x p e r i m e n t s 1, 2 , 3 and 4 . 32 15 Mean v a l u e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm. E x p e r i m e n t s 1, 2 , 3 and 4 . 33 16 Dominant w a v e l e n g t h ( D W L ) o f t h e mean v a l u e s f o r f a c t o r s and l e v e l s w i t h i n e x p e r i m e n t s . 34 17 E x c i t a t i o n p u r i t y (EP) o f t h e mean v a l u e s f o r f a c t o r s and l e v e l s w i t h i n e x p e r i m e n t s 35 A1 A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . E x p e r i m e n t 1. 5^ A2 A n a l y s i s o f v a r i a n c e for- c h r o m a t i c i t y c o o r d i n a t e ( y ) . E x p e r i m e n t 1. 55 A3 A n a l y s i s o f v a r i a n c e f o r ($Y). E x p e r i m e n t 1. 56 A4 A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm. E x p e r i m e n t 1. 57 A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm. Experiment 1. C o r r e l a t i o n m a t r i x and mean v a l u e . Experiment 1. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . Experiment 2. A n a l y s i s of v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 2. A n a l y s i s of v a r i a n c e f o r (#Y). Experiment 2. A n a l y s i s of v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm. Experiment 2. A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm. Experiment 2. C o r r e l a t i o n m a t r i x and mean v a l u e . Experiment 2. A n a l y s i s of v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x j . Experiment 5. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 5. A n a l y s i s of v a r i a n c e f o r (#Y). Experiment 5. A n a l y s i s of v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t at 450 nm. Experiment 3* A n a l y s i s of va r i a n c e f o r beta-carotene e q u i v a l e n t at 460 nm. Experiment 3. C o r r e l a t i o n m a trix and mean val u e . Experiment 3. A n a l y s i s of va r i a n c e f o r c h r o m a t i c i t y coordinate ( x ) . Experiment 4. A n a l y s i s of va r i a n c e f o r c h r o m a t i c i t y coordinate ( y ) . Experiment 4, A n a l y s i s of va r i a n c e f o r (%Y). Experiment 4. A n a l y s i s of va r i a n c e f o r beta-carotene e q u i v a l e n t a t 450 nm. Experiment 4. A n a l y s i s of va r i a n c e f o r beta-carotene e q u i v a l e n t at 460 nm. Experiment 4. C o r r e l a t i o n m a trix and mean val u e . Experiment 4. LIST OF FIGURES F i g u r e Page 1 S t r u c t u r a l f o r m u l a of b e t a - c a r o t e n e 1 2 The C.I.E, horseshoe shaped spectrum l o c u s showing fol 11 3 The inst r u m e n t f o r c o n t r o l l i n g temperature d u r i n g i r r a d i a t i o n 1 5 4 R e l a t i o n s h i p s between mean x, y, %1 and dose l e v e l s . Experiment 1 3 6 5 R e l a t i o n s h i p s between mean x, y, fol and dose l e v e l s . Experiment 2 3 7 6 R e l a t i o n s h i p s between mean x, y, $Y and dose l e v e l s . Experiment 3 3 8 7 R e l a t i o n s h i p s between mean x, y, fol and dose l e v e l s . Experiment 4 3 9 8 R e l a t i o n s h i p s between mean b e t a -carotene e q u i v a l e n t and dose l e v e l s a t 4-50 and 460 nm. Experiments 1 and 2 40 9 R e l a t i o n s h i p s between mean b e t a -carotene e q u i v a l e n t and dose l e v e l s a t 4 5 0 and 460 nm. Experiments 3 and 4 41 x i LIST OP ABBREVIATIONS AND DEFINITIONS r e p R o e n t g e n - e q u i v a l e n t - p h y s i c a l . - The amount of r a d i a t i o n t h a t would r e l e a s e 1 r o e n t g e n of energy i n 1 gram of t i s s u e . . Roentgen The q u a n t i t y o f i o n i z i n g i r r a d i a t i o n t h a t w i l l produce s u f f i c i e n t i o n s i n 1 cc of d r y a i r to c a r r y 1 e.s.u. of e l e c t r i c i t y . Rad Q u a n t i t y o f i o n i z i n g r a d i a t i o n which r e s u l t s i n the a b s o r p t i o n of 100 ergs per gram o f i r r a d i a t e d m a t e r i a l . Mrad 1 0 6 r a d . x i i ACKNOWLEDGEMENTS The w r i t e r wishes t o acknowledge the h e l p , guidance and encouragement g i v e n "by h i s s u p e r v i s o r , Dr. J . F . R i c h a r d s , Dept. o f Food S c i e n c e , U n i v e r s i t y o f B r i t i s h Columbia and to Miss Lynne Robinson f o r h e l p f u l guidance d u r i n g the w r i t i n g o f the computer program. A s p e c i a l acknowledgement i s extended t o P r o f e s s o r E.L. Watson, Dept. o f A g r i c u l t u r a l E n g i n e e r i n g , U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s a s s i s t a n c e i n o b t a i n i n g the .'. ins t r u m e n t f o r c o n t r o l l i n g temperature d u r i n g i r r a d i a t i o n . The w r i t e r wishes t o thank Dr. W.D. Powrie, Dept. o f Food S c i e n c e , U n i v e r s i t y o f B r i t i s h Columbia and Dr. C.W. Ro b e r t s , Dept. o f Poultry. S c i e n c e , U n i v e r s i t y o f B r i t i s h Columbia f o r s e r v i n g on the r e s e a r c h committee. INTRODUCTION I n c r e a s i n g importance i s b e i n g g i v e n t o the c o l o r of ©SS y o l k when j u d g i n g the q u a l i t y of eggs. One o f the most s t r i k i n g components of whole egg appearance i s the y o l k c o l o r . C o l o r i n egg y o l k s i s due t o the c a r o t e n o i d s which the hens absorb from f e e d and d e p o s i t as pigment. C a r o t e n o i d s are y e l l o w t o r e d pigments o f a l i p h a t i c o r a l i p h a t i c - a l i c y c l i c s t r u c t u r e composed o f i s o p r e n e groups, u s u a l l y '8, l i n k e d so t h a t the two methyl groups n e a r e s t the c e n t e r o f the molecule are i n p o s i t i o n s 1:6 and a l l o t h e r l a t e r a l methyl groups are i n p o s i t i o n 1:5» w i t h a s e r i e s o f conjugated C-C double bonds c o n s t i t u t i n g the chromophoric system of the c a r o t e n o i d s . The b a s i c s t r u c t u r e i s demonstrated by the formula f o r b e t a - c a r o t e n e a symmetrical hydrocarbon w i t h 40 carbon atoms as shown i n P i g . 1 F i g . 1 . S t r u c t u r a l f o r m u l a of b e t a - c a r o t e n e 2 The major c a r o t e n o i d subgroups are c a r o t e n e s and x a n t h o p h y l l s . The former i n c l u d e s a l l the hydrocarbon c a r o t e n o i d s , and the l a t t e r a l l the hydroxy, epoxy, and o x y - d e r i v a t i v e s o f the c a r o t e n e s . X a n t h o p h y l l s are a l s o f r e q u e n t l y e s t e r i f i e d , as, f o r example, p h y s a l i e n , which i s the d i p a l m i t o y l e s t e r o f z e a x a n t h i n . Many c a r o t e n o i d s were named by t h e i r d i s c o v e r e r f o r some s p e c i a l p r o p e r t y or f o r t h e i r source, e.g., ca r o t e n e (from c a r r o t s ) , c r y p t o x a n t h i n ( h i d d e n pigment), and z e a x a n t h i n (from Zea Maize) (Goodwin, 1954a; B o r e n s t e i n and B u n n e l l 1956). ^gg y o l k p r o d u c t s are t r a d e d by the food i n d u s t r y i n f r o z e n ( p l a i n , s a l t e d o r sugared), d r i e d o r f r e s h forms and are used i n the manufacture o f a number o f food products i n c l u d i n g mayonnaise, macaroni and bakery p r o d u c t s as an e m u l s i f i e r and to impart c o l o r . Thus, the e f f e c t o f p r o c e s s i n g on the c o l o r o f y o l k p r o d u c t s i s imp o r t a n t . The s p e c t r a l a b s o r p t i o n curves of the c a r o t e n o i d s , p a r t i c u l a r l y i n the v i s i b l e r e g i o n 400 - 500 nm are w i d e l y used f o r purposes of i d e n t i f i c a t i o n and a s s a y . The o f f i c i a l AOAC method of a s s e s s i n g egg y o l k c o l o r i n v o l v e s comparison of the absorbance (455nm) of an acetone e x t r a c t w i t h a be t a - c a r o t e n e s t a n d a r d curve (AOAC, 1969). C o l o r comparison c h a r t s are a l s o w i d e l y used. T o l k c o l o r assessment by 3 r e f l e c t a n c e measurement has a l s o been i n v e s t i g a t e d ( R i c h a r d s , 1970). I t was the primary purpose of t h i s r e s e a r c h t o study the e f f e c t o f v a r y i n g combinations of i r r a d i a t i o n treatment, f r e e z i n g methods, and s t o r a g e time on the c o l o r o f n a t u r a l l y and a r t i f i c i a l l y pigmented egg y o l k determined by two o b j e c t i v e methods. A secondary o b j e c t i v e was to determine the r e l a t i o n s h i p between the two methods of c o l o r e v a l u a t i o n . 4 RBVIEW OP LITERATURE The s t a b i l i t y of food carotenoids during and after processing has received considerable attention. The common unit operations of food processing i n general have only minor effects on the carotenoids. The naturally occuring carotenoid-protein complexes apparently are more stable than carotenoids per se. (Takamatsu, 1957). Freezing had l i t t l e effect on asparagus and lima beans carotenoids (Zimmerman et a l . , 194-1). Cryptoxanthin, and t o t a l carotenoids changed only s l i g h t l y i n corn stored for 9 months at 0 F° (Tichenor et a l . , 1965). Frozen broccoli showed no loss i n to t a l carotene during storage at 0 F° for 6.1 weeks (Martin et a l . , 1960). The effect of gamma-irradiation on carotenoids has been studied in a variety of systems. Lukton and Mackinney (1956) found a fil m of beta-carotene and lycopene i n the solid state to be surprisingly stable: 2% loss at 2 milli o n rep. Solutions of beta-carotene were unstable in petroleum ether, methyl stearate, methyl oleate, and methyl linoleate. S t a b i l i t y was greater i n stearate than i n oleate and linoleate. They concluded that destruction i s caused by secondary reactions and depends upon the extent to which free radicals or peroxides, formed i n the surrounding medium, are available for reaction with carotenoids. The same workers studied the effects of pcamma-irradiation on tomato purees, whole tomatoes, c a r r o t purees, and prawns. C a r o t e n o i d s t a b i l i t y was e x c e l l e n t a t doses up to 12 x 105 r e p i n v e g e t a b l e p r o d u c t s , but the a s t a x a n t h i n content o f prawns decreased as much as 60% a t 4 x 10 r e p . I r r a d i a t i o n o f green tomatoes r e t a r d e d the s y n t h e s i s of lycopene, and a t h i g h l e v e l s p r e v ented i t (Salunkhe e t a l . , 1959). The c o l o r o f tomatoes fadded a t doses o f from 5 x 105 t o 1 x 106 r a d s . Coses up t o 5.72 x 106 r a d s had no apparent e f f e c t on the c a r o t e n o i d s o f canned a p r i c o t n e c t o r , peach n e c t o r , o r peach h a l v e s (Salunkhe et a l . , 1959). F r a n c e s c h i n i e t a l . , (1959) s t u d i e d the e f f e c t o f gamma - i r r a d i a t i o n on the car o t e n o i d s . of c a r r o t s , sweet p o t a t o e s , green beans, and b r o c c o l i . Green bean c a r o t e n o i d s were u n s t a b l e when i r r a d i a t e d a t 1.86 megarads (nra d s ) a f t e r f r e e z i n g , but r e a s o n a b l y s t a b l e when i r r a d i a t e d a t room temperature. The o t h e r v e g e t a b l e s i n t h i s study d i d not e x h i b i t t h i s f r e e z i n g - r a d i a t i o n i n t e r r e l a t i o s h i p . The c a r o t e n o i d s of sweet p o t a t o e s showed r e l a t i v e l y l i t t l e d e s t r u c t i o n from i r r a d i a t i o n a t 1.86 Mrads. However, v i s u a l c o l o r changes v/ere g r e a t e r than pigment changes i n sto r a g e and h i g h l y dependent on s t o r a g e c o n d i t i o n s . C a r o t e n o i d d e s t r u c t i o n of b r o c c o l i was 25-50?o a t 1.86 Mrad. C a r o t e n o i d d e s t r u c t i o n of c a r r o t s was moderate a t 1.86 Mrad except when the c a r r o t s were i n an a i r atmosphere. P a c k i n g i n n i t r o g e n improved r e t e n t i o n o f c a r o t e n o i d pigments o f i r r a d i a t e d sweet c o r n ( T l c h e n o r et a l . , 1965). R e t e n t i o n o f b e t a - c a r o t e n e , c r y p t o x a n t h i n , and o t h e r c a r o t e n o i d s was good a t 1.0 mrad but decreased a t 3 to 5 mrads. L a i e t a l . (1959) gamma-irradiated both a h a r d r e d s p r i n g wheat and a hard r e d w i n t e r wheat. T o t a l c a r o t e n o i d s per 100g o f f l o u r d e c r e a s e d from 13.7 . to 10.5mg at 1.0 x 10 6rep. C a r o t e n o i d a d d i t i o n t o foods p r e d a t e d the commercial s y n t h e s i s o f b e t a - c a r o t e n e . C a r r o t e x t r a c t s , palm o i l e x t r a c t s , annatto e x t r a c t s , and o l e o r e s i n papr.il.ca have been used f o r g e n e r a t i o n s to c o l o r cheese, b u t t e r , soups, sausage p r o d u c t s , e t c . The advent o f pure s y n t h e t i c c a r o t e n o i d s has i n c r e a s e d i n t e r e s t i n c o l o r i n g foods v/ith these compounds because of the obvious advantages of working with, w e l l -c o n t r o l l e d , r e p r o d u c i b l e c o l o r s o u r c e s . C a r o t e n o i d s are added to f o o d s t u f f s f o r b o t h n u t r i t i o n a l enrichment and c o l o r improvement ( B u n n e l l et a l . , 1966). The major c a r o t e n o i d s , n a t u r a l and s y n t h e t i c , used t o c o l o r foods a r e , b i x i n , a l p h a - c a r o t e n e , b e t a - c a r o t e n e , b e t a - a p o - 8 - c a r o t e n a l , c a n t h a x a n t h i n , b e t a - a p o - 8 - c a r o t e n o i c a c i d e t h y l e s t e r , c a p s a n t h i n , and c a p s o r u b i n ( B u n n e l l et a l . , 1966). B e t a - c a r o t e n e i s p r o b a b l y . t h e most w i d e l y used s y n t h e t i c c a r o t e n o i d . The major uses i n North America are t o c o l o r and 7 f o r t i f y margarine, s h o r t e n i n g , f r u i t d r i n k s , popcorn, and baked goods ( B a u e r n f e i n d et a l . , 1 9 5 8 ) . B e t a - c a r o t e n e used i n c i t r u s beverages, p r i m a r y cheese, egg y o l k p r o d u c t s , i c e cream, and cake mixes ( B u n n e l l e t a l . , 1 9 5 8 ) . An i n t e r e s t by f o o d p r o c e s s o r s i n s t a n d a r i z e d egg y o l k s of d a r k e r - c o l o r f o r use i n bakery p r o d u c t s , macaroni, and mayonnaise has prompted i n v e s t i g a t i o n o f the a d d i t i o n o f be t a - c a r o t e n e t o f r o z e n and d r i e d y o l k p r o d u c t s . The c o l o r o f y o l k p r o d u c t s has been expressed i n terms o f carot e n e c o n c e n t r a t i o n by the t e c h n i c a l committee of the N a t i o n a l Egg Products A s s o c i a t i o n (NEPA). NATURAL EGG YOLK EXPRESSED AS CAROTENE UNITS NEPA y o l k c o l o r ( s t a n d a r d s ) 1 ( v e r y l i g h t y e l l o w ) 2 3 4 5 6 (orange) C o l o r e q u i v a l e n t s i n terms of carotene(P.P.M) 15 40 70 90 120 150 Canthaxanthin, which i s not y e t approved i n the U.S., i s used i n Europe t o c o l o r tomato p r o d u c t s . Beta-ape—8-c a r o t e n o i c a c i d e t h y l e s t e r i s used i n Europe t o pigment egg y o l k . 8 A c o l o r assay method i n which the absorbance of an acetone extract of yolk products i s determined at 4-55 nm and then converted to equivalent beta-carotene concentration i s used by the AOAC (1960). Borenstein and Bunnell, (1966) studied the e f f e c t of storage time at -5 F° on the r e t e n t i o n of carotenoid content of natural and synthetic beta-carotene pigmented egg yolk ; and found no loss of carotenoid content a f t e r 3 months storage but s l i g h t decrease a f t e r 15 months. A.very simple and widely used method of measuring color i n egg yolks i s v i s u a l scoring. The Heiman-Carver color r o t o r (1935) has been used extensively i n the U.S. but i s unavailable today (Heiman and Carver, 1935). This device consisted of a black wheel containing 24 d i f f e r e n t yolk shaped c o l o r samples so that a co l o r number could be assigned to any yolk a f t e r a d i r e c t v i s u a l comparison. The Hoffman La Roche color fan i s a les s cumbersome a p p l i c a t i o n of the same idea (Hoffman La Roche & Co. Ltd. 1962). I t contains a uniform progression of increas i n g c o l o r from i v o r y to orange-yellow, numbered 1 to 12. In Canada, an enamelled r i n g from (NRC discs) i s used (Ashton and Flet c h e r , 1962). Much research was done i n preparing the standards, i n mixing the enamel to (CIE) t r i s t i m u l u s values, i n shaping the surface of the di s c so that the method of viewing would be the same f o r the di s c as the act u a l yolk. This system consists of 15 discs from l i g h t yellow to deep orange. 9 The measurement o f c o l o r , or i n o t h e r words the d e t e r m i n a t i o n of v a l u e s c h a r a c t e r i z i n g a c o l o r p e r c e p t i o n , t r a n s l a t e d the f u n c t i o n s of human v i s i o n w i t h the h e l p o f p h y s i c a l measuring i n s t r u m e n t s . The laws of a d d i t i v e c o l o r mixing show t h a t any c o l o r p e r c e p t i o n can be produced by m i x i n g t h r e e s u i t a b l e s p e c t r a l s t i m u l i or p r i m a r i e s i n the p r o p e r r a t i o . The human seems to judge the v i s i b l e p a r t of the e l e c t r o m a g n e t i c spectrum by o n l y t h r e e d i f f e r e n t s p e c t r a l s e n s i t i v i t y f u n c t i o n s , which are e x p e r i e n c e d by the o b s e r v e r as a s i n g l e e f f e c t or c o l o r p e r c e p t i o n . Thus, t h r e e numbers or q u a n t i t i e s are r e q u i r e d i n o r d e r to c l e a r l y d e f i n e any one c o l o r p e r c e p t i o n . T h e r e f o r e , c o l o r may be understood as l o c a l v e c t o r s i n a c o l o r space. Each p o i n t w i t h i n t h i s c o l o r space c h a r a c t e r i z e s c o l o r a c c o r d i n g to hue (wavelength), s a t u r a t i o n ( p r o p o r t i o n of s p e c t r a l l i g h t i n the mixture o f s p e c t r a l and white l i g h t ) and l u m i n o s i t y ( i n t e n s i t y of l i g h t p e r c e p t i o n connected with, c o l o r p e r c e p t i o n ) . These are the t h r e e p r o p e r t i e s which any o b s e r v e r w i t h normal v i s i o n would a t t r i b u t e t o c o l o r as a sensory p e r c e p t i o n (Vuilleumier,1969). The t r i s t i m u l u s v a l u e s of the s t a n d a r d C L E . system are d e f i n e d by the I n t e r n a t i o n a l Commission on I l l u m i n a t i o n ( C . I . E ) ( 1 9 3 1 ) as a c o n v e n t i o n a l r e f e r e n c e system f o r c o l o r measurements (Mackinney and L i t t l e , 1962). The t h r e e q u a n t i t i e s which c h a r a c t e r i z e a c o l o r p e r c e p t i o n w i t h a d e f i n e d i l l u m i n a n t are symbolized by X, Y and Z ( t r a n s f o r m e d , u n r e a l p r i m a r i e s ) . The v a l u e of X ( o r x i n the case of spectrum 10 c o l o r s ) r e p r e s e n t s the amount of p r i m a r y which i s r e d d i s h p u r p l e o f h i g h e r s a t u r a t i o n than any o b t a i n a b l e c o l o r h a v i n g t h i s hue. The v a l u e o f Y ( o r y) r e p r e s e n t s the amount o f green primary c o n s i d e r a b l y more s a t u r a t e d than the spectrum c o l o r whose wavelength i s 520 nm. The v a l u e o f Z ( o r z) r e p r e s e n t s the amount of b l u e primary t h a t i s c o n s i d e r a b l y more s a t u r a t e d than the spectrum c o l o r whose wavelength i s 4-77 nm (Hardy, 1956). The t r i s t i m u l u s v a l u e s X, Y and Z of a c o l o r were d e f i n e d as the r e l a t i v e amounts of each o f the t h r e e u n r e a l p r i m a r i e s needed t o e f f e c t a c o l o r match under s p e c i f i e d c o n d i t i o n s . The s p e c i f i c a t i o n of a c o l o r i n terms of i t s t r i s t i m u l u s v a l u e s i s cumbersome. I t t h e r e f o r e became a matter of convenience t o express r e s u l t s more si m p l y . The e n t i r e l u m i n o s i t y i s a s c r i b e d t o Y, and the c h r o m a t i c i t y can be expressed by c o - o r d i n a t e s x, y and z, such t h a t : X+ Y+ Z ' X+ Y+ Z ' X+ Y+ Z and c o n s e q u e n t l y : x+y+z = 1 T h e r e f o r e a c o l o r i s u n i q u e l y d e f i n e d by i t s c h r o m a t i c i t y c o o r d i n a t e s x and y, and i t s l i g h t n e s s , the t r i s t i m u l u s Y v a l u e . T r i c h r o m a t i c c o e f f i c i e n t s x, y and z are the p r o p o r t i o n s o f each of the p r i m a r i e s i n the t o t a l m ixture (Mackinney and 11 L i t t l e , 1962). I f the d e r i v e d values of x and y are p l o t t e d i n a r e c t a n g u l a r coordinate system a very convenient r e p r e s e n t a t i o n a c c o r d i n g to hue and s a t u r a t i o n r e s u l t s . The c h r o m a t i c i t y diagram of the G.I. 7,. i s shown i n F i g . 2 ( F r a n c i s , 1969). -TOO - 90 . 80 • 70 • 60 Y,% • 50 - AO • 30 - 10 • 0 F i g . 2. The G.I.E. horseshoe shaped spectrum lo c u s showing %Y, The r e l a t i v e b r i g h t n e s s of a sample i s i n d i c a t e d d i r e c t l y by the value of Y on a s c a l e t h a t r e p r e s e n t s an absolute b l a c k by zero and a p e r f e c t white by 100 (Hardy, 1936). 12 MATERIALS AND METHODS EXPERIMENT 1 Eggs were o b t a i n e d from the U.B.C. s t r a i n o f S i n g l e Comb White Leghorn p u l l e t s housed i n i n d i v i d u a l cages on the U.B.C. p o u l t r y farm. A l l p u l l e t s were f e d the same d i e t d u r i n g the experiment t o o b t a i n as u n i f o r m egg y o l k c o l o r as p o s s i b l e . Eggs were i n d i v i d u a l l y broken and egg albumen s e p a r a t e d from y o l k . A l l egg y o l k s were mixed t o g e t h e r and 10$ sodium c h l o r i d e (w/w) was added to p r e v e n t g e l a t i o n d u r i n g f r e e z i n g and s t o r a g e . Each experiment r e q u i r e d about 200 eggs. F i f t y -seven p l a s t i c c o n t a i n e r s (113gm c a p a c i t y ) v/ere f i l l e d v/ith the homogeneous y o l k m a t e r i a l and then randomly a s s i g n e d t o treatment groups. D u p l i c a t e samples of 55gm each v/ere drawn and e v a l u a t e d f o r each e x p e r i m e n t a l u n i t ( c o n t a i n e r ) . The f a c t o r s s t u d i e d i n t h i s experiment c o n s i s t e d o f : 7 i r r a d i a t i o n treatments (zero-dose c o n t r o l , and a l l combinations of 0.5, 1.0 and 2.0Mrad a d m i n i s t e r e d b e f o r e o r a f t e r f r e e z i n g ) ; 2 f r e e z i n g and s t o r a g e temperature (-10 and -35 F ° ) ; 2 atmospheres d u r i n g i r r a d i a t i o n ( a i r and n i t r o g e n ) and 2 pos t - t r e a t m e n t s t o r a g e time (10 and 30 d a y s ) . The treatments v/ere arranged as a 7 x 2 x 2 x 2 f a c t o r i a l i n a co m p l e t e l y randomized d e s i g n . In a d d i t i o n , an u n t r e a t e d sample was e v a l u a t e d and served as a f r e s h c o n t r o l . Gamma-radiation v/as a d m i n i s t e r e d i n a Gammacell 220 13 (Atomic Energy of Canada L t d . ) a t a dose r a t e o f about 1.0 Mrad per hour. Unfrozen samples were i r r a d i a t e d under ambient temperatures. An attempt was made to m a i n t a i n the temperature of f r o z e n samples d u r i n g i r r a d i a t i o n by p a c k i n g the c o n t a i n e r s w i t h crushed i c e i n a doubly i n s u l a t e d v e s s e l . I n i t i a l f r e e z i n g o f samples was accomplished i n b l a s t a i r f r e e z e r s a t -10 or -35 F ° . A n i t r o g e n atmosphere was a t t a i n e d by a l l o w i n g n i t r o g e n gas to f l o w i n t o the headspace of the c o n t a i n e r s f o r about 10 sec. b e f o r e c l o s i n g . Samples • were s t o r e d f o r 10 or 30 days a t -10 or -35 F ° a f t e r which time y o l k c o l o r was e v a l u a t e d by two methods. The f i r s t method, an e x t r a c t i o n - c o l o r i m e t r i c procedure was c a r r i e d out a c c o r d i n g t o the method o u t l i n e d by the A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists (AOAC, 1?65). In t h i s procedure 2.5gfn of l i q u i d y o l k was mixed w i t h one to-two ml of acetone and s t i r r e d t o a smooth p a s t e . A p p r o x i m a t e l y 50 ml of acetone, was added and the mixture was washed onto Whatman No. 4 f i l t e r paper w i t h s u c c e s s i v e s m a l l p o r t i o n s of acetone. The f i l t r a t e was c o l l e c t e d i n a ground g l a s s -s toppered, 100 ml v o l u m e t r i c f l a s k and d i l u t e d t o volume w i t h acetone. The c o n t e n t s o f the f l a s k were shaken, and the absorbance o f an a l i q u o t r e a d a t 10 nm i n t e r v a l s between 400 - 500 nm on a p r e v i o u s l y s t a n d a r i z e d Bausch and Lomb S p e c t r o n i c 20. A p r e v i o u s l y developed s t a n d a r d curve f o r be t a - c a r o t e n e was used t o es t i m a t e the b e t a - c a r o t e n e e q u i v a l e n t c ontent of the sample. 1 4 The second method, an H i t a c h i , P e r k i n - E l m e r spectrophotometer v/ith a d i f f u s e r e f l e c t a n c e attachment v/as s t a n d a r i z e d to 100fo r e f l e c t a n c e a g a i n s t MgO. The sample was determined a t 10 nm i n t e r v a l s from 400 - 630 nm. From t h e s e v a l u e s the C.I.E. t r i s t i m u l u s v a l u e s X, Y and Z, and the t r i c h r o m a t i c c o e f f i c i e n t s x* y and °/6l were c a l c u l a t e d by the weighted o r d i n a t e method i n c o r p o r a t i n g i l l u m i n a n t C a t a c o n s t a n t wavelength i n t e r v a l s o f 10 nm (Mackinney and L i t t l e , 1962) . Dominant Wavelength (DWL) and E x c i t a t i o n P u r i t y (EP) were c a l c u l a t e d a c c o r d i n g t o the method by McCarley e t a l . , 1965. EXPERIMENT 2 The methods used were s i m i l a r t o those o f Experiment 1 v/ith 2 e x c e p t i o n s . F i r s t l y , a Unicam SP . 800 B r e c o r d i n g spectrophotometer w i t h r e f l e c t a n c e attachment v/as used f o r r e f l e c t a n c e d e t e r m i n a t i o n s . The sample was c o n t a i n e d i n an o p t i c a l l y c l e a r p e t r i d i s h (3.5 x 10 mm). R e f l e c t a n c e was r e a d from the r e c o r d a t 10 nm i n t e r v a l s from 400 - 680 nm. Secondly, a l i q u i d n i t r o g e n system was used to c o n t r o l temperature d u r i n g r a d i a t i o n a t -10 and -35 I n t h i s method l i q u i d n i t r o g e n was f o r c e d by compressed a i r i n t o a c o n t a i n e r i n the r a d i a t i o n drawer. Rubber tube i n the c o n t a i n e r p e r m i t t e d r e g u l a t i o n of f l o w . Temperature was F i g . 3 . The instrument f o r c o n t r o l l i n g temperature d u r i n g i r r a d i a t i o n . 16 c o n t r o l l e d a t -10 and -35 F° t o w i t h i n ± 2 F ° . The system i s shown i n F i g . 3. EXPERIMENT 3 The methods were s i m i l a r t o those o f Experiment 2. S y n t h e t i c b e t a - c a r o t e n e was used f o r c o l o r i n g egg y o l k . Two and one h a l f gm o f 10% b e t a - c a r o t e n e water d i s p e r s i b l e b e a d l e t s were weighed i n t o a s m a l l beaker and 25 ml of d i s t i l l e d water was added. An a l i q u o t o f 0.53 ml of b e t a - c a r o t e n e s o l u t i o n was added per 100 gm o f egg y o l k . EXPERIMENT 4-The methods were s i m i l a r t o those o f Experiment 2. Canthaxanthin was used t o pigment the egg y o l k . T h i s was accomplished i n the f o l l o w i n g manner: Two and one h a l f gm of 10% c a n t h a x a n t h i n water d i s p e r s i b l e b e a d l e t s were weighed i n t o a s m a l l beaker and 25 ml of d i s t i l l e d water was added. An a l i q u o t o f 0.23 ml of c a n t h a x a n t h i n s o l u t i o n was added per 100 gm o f egg y o l k . STATISTICAL METHODS The raw r e f l e c t a n c e d a t a were reduced t o t r i s t i m u l u s v a l u e s (X,Y,Z) and c h r o m a t i c i t y c o o r d i n a t e s (x,y,#Y) 17 c a l c u l a t e d by the use o f the w e i g h t e d - o r d i n a t e method. The i n d i v i d u a l c o l o r parameters were s u b j e c t e d t o a n a l y s e s of v a r i a n c e and simple l i n e a r c o r r e l a t i o n s between each p a i r of v a r i a b l e s was determined. A l l c a l c u l a t i o n s were performed by an IBM 360 d i g i t a l computer. 18 RESULTS Numerous z e r o - o r d e r i n t e r a c t i o n terms were found t o be s i g n i f i c a n t i n these experiments. Most of t h e s e i n t e r a c t i o n s i n v o l v e d e i t h e r r a d i a t i o n treatment o r temperature and f r e q u e n t l y both, Hov/ever, g r a p h i c a l e x amination of the apparent i n t e r a c t i o n s r e v e a l e d t h a t almost without e x c e p t i o n d i f f e r e n c e s i n response among the l e v e l s of one f a c t o r were q u a l i t a t i v e l y the same f o r each l e v e l o f the second f a c t o r , T h e r e f o r e , the means f o r the main e f f e c t s were c o n s i d e r e d t o be v a l u a b l e i n d i c a t i o n s of treatment responses a t l e a s t q u a l i t a t i v e l y (Cox, 1958). REFLECTANCE MEASUREMENTS C h r o m a t i c i t y C o o r d i n a t e s Mean v a l u e s f o r c h r o m a t i c i t y c o o r d i n a t e s x and y are shown i n t a b l e s 1 to 4, 11 and 12. R e l a t i o n s h i p s between means x, y and dose l e v e l s are shown i n F i g . 1, 2, 3 and 4. A n a l y s e s of v a r i a n c e are r e p r e s e n t e d i n Tables A1, A2, B1, B2, C1, 02, D1 and D2. The mean (x) v a l u e and mean (y) v a l u e o f : f r e s h samples were h i g h l y s i g n i f i c a n t l y (P<0.01) d i f f e r e n t t han those o f c o r r e s p o n d i n g t r e a t e d samples i n a l l experiments. 1 9 I r r a d i a t i o n treatment had a h i g h l y s i g n i f i c a n t ( P < 0 . 0 1 ) e f f e c t on both mean (x) and ( y ) v a l u e i n a l l experiments. The mean v a l u e s were s i g n i f i c a n t l y h i g h e r f o r c o n t r o l samples than f o r i r r a d i a t e d samples and h i g h e r f o r samples i r r a d i a t e d a f t e r f r e e z i n g than b e f o r e f r e e z i n g . The c h r o m a t i c i t y c o o r d i n a t e s d e c r e a s e d c o n s i s t e n t l y w i t h i n c r e a s i n g dose o f r a d i a t i o n . The mean (x) v a l u e was lower a f t e r 30 days s t o r a g e • t h a n a f t e r 10 days i n a l l experiments. The d i f f e r e n c e was s i g n i f i c a n t i n a l l but Experiment 2. C o n v e r s e l y , mean (y) v a l u e s were h i g h e r a f t e r 30 days s t o r a g e t h a n 10 days s t o r a g e . F r e e z i n g and sto r a g e a t -10 P° r e s u l t e d i n h i g h e r mean (x) and (y) v a l u e s than a t -35 F° i n a l l experiments. Atmosphere had no s i g n i f i c a n t e f f e c t on (x) v a l u e i n any o f the experiments. Mean ( y ) v a l u e was s i g n i f i c a n t l y h i g h e r f o r n i t r o g e n - p a c k e d than a i r - p a c k e d samples i n Experiment 1 o n l y . (Atmosphere had no s i g n i f i c a n t e f f e c t on (y) v a l u e i n Experiments 2, 3 and 4) L i g h t n e s s Mean v a l u e s f o r (%Y) are shown i n T a b l e s 5* 6 and 13. R e l a t i o n s h i p s between mean (?&I) and dose l e v e l s are shown i n E i g . 1, 2, 3 and 4. An a l y s e s o f v a r i a n c e a re r e p r e s e n t e d 20 i n T a b l e s A3, B3, G3 and D3. F r e s h samples were s i g n i f i c a n t l y l i g h t e r i n c o l o r ( h i g h e r mean %T) than t r e a t e d samples i n a l l experiments. S i m i l a r l y , i r r a d i a t e d samples v/ere d a r k e r than u n i r r a d i a t e d c o n t r o l s i n a l l experiments. In Experiments 3 and 4 the darkness o f samples i n c r e a s e d s i g n i f i c a n t l y w i t h i n c r e a s i n g i r r a d i a t i o n dose, and samples i r r a d i a t e d b e f o r e f r e e z i n g were s i g n i f i c a n t l y d a r k e r than those i r r a d i a t e d i n the f r o z e n s t a t e . S i m i l a r i n s i g n i f i c a n t t r e n d s were e v i d e n t i n Experiment 2. The e f f e c t o f st o r a g e time on l i g h t n e s s was i n c o n s i s t e n t among experiments. In Experiments 1 and 4 samples s t o r e d f o r 10 days were s i g n i f i c a n t l y d a r k e r than those s t o r e d f o r 30 days. In Experiments 2 and 3 the samples s t o r e d f o r 10 days were l i g h t e r but t h i s e f f e c t was s i g n i f i c a n t o n l y i n Experiment 2. I n Experiment 1 and 2 samples s t o r e d a t -35 F° were s i g n i f i c a n t l y l i g h t e r t h an samples s t o r e d a t -10 F ° . No s i g n i f i c a n t e f f e c t o f temperature was found i n Experiments 3 and 4. I n Experiment $ a h i g h l y s i g n i f i c a n t i n t e r a c t i o n betv/een s t o r a g e time and temperature was found. T h i s may account f o r the l a c k of s i g n i f i c a n c e o f the 2 c o r r e s p o n d i n g main e f f e c t s i n t h i s experiment. 21 Atmosphere had no s i g n i f i c a n t e f f e c t on lightness i n any of the experiments. Dominant Wavelength (DWL) Mean DWL was almost i n v a r i a b l e among treatments within experiments (Table 16). The values ranged only from 577 to 590 nm. E x c i t a t i o n Purity (EP) EP was markedly aff e c t e d by dose l e v e l . Increasing the dose l e v e l r e s u l t e d i n a decrease i n EP. Also samples which were i r r a d i a t e d a f t e r f r e e z i n g or stored at -10 P° had higher EP values than corresponding samples i r r a d i a t e d before f r e e z i n g or stored at - 3 5 P°» EP tended to decline with length of storage but the e f f e c t was generally s l i g h t . Atmosphere had no e f f e c t on EP (Table 1 7 ) . ABSORBANCE MEASUREMENTS Mean values f o r beta-carotene equivalent (BCE) at 450 and 460 nm are shown i n Tables 7 to 10, 14 and 1 5 . Relationships; between mean BCE at 450, 460 nm and dose l e v e l are shown i n P i g . 8 and 9 . Analyses of variance are represented i n Tables A4, A5, B4, B5, C4, C5, D4 and D5. 22 Reading a t 450 nm Bet a - c a r o t e n e e q u i v a l e n t (BCE) v a l u e s o f f r e s h samples were h i g h l y s i g n i f i c a n t l y ( P ^ O . 01) d i f f e r e n t t h an c o r r e s p o n d i n g t r e a t e d samples i n a l l experiments. I r r a d i a t i o n treatment had a h i g h l y s i g n i f i c a n t ( P C 0 . 0 1 ) e f f e c t on BCE. Mean BCE v a l u e s were h i g h e r f o r c o n t r o l than f o r i r r a d i a t e d samples and h i g h e r f o r samples i r r a d i a t e d a f t e r f r e e z i n g than before.. Mean BCE v a l u e s d e c r e a s e d c o n s i s t e n t l y w i t h i n c r e a s i n g dose o f r a d i a t i o n . The e f f e c t of s t o r a g e time on BCE v a l u e was not c o n s i s t e n t among experiments. BCE v a l u e s were h i g h e r a f t e r 50 days than 10 days s t o r a g e i n Experiments 1 and 4 but the o p p o s i t e was t r u e i n Experiments 2 and 3 . The d i f f e r e n c e s were h i g h l y s i g n i f i c a n t (P^rO.01) i n the f i r s t 3 experiments. Samples f r o z e n and s t o r e d a t - 3 5 E° had s i g n i f i c a n t l y (P-^0.01) h i g h e r average BCE v a l u e s t h a n c o r r e s p o n d i n g samples a t -10 P° i n Experiments 2 and 4. No s i g n i f i c a n t temperature e f f e c t was found i n Experiments 1 and 3 . Mean BCE v a l u e s were h i g h e r f o r a i r - p a c k e d than n i t r o g e n - p a c k e d samples i n a l l experiments and t h e s e d i f f e r e n c e s were s i g n i f i c a n t ( P < 0 . 0 5 ) i n Experiments 2, 3 and 4. Reading a t 460 nm The r e s u l t s a t 460 nm were s i m i l a r t o those a t 460 nm. 2 3 CORRELATION MATRIX Simple l i n e a r c o r r e l a t i o n c o e f f i c i e n t s among absorbance and r e f l e c t a n c e v a r i a b l e s a re shown i n Ta b l e s A6, B6, C6 and D6 f o r Experiments 1, 2 , 3 and 4 r e s p e c t i v e l y . The c o r r e l a t i o n c o e f f i c i e n t s were h i g h l y s i g n i f i c a n t ( P^O.01) i n a l l experiments except those i n Experiment 1 between primary T and BCE v a l u e s a t 450 and 460 nm. 24 T a b l e 1. Mean (x) v a l u e f o r f r e s h samples and a l l treated samples by experiment. Experiment F r e s h (n=2) Other (n=112) 1 0.4700 0.4420 2 0.4690 * * 0.4427 5 0.4997 * * 0.4930 4 0.5235 * * 0.4954 ** Means are s i g n i f i c a n t l y d i f f e r e n t a t p^.0.01 (see T a b l e s A1, B-J, C1 and D1 Tab l e 2. Mean (x) v a l u e f o r c o n t r o l samples and t r e a t e d samples by experiment. Experiment C o n t r o l (n=16) T r e a t e d (n=96) * * 1 0.4637 0.4384 * * 2 0.4699 0.4381 * * 3 0.5092 0.4982 ** 4 0.5134- 0.4924 ** Means a r e s i g n i f i c a n t l y d i f f e r e n t a t P^.0.01 (see Tables A1, B1, C1 and D1 25 T a b l e 3. Mean (y) v a l u e f o r f r e s h samples and a l l t r e a t e d samples by experiment. Experiment F r e s h (n=2) Other (n=112) 1 0.4-770 0.4-609** 2 0.4-770 0.4-653** 3 0.4-325 0.4-220** 4- 0.4-125 0.3957** ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P<0.01 (see T a b l e s A2, B2, 02 and D2 Table 4-. Mean (y) v a l u e f o r c o n t r o l samples and t r e a t e d samples by experiment. Experiment C o n t r o l (n=16) T r e a t e d (n=96) * * 1 . 0.4684 0.4596 2 * * 0.474-8 0.4637 * * 0.4-577 0.4316 4 * * 0.4077 0.3937 ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P^ .0.01 (see T a b l e s A2, B2, C2 and D2 26 Table 5 . Mean (#Y) for fresh samples and a l l treated samples by experiment. Experiment Fresh (n=2) Other (n=112} 1 4 4 . 5 6 * * 55.72 2 44.10 30 .98* 3 42.57 * * 28.22 4 26.96 _ * * 23.00 ** Means are sig n i f i c a n t l y different at P<0.01 * Means are s i g n i f i c a n t l y different at P<^0.05 (see Tables A 3 , B3, 03 and D3) Table 6 . Mean (#Y) value for control samples and treated samples by experiment. Experiment Control (n=16) Treated (n=96) 1 37. OS 3 5 . 4 9 N S 2 3 5 . 0 0 30.31 * 3 33.04 * * 27.42 4 24.73 * * 22.71 ** Means are si g n i f i c a n t l y different at P<.0.01 * Means are si g n i f i c a n t l y different at P $ 0 . 0 5 N8 Means are insignificant (see Tables A3, B3, 03 and D3) 27 Table 7. Mean beta-carotene equivalent at 4-50 nm for fresh samples and a l l treated samples by-experiment . Experiment Presh (n=2) Other (n=112) * * 1 0.5070 0.2742 * * 2 0.5070 0.3790 * * 3 1.4575 1.0982 * * 4 1.1930 0.7830 T ** Means are si g n i f i c a n t l y different at P^O.01 (see Tables A4, B4, 04 and D4) Table 8. Mean beta-carotene equivalent at 450 nm for control samples and treated samples by experiment. Experiment Control (n=16) Treated (n=96) * * 1 0.4258 0.2489 * * P 0.6251 0.3380 * * 3 1.2894 1.0663 * * 4 1.0731 0.734-6 . ** Means are si g n i f i c a n t l y different at P<.0.01 (see Tables A4, B4, 04 and D4) 2 8 T a b l e 9. Mean b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm f o r f r e s h samples and a l l t r e a t e d samples by experiment. Experiment F r e s h (n=2) Other (n=112) 1 0.4680 * * 0.2591 2 0.4680 0.3559* 1.4075 * * 1.0734 4 1.2000 * * 0.7948 ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P^ . 0 . 0 1 * Means are s i g n i f i c a n t l y d i f f e r e n t a t P 4 0 . 0 5 (see T a b l e s A5, B 5 , C5 and D5) T a b l e 10. Mean b e t a - c a r o t e n e e q u i v a l e n t a t 460nm f o r c o n t r o l samples and t r e a t e d samples by experiment. Experiment C o n t r o l (n=16) T r e a t e d (n=96) 1 0.3996 0.2357 ** * * 2 0.5325 0.3181 * * 3 1.2563 1.0430 4 * * 1.0724 0.7486 ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P40.01 ( see T a b l e s A5, B 5 , C5 and D5) 2 9 Table 1 1 . Mean v a l u e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . Experiments 1 , 2 , 3 and 4 . I r r a d i a t i o n , Dose (Mrad)* Experiment 1 2 3 4 0 . 0 0.5 0 . 4 6 3 7 t 0 . 4 5 0 9 ? 0 . 4 6 9 9 1 0 . 4 5 3 3 ^ 0.5092-? 0 . 5 0 3 3 ^ 0.5134A 0.5022^ 1 . 0 2 . 0 0 . 4 4 1 1 ^ 0.423l5 0 . 4 4 2 2 ° 0 . 4 1 8 9 ^ 0 . 4 9 8 9 ^ 0 . 4 9 1 7 ^ 0 . 4 9 3 4 ° 0 . 4 8 1 7 I r r a d i a t i o n Time 1 2 3 4 B e f o r e F r e e z i n g 0 . 4 3 4 8 0 . 4 2 9 9 0 . 4 9 6 3 0 . 4 9 0 5 A f t e r F r e e z i n g 0 . 4 4 2 0 * t 0 . 4 4 6 4 * * 0.5000** 0 . 4 9 4 4 Storage Time 1 2 3 4 1 0 days 0 . 4 4 2 7 0 . 4 4 3 3 0 . 5 0 2 5 0 . 5 0 1 6 30 days 0 . 4 4 1 2 * 0 . 4 4 2 0 * % 0 . 4 9 7 0 * * 0 . 4 8 9 2 Temperature - 1 0 F° - 3 5 F° 1 2 3 4 . 0 . 4 5 3 2 0 . 4 4 8 5 0.5057 0.5013 0 . 4 3 0 8 * * 0 . 4 3 6 9 * * 0 . 4 9 3 7 * * 0 . 4 8 9 6 Atmosphere A i r N i t r o g e n 1 2 3 4 0 . 4 4 2 1 0 . 4 4 2 7 0 . 4 9 9 5 0.4952 0 . 4 4 1 9 ^ 0 . 4 4 2 6 ™ ! 0.4999^ 0 . 4 9 5 7 s Duncan new m u l t i p l e range t e s t f o r dose mean values.; any two means not s h a r i n g the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at P < £ 0 . 0 1 . ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P * > 0 . 0 1 . * Means are s i g n i f i c a n t l y d i f f e r e n t a t P ^ " 0 . 0 5 . ns Means are i n s i g n i f i c a n t . (see T a b l e s A 1 , B 1 , C1 and D 1 ) . 30 T a b l e 12. Mean v a l u e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiments 1, 2, 3 and 4. I r r a d i a t i o n , Dose (Mrad)* Experiment 1 2 3 4 0.0 0 .5 0.4684f 0.4664^ 0.4748? 0.4?07i 0.43777 0.4343B 0.4077 0.4010^ 1.0 2.0 0.4619? 0.4506S 0.4670^ 0 . 4 5 3 4 i ; 0.4319p 0.4287^ 0.3942^ 0.3360^ I r r a d i a t i o n Time 1 2 3 4 B e f o r e F r e e z i n g 0.4567 0.4616 0.4504 0.3918 A f t e r F r e e z i n g 0.4626** 0.4658** 0.4329** 0.3956 Storage Time 1 2 3 4 10 days 0.4608 0.4637 0.4321 0.3942 30 days 0.4609*1*5 0.4668** 0.4330** 0 .3972 Temperature -10 F° - 3 5 P° 1 2 3 4 0.4678 0.4683 0.4341 0 .3961 * * 0.4540** 0.4622** 0.4310 0.3953 n S Atmosphere A i r N i t r o g e n 1 2 3 4 0.4600 0.4651 0.4325 0.3957 0.4617*g 0.4655^ 0.4326^ 0.3957 * Duncan new m u l t i p l e range t e s t f o r dose mean v a l u e s ; any two means not s h a r i n g the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t a t P^O.01. ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P<0.01. * Means are s i g n i f i c a n t l y d i f f e r e n t a t P C 0 . 0 5 . ns means are i n s i g n i f i c a n t . (see T a b l e s A2, B2, 02 and D2). 31 Table 13. Mean v a l u e f o r (#Y). Experiments 1, 2, 3 and 4. I r r a d i a t i o n ^ Dose (Mrad)* Experiment 1 2 3 4 0.0 0.5. 37.08A 35.18^ 35.00^ 31.50? 33.04? 28.92? R 24.73 24.03 1.0 2.0 35.40^ 35.89^ 31.097 28.34-p 28.04^ 25.30° 23.29 20.82° I r r a d i a t i o n Time 1 2 3 4 Before F r e e z i n g 35.96 29.62 26.84 21.86 A f t e r F r r e z i n g 35.01™! 31.00?? 28.00** 23.57 Storage Time 1 2 3 4 10 days 34.91 33.01 28.48 22.35 30 days 36.53** 28.00** 23.66 Temperature -10 F° -35 F° 1 2 3 4 31.00 29.21 28.44 23.24 * * 40.43* 2 8 . 0 0 ^ 22.76ns Atmosphere A i r N i t r o g e n 1 2 3 4 35.63 31.31 28.31 23.03 35.80^ 2 8 . 1 4 ^ 22.97 * Duncan new m u l t i p l e range t e s t f o r dose mean v a l u e s ; any two means not s h a r i n g the same l e t t e r a re s i g n i f i c a n t l y d i f f e r e n t a t P^'0.01. ** Means are s i g n i f i c a n t l y d i f f e r e n t a t P^O.01. * Means are s i g n i f i c a n t l y d i f f e r e n t a t P^0.05. ns Means are i n s i g n i f i c a n t . (see T a b l e s A3, B3, C3 and D3). 32 T a b l e 14. Mean v a l u e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm. Experiments 1, 2, 3 and 4. I r r a d i a t i o n / Dose (Mrad)* Experiment 1 2 3 4 0.0 0.5 0.4258^ 0.3042^ 0.6251A 0.4121 ^  1.289< 1.1707^ 1.0731 0.8502^ 1.0 2.0 0.2568S 0.1857? 0.3373r 0.2646^ 1.0655^  0.9628^ 0.7336° 0.6199 I r r a d i a t i o n Time 1 2 3 4 B e f o r e F r e e z i n g 0.2128 0.2921 1.0367 0.7045 A f t e r F r e e z i n g 0.2849*=*= 0.3839** 1.0959** 0.7647 Storage Time 1 2 3 4 10 days 0.2582 0.4123 1.1120 0.7727 50 days 0.2901** 0.3458* 1.0844 0.7932ns Temperature -10 F° -35 F° ' 1 2 3 4 0.2689 0.3656 1.1006 0.7492 0.2794*s 0.3925ns 1.0958**3 0.8167 Atmosphere A i r N i t r o g e n 1 2 3 4 0.2782 0.3908 1.1169 0.8173 0.2702*s 0.3672** 1.0795** 0.7486 / Duncan new m u l t i p l e range t e s t f o r dose mean v a l u e s ; any two means not s h a r i n g the same l e t t e r a re s i g n i f i c a n t l y d i f f e r e n t a t P<0.01. ** Means are s i g n i f i c a n t l y d i f f e r e n t a t PC0.01. * Means are s i g n i f i c a n t l y d i f f e r e n t a t P^rO.05. ns , Means are i n s i g n i f i c a n t . (see T a b l e s A4, B4, 04 and D4). 33 Table 1 5 . Mean v a l u e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 4 6 0 nm. Experiments 1 , 2 , 3 and 4 . I r r a d i a t i on / Dose (Mr-ad)* Experiment 0 . 0 1 2 3 0 . 3 9 9 6 -0 . 5 8 2 5 ? 1.25637 1 . 0 ? 2 4 A 0 . 5 0 . 2 8 5 7 ? 0 . 3 8 6 4 g 1 . 1 4 1 2 * 0 . 8 6 1 7 1 . 0 0.2453S 0.3172X, 1 . 0 3 9 6 ° 0 . 7 4 7 3 2 . 0 0 . 1 7 4 9 ? 0 . 2 5 0 8 ^ 0 . 9 4 8 1 ^ 0 . 6 5 6 7 I r r a d i a t i o n Time 1 2 3 4 B e f o r e F r e e z i n g 0 . 2 0 1 2 0 . 2 7 1 8 1 . 0 1 8 2 0 . 7 2 1 4 A f t e r F r e e z i n g 0.2702*=* 0 . 3 6 4 5 * * 1 . 0 6 7 7 * * 0 . 7 7 5 7 Storage Time 1 2 3 4 1 0 days 0 . 2 4 6 2 0 . 3 8 6 7 1 . 0 8 5 0 0 . 7 8 4 7 30 days 0 . 2 7 2 0 * * 1 . 0 6 1 9 ^ 0 . 8 0 4 9 Temperature - 1 0 F° - 3 5 F° 1 2 3 4 0 . 2 5 6 5 0 . 3 3 7 8 1 . 0 7 7 0 0 . 7 6 0 6 0 . 2 6 1 7 * 1 0 . 3 7 4 0 1 . 0 6 9 9 * ? 0 . 8 2 9 0 Atmosphere A i r N i t r o g e n 1 2 3 4 0 . 2 6 2 6 0 . 3 6 7 4 1 . 0 8 9 3 0 . 8 2 8 3 0 . 2 5 5 6 * 1 3 0 . 3 4 4 4 * 1 . 0 5 7 6 * * 0 . 7 6 1 3 Duncan new m u l t i p l e range t e s t f o r dose mean v a l u e s ; any two means not s h a r i n g the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t a t P.<0 . 0 1 . Means a r e s i g n i f i c a n t l y d i f f e r e n t a t P - < 0 . 0 1 . Means are s i g n i f i c a n t l y d i f f e r e n t a t P ^ 0 . 0 5 . Means are i n s i g n i f i c a n t , (see T a b l e s A5, B 5 , 05 and D 5 ) . * * * ns 34 Table 16 . Dominant Wavelength (DWL) of the mean values for factors and level within experiments. Factor Level 1 Experiment 2 3 4 Fresh 577 577 589 0.0 573 577 534 589 Dose (Mrad) 0.5 1.0 577 577 577 577 534 584 589 589 2.0 577 577 534 590 I r r a d i a t i o n Time Before F r e e z i n g • A f t e r F r e e z i n g 577 577 577 577 584 334 590 539 Stora-e Time 10 days 30 days 577 577 577 577 585 584 590 y •• Temperature -10 F° -35 F° 577 577 577 577 535 584 590 589 Atmosphere A i r N i t r o g e n 577 577 577 577 584 584 58 Q 589 35 T a b l e 1 7 . E x c i t a t i o n P u r i t y (EP) of the mean v a l u e s f o r f a c t o r s and l e v e l s w i t h i n experiments* Experiment F a c t o r L e v e l 1 2 3 4 Fresh 82.03 85.76 82.30 32.72 0.0 82.04 85.41 36.30 78.77 Dose 0.5 78.09 7C91 33.94 74.19 (Mrad) 1.0 74.32 75.05 81.93 70.33 2.0 66.60 66.42 79.04 64.71 I r r a d i a t i o n Before F r e e z i n g 71.23 71.48 80.82 68.33 Time A f t e r F r e e z i n g 74.75 76.77 82.47 70.89 Storage 10 days 74.42 75.39 82.63 72.23 Time 30 days 74.07 75.94 81.37 69.44 -10 F° 79.08 78.01 83.85 72.39 Temperature -35 P 69.48 73.35 80.11 69.30 A i r ?4.04 75.63 82.25 71.17 Atmosphere Ni t r o g e n 74.47 75.72 82.33 71.34 37 0 0.5 2.0 D O S E F i g . 5. R e l a t i o n s h i p s dose l e v e l s . — i 1 j 1.0 2.0 1.0 0.5 0 CmradD between mean x, y, #Y and Experiment 2. 38 P i g . 6 . R e l a t i o n s h i p s between mean x, y, %1 and dose l e v e l s . Experiment 3 . 0.5 0.4 X 0,3 mm, mmm *• . 0 0.5 1.0 2.0 2.0 1.0 . 0 ^ D O S E ( m r a d ) 50 40 o\o 30 0 F i g . 7. R e l a t i o n s h i p s between mean x, y, %Y and dose l e v e l s . Experiment 4. 40 0 0.5 1.0. 2.0 DOSE(mrad) 8. R e l a t i o n s h i p s between mean b e t a - c a r o t e n e e q u i v a l e n t and dose l e v e l s ; A&B= b e t a - c a r o t e n e e q u i v a l e n t a t 4-50 and 460 nm r e s p e c t i v e l y . Experiment 1; C&D= b e t a -c a r o t e n e e q u i v a l e n t a t 4 5 0 and 460 nm r e s p e c t i v e l y . Experiment 2. 41 DOSE (mrad) 9 . R e l a t i o n s h i p s between mean b e t a - c a r o t e n e e q u i v a l e n t and dose l e v e l s ; A&B= b e t a - c a r o t e n e e q u i v a l e n t a t 450 and 460 nm r e s p e c t i v e l y . Experiment 3; C&D= b e t a -c a r o t e n e e q u i v a l e n t a t 4 5 0 and 460 nm r e s p e c t i v e l y . Experiment 4. 42 DISCUSSION Both i r r a d i a t i o n dose and time of i r r a d i a t i o n had marked e f f e c t s on c h r o m a t i c i t y c o o r d i n a t e s , l i g h t n e s s and BCE v a l u e s . Changes i n the c h r o m a t i c i t y c o o r d i n a t e s (x and y) i n d i c a t e d a decrease i n e x c i t a t i o n p u r i t y ( d e c l i n e i n b o t h x and y) but l i t t l e change i n dominant wavelength (DWL) i n a l l experiments. The r e s u l t s o f Experiments 3 and 4 c l e a r l y i n d i c a t e t h a t i n c r e a s i n g i r r a d i a t i o n dose and i r r a d i a t i o n b e f o r e f r e e z i n g r e s u l t s i n an i n c r e a s e i n darkness of samples. In these two experiments b e t a - c a r o t e n e and c a n t h a x a n t h i n r e s p e c t i v e l y were added t o the y o l k as c o l o r a n t s . The l a c k of a s i g n i f i c a n t e f f e c t of i r r a d i a t i o n dose and time on darkness i n the f i r s t two experiments suggests t h a t e i t h e r the pigments added i n Experiments 3 and 4 are more r a d i o - s e n s i t i v e p e r se than the n a t u r a l c o l o r a n t o f y o l k or t h a t the n a t u r a l c o l o r a n t s are made more r a d i o r e s i s t a n t by i n t e r a c t i o n w i t h components of the m i l i e u a t l e a s t t o m o d i f i c a t i o n s r e s u l t i n g i n changes i n l i g h t n e s s ($Y). The marked and s i g n i f i c a n t d e crease i n BCE v a l u e s w i t h . i n c r e a s i n g r a d i a t i o n dose and the lower BCE v a l u e s f o r samples i r r a d i a t e d b e f o r e f r e e z i n g i n a l l experiments c l e a r l y i m p l i c a t e changes i n the pigments themselves, or i n t h e i r e x t r a c t a b i l i t y by acetone i n the c o l o r changes e v i d e n t by r e f l e c t a n c e measurements. 43 The e f f e c t s o f i r r a d i a t i o n dose and time are c o n s i s t e n t w i t h the h y p o t h e s i s t h a t f r e e r a d i c a l s and p e r o x i d e s are induced by r a d i a t i o n i n p r o p o r t i o n t o dose l e v e l and r e a c t w i t h the c a r o t e n o i d s t o an e x t e n t dependent upon p h y s i c a l s t a t e ( f r o z e n or u n f r o z e n ) d u r i n g i r r a d i a t i o n . The r e s u l t s i n d i c a t e t h a t low doses o f r a d i a t i o n and i r r a d i a t i o n i n the f r o z e n s t a t e minimize the c o l o r and pigment changes i n d u c e d . The e f f e c t o f temperature on c h r o m a t i c i t y c o o r d i n a t e s was c o n s i s t e n t f o r a l l experiments. Samples f r o z e n and h e l d a t -10 E ° had c o n s i s t e n t l y h i g h e r mean (x) and ( y ) v a l u e s than t h o s e a t -35 F ° . Only the n a t u r a l l y - p i g m e n t e d samples (Experiments 1 and 2) showed a c o r r e s p o n d i n g d a r k e n i n g a t -10 F° compared t o -35 P ° . However, h i g h l y s i g n i f i c a n t i n t e r a c t i o n s • b e t w e e n s t o r a g e temperature and i r r a d i a t i o n treatment occured i n both Experiments 3 and 4 f o r l i g h t n e s s and may account f o r the l a c k o f a s i g n i f i c a n t temperature e f f e c t . The i n t e r a c t i o n s i n d i c a t e d t h a t darkness i n c r e a s e d more r a p i d l y w i t h i r r a d i a t i o n dose a t -10 F° than a t -35 E ° . S i m i l a r l y , the presence o f i n t e r a c t i o n s i n v o l v i n g temperature i n the Experiment 3 may account f o r the l a c k o f a s i g n i f i c a n t temperature e f f e c t on BCE i n t h i s experiment. The e f f e c t o f s t o r a g e time on the mean c h r o m a t i c i t y v a l u e s was c o n s i s t e n t i n a l l experiments but the e f f e c t s on l i g h t n e s s and BCE were not. However, mean l i g h t n e s s and BCE v a l u e s were p o s i t i v e l y r e l a t e d . S i m i l a r s i g n i f i c a n t c o r r e l a t i o n s 44 from a n a l y s i s of i n d i v i d u a l sample d a t a i n d i c a t e t h a t pigment d e s t r u c t i o n o r m o d i f i c a t i o n (lower BCE v a l u e ) can be expected to produce a decrease i n l i g h t n e s s of samples r e g a r d l e s s o f pigment s o u r c e a l t h o u g h the r e l a t i o n s h i p i s f a r from p e r f e c t . 4 5 SUMMARY AND CONCLUSIONS Egg y o l k c o l o r was s t u d i e d i n r e l a t i o n t o r a d i a t i o n dose, r a d i a t i o n time, f r e e z i n g p r o c edure, s t o r a g e time and headspace atmosphere. 1. I r r a d i a t i o n dose had a h i g h l y s i g n i f i c a n t e f f e c t on y o l k c o l o r . S i g n i f i c a n t d i f f e r e n c e s were found i n r e f l e c t a n c e and absorbance measurements among doses i n a l l experiments. 2. Samples i r r a d i a t e d a f t e r f r e e z i n g had h i g h e r mean c h r o m a t i c i t y v a l u e s , BCE v a l u e s and e x c i t a t i o n p u r i t y t han samples i r r a d i a t e d b e f o r e f r e e z i n g i n a l l experiments. 3. A f t e r 50 days s t o r a g e mean x - v a l u e s were lower and mean y - v a l u e s were h i g h e r than a f t e r 10 days s t o r a g e . These changes w e r e ' a s s o c i a t e d w i t h almost no change i n DWL or EP. 4. Samples s t o r e d a t -10 P° had h i g h e r mean c h r o m a t i c i t y v a l u e s and lower e x c i t a t i o n p u r i t y i n a l l experiments. N a t u r a l l y - p i g m e n t e d samples showed a c o r r e s p o n d i n g d a r k e n i n g (lower %Y) a t -10 F° compared t o -55 P ° . 5. With o n l y one e x c e p t i o n , atmosphere had no s i g n i f i c a n t e f f e c t on mean x, y and %T. Samples under a i r atmosphere had c o n s i s t e n t l y h i g h e r mean BCE v a l u e s t h a n those under n i t r o g e n atmosphere. These d i f f e r e n c e s were s i g n i f i c a n t i n Experiments 2, 5 and 4. 6. Dominant wavelength was not a f f e c t e d by the v a r y i n g treatments w i t h i n experiments. Mean DV/L v a l u e s were 577-578 and 589-590 nm f o r n a t u r a l l y and a r t i f i c i a l l y pigmented 46 samples r e s p e c t i v e l y . 7 . C o r r e l a t i o n a n a l y s e s r e v e a l e d h i g h l y s i g n i f i c a n t ( P ^ O . 0 1 ) l i n e a r r e l a t i o n s h i p s between BCE and b o t h c h r o m a t i c i t y c o o r d i n a t e s and l i g h t n e s s r a n g i n g from + 0 . 0 9 t o + 0 . 7 9 . 8. I t i s concluded t h a t i r r a d i a t i o n dose and p h y s i c a l s t a t e of the product have important e f f e c t s on the acetone s o l u b l e pigments.and c o l o r of n a t u r a l l y o r a r t i f i c i a l l y pigmented egg y o l k such t h a t the e f f e c t i n c r e a s e s w i t h dose and i s g r e a t e r f o r i r r a d i a t i o n of l i q u i d compared to f r o z e n y o l k . 9 . I t i s f u r t h e r concluded t h a t a l t h o u g h s t a t i s t i c a l s i g n i f i c a n c e ( P < 0 . 0 1 ) o f the r e l a t i o n s h i p s between BCE and each of the c h r o m a t i c i t y c o o r d i n a t e s and l i g h t n e s s was demonstrated the magnitude o f the c o r r e l a t i o n c o e f f i c i e n t s i s not g r e a t enough t o permit a c c u r a t e p r e d i c t i o n . 47 LIST OP REFERENCES Ashton, E.H., and Fletcher, D.A. 1962, Development and use of color standards for egg yolks. Poultry S c i . 41, 1903. A.O.A.C. 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Poultry Sci. 48, 3. Zimmerman, W.I,, Tressler, D.K., and Maynard, L.A. 1941. Determination of carotene i n fresh and frozen vegetables by an improved method. I I . carotene content of asparagus and green lima beans. Food Res. 6 ; 57. 53 APPENDIX A 54 T a b l e A1. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . Experiment 1. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 O.15434 X 10" 2 <5 x 10~ 7 R a d i a t i o n Treatment 6 0.38676 X 10 ^  <5 x 10~ 7 C o n t r o l vs Treatment 1 0.88015 X 10~ 2 <5 x 10" 7 Dose 2 0.63822 X 10 eL <5 x 1Q~7 I r r a d i a t i o n Time 1 0.12615 X 10~ 2 <5 x i c f7 Dose x I r r a d i a t i o n Time 2 0.18884 X 10""5 0.0002 Storage 1 0.67580 X 10""4 0.0389 Temperature 1 0.14018 X 10 ' <5 x 10~ 7 Atmosphere 1 0.10804 X 10~ 5 0.7773 R a d i a t i o n Treatment x S t o . 6 0.17893 X -4 10 ^  0.3240 R a d i a t i o n Treatment x Temp. 6 0.50792 X 10""4 0.0120 R a d i a t i o n Treatment x Atmo. 6 0.23512 X 10~ 5 0.9831 Storage x Temperature 1 0.97232 X 10~ 5 0.4262 Storage x Atmosphere 1 0.21438 X 10"^ 0.2343 Temperature x Atmosphere 1 0.94723 X -4 10 0.0165 E r r o r 25 0.14535 X 10~4 0.0016 E r r o r between d u p l i c a t e s 57 0.56404 X 10~ 5 T o t a l 115 55 T a b l e A2. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 1. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.50978 X 10~ 5 <5 x 10" •5 R a d i a t i o n Treatment 5 0.10526 X 10 * <5 x 10" •7 C o n t r o l v s Treatment 1 0.10625 X 10"*2 <5 x 10" •7 Dose 2 0.21128 X -2 10 * <5 x 10" •7 I r r a d i a t i o n Time 1 0.84609 X 10~ 5 <5 x 10" -5 Dose x I r r a d i a t i o n Time 2 0.90594 X 10~ 4 0.0050 Storage 1 0.32143 X 10~ 6 0.8522 Temperature 1 0.53213 X 10 * <5 x 10" -7 Atmosphere 1 0.82286 X -4 10 0.0205 R a d i a t i o n Treatment x S t o . 6 0.23839 X 10" 5 0.9799 R a d i a t i o n Treatment x Temp. 6 0.35265 X 10""4 0.0437 R a d i a t i o n Treatment x Atmo. 6 0.10348 X 10~ 4 0.6114 Storage x Temperature 1 0.58893 X 10~ 4 0.1004 Storage x Atmosphere 1 0.15750 X 10" 4 0.2937 Temperature x Atmosphere 1 0.91429 X 10~ 5 0.4262 E r r o r 25 0.15669 X 10" 4 0.0002 E r r o r between d u p l i c a t e s 57 0.45158 X 10~ 5 T o t a l 113 56 T a b l e A3. A n a l y s i s o f v a r i a n c e f o r (%Y). Experiment 1• Source DP Mean Square P r o b a b i l i t y F r e s h vs A l l 1 153.69 0.0006 R a d i a t i o n Treatment 5 14.15 0.2267 C o n t r o l vs Treatment 1 34-. 83 0.0653 Dose 2 4.22 , 0.6544-I r r a d i a t i o n Time 1 21.71 0.1415 Dose x I r r a d i a t i o n Time 2 9.97 0.3702 Storage 1 73.55 0.0101 Temperature 1 2491.2 <5 x 10' Atmosphere 1 0.79 0.7678 R a d i a t i o n Treatment x S t o . 6 4.52 0.8240 R a d i a t i o n Treatment x Temp. 6 3.97 0.8655 R a d i a t i o n Treatment x Atmo. 6 2.32 0.9572 Storage x Temperature 1 6.22 0.4535 Storage x Atmosphere 1 36.89 0.0584 Temperature x Atmosphere 1 192.15 0.0002 E r r o r 25 9.59 < 5 x 10' E r r o r between d u p l i c a t e s 57 0.90 T o t a l 113 ,-7 -7 57 T a b l e A4. A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 4-50 nm. Experiment 1. Source DP Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.10655 < 5 x 1 0 7 R a d i a t i o n Treatment g 0 . 1 3 3 0 3 < 5 x 10"' C o n t r o l ys Treatment 1 0.42930 < 5 x 10"' Dose 2 0 . 1 1 3 7 9 < 5 x 10"' I r r a d i a t i o n Time 1 0.12478 < 5 x 10" Dose x I r r a d i a t i o n Time 2 0.82726 X 1 0 " 2 0 . 0 0 7 9 Storage 1 0.28608 X 10" 1 0.0002 Temperature 1 0.31291 X 1 0 " 2 0.1438 Atmosphere 1 0 . 1 7 9 2 0 X 10 * 0.2681 R a d i a t i o n Treatment x S t o . 6 0 . 2 9 9 9 3 X 1 0 ~ 2 0 . 0 8 3 3 R a d i a t i o n Treatment x Temp. 6 0 . 1 3 7 5 1 X 1 0 " 2 0 . 4 5 8 5 R a d i a t i o n Treatment x Atmo. 6 0.53491 X 1 0 ~ 2 0 . 0 0 7 8 Storage x Temperature 1 0.68014 X 1 0 ~ 5 0 . 4 9 8 8 Storage x Atmosphere 1 0 . 1 5 5 5 7 X 1 0 ~ 3 0 . 7 3 7 7 Temperature x Atmosphere 1 0.27032 X 1 0 " 5 0 . 6 6 7 0 E r r o r 2 5 0.13998 X 1 0 ~ 2 0.0582 E r r o r between d u p l i c a t e 57 0.84382 X 1 0 " 5 T o t a l , 113 58 T a b l e A 5 . A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm. Experiment 1 . Source DF Mean Square P r o b a b i l i t y Fresh vs A l l 1 0.85756 x 1 0 " 1 <5 x 10" •5 Radiation Treatment 6 0 . 1 1 7 1 9 <5 x 10" •7 Control vs Treatment 1 0.36867 <5 x 10" •7 Dose 2 0 . 1 0 2 2 5 <5 x 10" -7 Irradiation Time 1 0.11426 <5 x 10" •7 Dose x Irradiation Time 2 0.78665 X 10 * 0 . 0 1 0 5 Storage 1 0.18721 X 10 ' 0.0014 Temperature 1 0 . 7 5 0 8 9 X 10~ 5 0.4821 Atmosphere 1 0 . 1 3 5 8 0 X 10" 2 0.3416 Radiation Treatment x Sto. 6 0 . 2 5 5 2 2 X 10~ 2 0.1431 Radiation Treatment x Temp. 6 0.12832 X 10~ 2 0 .5139 Radiation Treatment x Atmo. 6 0.45692 X 10 ^  0.0183 Storage x Temperature 1 0.24891 X 10~ 2 0.1966 Storage x Atmosphere 1 0 . 1 5 7 2 9 X 10~ 5 0 . 7532 Temperature x Atmosphere 1 0.28289 X 10~ 5 0.6637 Error 25 0.14318 X 10~ 2 0 .0155 Error between duplicates 5? 0.714-09 X 10" 5 Total 113 5° T a b l e AS. C o r r e l a t i o n m a t r i x and mean v a l u e . Experiment 1» V a r i a b l e X y • % Y 450nm 460nm Mean X 1.0000 0.4425 y * * 0.9434 1.0000 0.4612 Y -O.5047 -0.5394 1.0000 3587.0 450 nm * * 0.6574 * * 0.5644 0.1265 1.0000 0.2782 460 nm ** 0.6764 * * 0.5833 0.0906 * * 0.9888 1.0000 0.2623 C o r r e l a t i o n c o e f f i c i e n t s a re s i g n i f i c a n t l y d i f f e r e n t a t P<0.01. APPENDIX B 61 T a b l e B1. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . Experiment 2. Source DE Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.13622 X -P 10 < 5 x 10" •5 R a d i a t i o n Treatment 6 0 . 7 5 9 0 3 X 1 0 " 2 < 5 x 10" •7 C o n t r o l vs Treatment 1 0.13816 X 1 0 " 1 <C5 x 10" •7 Dose 2 0 . 9 8 3 0 0 X 10 * < 5 x 10" -7 I r r a d i a t i o n Time 1 0 . 6 5 8 3 6 X 10"" 2 < 5 x 10" •7 Dose x I r r a d i a t i o n Time 2 0.274-13 X 10 * < 5 x 10" -7 Storage 1 0 . 5 0 2 2 3 X 0.1182 Temperature 1 0 . 3 7 6 0 7 X 10 * < 5 x 10" •7 Atmosphere 1 0 . 7 2 3 2 1 X 1 0 ~ 6 0.8284 R a d i a t i o n Treatment x S t o . 6 0 . 1 0 5 8 9 X 1 0 ~ 5 0 . 0 0 1 1 R a d i a t i o n Treatment x Temp. 6 0 . 9 0 9 7 3 X 1 0 ~ 4 0 . 0 0 2 7 R a d i a t i o n Treatment x Atmo. 6 0 . 2 9 3 0 7 X 1 0 " 4 0 . 2 1 9 6 Storage x Temperature 1 0 . 7 2 3 2 1 X 1 0 " 6 0.8284 Storage x Atmosphere 1 0 . 2 5 0 8 0 X 1 0 " 4 0.2681 Temperature x Atmosphere 1 0 . 1 5 0 0 9 X 10"" 4 0 . 3 9 3 8 E r r o r 25 0 . 1 9 5 8 7 X 10"* 4 < 5 x 1 0 ' •7 E r r o r between d u p l i c a t e s 57 0 .15351 X 1 0 " 5 T o t a l 115 62 T a b l e B2. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 2. Source DE Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.26963 X 10~5 <5 x 10" •5 R a d i a t i o n Treatment g 0.15995 X 10 * <5 x 10" -7 C o n t r o l vs Treatment 1 0.16942 X I D " 2 <5 x 10" •7 Dose 2 0.26472 X 10 <15 x 10" •7 I r r a d i a t i o n Time 1 0.4-1251 X 10~5 <5 x 10" •5 Dose x I r r a d i a t i o n Time 2 0.10980 X 10"2 <5 x 10" •7 S t o r a g e 1 0.26414- X 10~5 <C5 x 10" •5 Temperature 1 0.1044-3 X 10~2 <5 x 10" •7 Atmosphere 1 0.35714 X 10~5 0.4-991 R a d i a t i o n Treatment x S t o . 6 0.29018 X 10"* 0.0066 R a d i a t i o n Treatment x Temp. 6 0.65655 X 10~4 <5 x 10" •5 R a d i a t i o n Treatment x Atmo. 6 0.10363 X 10~4 0.2503 Storage x Temperature 1 0.60357 X 10~5 0.3773 S t o r a g e x Atmosphere 1 0.91429 X 10~5 0.2754 Temperature x Atmosphere 1 0.10321 X -4-10 ^  0.24-62 E r r o r 25 0.73600 X 10~5 <5 x 10" •7 E r r o r between d u p l i c a t e s 57 0.80702 X 10"6 T o t a l 113 63 T a b l e B 3 . A n a l y s i s o f v a r i a n c e f o r (%Y). Experiment 2. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 3 3 8 . 2 9 0 . 0 1 1 1 R a d i a t i o n Treatment 6 1 1 1 . 6 8 0 . 0 5 2 1 C o n t r o l vs Treatment 1 301.18 0 . 0 1 5 7 Dose 2 94.4-7 0.1442 I r r a d i a t i o n Time 1 4-5.73 0 . 3 2 6 9 Dose x I r r a d i a t i o n Time 2 6 7 . 1 0 0.2466 Storage 1 461 . 3 9 0 . 0 0 3 9 Temperature 1 3 5 2 . 1 1 0.0098 Atmosphere 1 1 1 . 9 3 0.6182 R a d i a t i o n Treatment x S t o . 6 3 2 . 9 5 0.634-7 R a d i a t i o n Treatment x Temp. 6 13.16 0 . 9 3 5 5 R a d i a t i o n Treatment x Atmo, 6 18.95 0.8610 Storage x Temperature ^ 1 101 .23 0.1444 Storage x Atmosphere 1 4.06 0 . 7 6 0 3 Temperatur x Atmosphere 1 1 . 3 9 0 . 8 3 9 0 E r r o r 25 45.42 < 5 x 10' E r r o r between d u p l i c a t e s 57 0 . 4 9 7 0 T o t a l 113 -7 64 Table B4. Analysis of variance for beta-carotene equivalent at 4-50 nm. Experiment 2. Source DF Mean Square Probability Fresh vs A l l 1 0.32184 X 10~ 1 0.0048 Radiation Treatment s 0.28803 <5 x 10" •7 Control vs Treatment 1 0.11306 X 10~ 1 <5 x 10" •7 Dose 2 0 . 17391 < 5 x 10" •7 Irradiation Time 1 0.20240 O x 10" •5 Dose x Irradiation Time 2 0.23670 X IO" 1 0.0038 Storage 1 0.12382 < 5 x 10" -5 Temperature 1 0.20304 X 10~1 0 . 0 2 0 3 Atmosphere 1 0.15604 X 10" 1 0.0391 Radiation Treatment x Sto. 6 0.54923 X i o "2 0.1790 Radiation Treatment x Temp. 6 0.55904 X 10~ 2 0.4081 Radiation Treatment x Atmo. 6 0.21043 X 10~ 2 0.7096 Storage x Temperature 1 0.87509 X 10~ 2 0 . 1155 Storage x Atmosphere 1 0.92893 X 10""2 0 . 1053 Temperature x Atmosphere 1 0.15156 X -2 10 0.5149 Error 25 0.53626 X 1 0 ~ 2 0.0026 Error between duplicates 57 0.13674 X 10~ 2 Total 115 65 Table B 5 . Analysis of variance for beta-carotene equivalent at 460 nm. Experiment 2. Source DF Mean Square Probability Fresh vs A l l 1 0.24687 X 10~ 1 0 .0115 Radiation Treatment g 0.24953 <5 x 10" -7 Control vs Treatment 1 0.95840 <5 x 10" •7 Dose 2 0.14697 <5 x 10" •7 Irradiation Time 1 0.20628 <5 x 10" -5 Dose x Irradiation Time 2 0.19295 X 1 0 " 1 0.0088 Storage 1 0.10640 <5 x 10" •5 Temperature 1 0.36577 X 10~ 1 0.0029 Atmosphere 1 0 . 1 4 7 6 6 X 10" 1 0.0439 Radiation Treatment x Sto. 6 0.48347 X 10~ 2 0.2380 Radiation Treatment x Temp. 6 0 .25612 X 10 eL 0.6064 Radiation Treatment x Atmo. 6 0 . 1 9 3 8 6 X -2 10 * 0.7454 Storage x Temperature 1 0.66343 X 10" 2 0.1685 Storage x Atmosphere 1 0.74263 X 10~ 2 0.1456 Temperature x Atmosphere 1 0.64129 X 10~ 5 0.6684 Error 25 0.33524 X -2 10 0 . 0 0 2 7 Error betv/een duplicate 57 0.13680 X -2 10 Total 113 66 Table B5. Correlation matrix and mean value. Experiment 2. V a r i a b l e y %Y 450nm 460nm Mean X 1.0000 0.4431 7 * * 0.9291 1.0000 0.4655 °/oY * * 0.4130 * * 0.2238 1.0000 31.21 450 nm * * 0.7654 * * 0.5895 * * 0.5304 1.0000 0.5S13 450 nm * * 0.7459 0.5650 * * 0.54-49 ** •• 0 . 9 9 5 9 1.0000 0.3579 C o r r e l a t i o n c o e f f i c i e n t s a r e s i g n i f i c a n t l y d i f f e r e n t a t P<0.01. 67 A P P E N D I X C 68 Table C1. Analysis of variance for chromaticity coordinate (x). Experiment 3« Source DF Mean Square Probability Fresh vs A l l 1 0.89290 X 10"* 0.0010 Radiation Treatment 6 0.76426 X 10" 5 <5 x 10" -7 Control vs Treatment 1 0.16878 X 10 ^  <5 x 10" -7 Dose 2 0.11825 X -2 10 <5 x 10" -7 Irradiation Time 1 0.31901 X 10" 5 <5 x 10' -5 Dose x Irradiation Time 2 0.10689 X 10~ 5 <5 x 10' -5 Storage 1 0.86358 X io~ 5 <5 x 10' -7 Temperature 1 0.40200 X _2 10 * <5 x 10" -7 Atmosphere 1 0.47232 X 10"5 0.4002 Radiation Treatment x Sto. 6 0.32054 X 10" 5 0.7988 Radiation Treatment x Temp. 6 0.23051 X 10"* 0.0099 Radiation Treatment x Atmo. 6 0.28482 X 10~ 5 0.8389 Storage x Temperature 1 0.26722 X 10" 5 < 5-X 10" -5 Storage x Atmosphere 1 0.20089 X 10~ 5 0.5847 Temperature x Atmosphere 1 0.89286 X 10" 8 0.9213 Error 25 0.63304 X 10"5 < 5 x 10' -5 Error between duplicate 57 0.13596 X 10" 5 Total 113 69 T a b l e C2. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 3. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.15664 x 1 0 ~ 5 < 5 x 10" •5 R a d i a t i o n Treatment 6 0.19734 X 1 0 ~ 5 < 5 x 10" •7 C o n t r o l vs Treatment 1 0.50060 X 1 0 ~ 5 < 5 x 10" •7 Dose 2 0 .25517 X 1 0 ~ 5 < 5 x 10" •7 I r r a d i a t i o n Time 1 0.14504 X 1 0 ~ 5 < 5 x 10" •5 Dose x I r r a d i a t i o n Time 2 0.14042 X 10"~4 0 .0095 Storage 1 0 .23223 X 10""4 0 .0053 Temperature 1 0.26722 X 1 0 ~ 5 <C3 x 10" •7 Atmosphere 1 0.22321 X 1 0 ~ 6 0.7599 R a d i a t i o n Treatment x S t o . 6 0.51815 X 10""5 0.0913 R a d i a t i o n Treatment.x Temp. 6 0.78065 X 1 0 ~ 5 0 .0197 R a d i a t i o n Treatment x Atmo. 6 0.12649 X 1 0 ~ 5 0 .7975 Storage x Temperatur : 1 0.21457 X 1 0 " 4 0.0069 Storage x Atmosphere 0.15089 X 1 0 ~ 5 0.4492 Temperature x Atmosphere 1 0.80557 X 1 0 ~ 7 0.8361 E r r o r 25 0.24889 X 1 0 ~ 5 0.0016 E r r o r between d u p l i c a t e s 57 0.96491 X 1 0 - 5 T o t a l 113 70 Table C3. Analysis of variance for (#Y). Experiment 3» Source DF Mean Square Probability Fresh vs A l l 1 404.36 <5 x 10" •7 Radiation Treatment 5 118.11 <5 x 10" -7 Control vs Treatment 1 432.82 < 5 x 10" -7 Dose 2 113.93 < 5 x 10" -7 Irradiation Time 1 31.93 0.0018 Dose x Irradiation Time 2 8.03 0.0606 Storage 1 7.37 0.0995 Temperature 1 5.41 0.1562 Atmosphere 1 0.78 0.5930 Radiation Treatment x Sto. 6 19.41 0.0001 Radiation Treatment x Temp. 6 17.36 0.0003 Radiation Treatment x Atmo. 6 0.47 0.9774 Storage x Temperature 1 21.47 0.0077 Storage x Atmosphere 1 0.20 0.7742 Temperature x Atmosphere 1 0.18 0.7832 Error 25 2.58 <5 x 10" •7 Error between duplicates 57 0.36 Total 113 71 T a b l e C4. A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 450 nm. Experiment 3 . Source DP Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.25367 < 5 x 10" -5 R a d i a t i o n Treatment 6 0.24488 < 5 x 10" -7 C o n t r o l vs Treatment 1 0.68270 *C 5 x 10" -7 Dose 2 0 . 3 4 5 5 1 < 5 x 10" •7 I r r a d i a t i o n Time 1 0.83958 X 10" -1 0.0002 Dose x I r r a d i a t i o n Time 2 0.58167 X 10" -2 0.2629 Storage 1 0.21285 X 10" -1 0.0306 Temperature 1 0.62229 X 10" -3 0.7014 Atmosphere 1 0 . 3 9 0 0 1 X 10" -1 0 . 0 0 5 0 R a d i a t i o n Treatment x S t o . 6 0.13629 X 10" -1 0 . 0 1 5 7 R a d i a t i o n Treatment x Temp. 6 0.12288 X 10" -1 0.0248 R a d i a t i o n Treatment x Atmo. 6 0 . 3 1 1 9 4 X 10" -1 0.0001 Storage x Temperature 1 0.26846 X 10" -1 0.0166 Storage x Atmosphere 1 0.19223 X 10" -2 0.5082 Temperature x Atmosphere 1 0.17286 X 10" -4 0.9050 E r r o r 2 5 0.41324 X 10" -2 0.2112 E r r o r between d u p l i c a t e 57 0 . 3 2 0 3 3 X 10" -2 T o t a l 113 72 T a b l e C5. A n a l y s i s o f v a r i a n c e f o r b e t a - c a r o t e n e e q u i v a l e n t a t 460 nm. Experiment 5. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0 . 2 1 9 2 7 < 5 x 10" •5 R a d i a t i o n Treatment 6 0.21484 < 5 x 10" •7 C o n t r o l vs Treatment 1 0.62421 < 5 x 10" •7 Dose 2 0.29856 < 5 x 10" •7 I r r a d i a t i o n Time 1 0.58658 X 10' -1 0.0014 Dose x I r r a d i a t i o n Time 2 0.45271 X 10' -2 0.5814 Storage 1 0.14858 X 10' -1 0.0778 Temperature 1 0.14429 X 10' -2 0.5826 Atmosphere 1 0.28289 X 10" -1 0.0181 R a d i a t i o n Treatment x S t o . 6' 0.14006 X 10' -1 0.0203 R a d i a t i o n Treatment x Temp. 6 0.12906 X 10' -1 0.0287 R a d i a t i o n Treatment x Atmo. 6 0.26599 X 10' -1 0.0006 Storage x Temperature 1 0.50956 X 10' -1 0.0140 Storage x Atmosphere 1 0.17286 X 10' -2 0 . W 7 Temperature x Atmosphere 1 0.91429 X 10' -5 0.9167 E r r o r 25 0.44952 X 10' -2 0.1463 E r r o r between d u p l i c a t e 57 0.32083 X 10' -2 T o t a l 113 73 Table C6. C o r r e l a t i o n matrix and mean value. Experiment 3. Variable X -tr fol 450nm 460nm Mean X 1.0000 0.4996 7 * * 0.8760 1.0000 0.4325 fol 0.4420 * * 0.5628 1.0000 28.47 450 nm * * 0.5502 0.6836 0.6867 1.0000 1.104 460 nm ** 0.5547 0.6812 * * 0 . 6 7 9 7 ** 0.9938 1.0000 1.079 Cor r e l a t i o n c o e f f i c i e n t s are s i g n i f i c a n t l y d i f f e r e n t at P^O.01. 74-APPENDIX D 75 Table D1. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( x ) . Experiment 4. Source DF Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0.15484 X 1 0 ~ 2 < 5 x 10" •5 R a d i a t i o n Treatment g 0.23442 X 1 0 ~ 2 <5 x 10" •7 C o n t r o l vs Treatment 1 0.60121 X i o '2 < 5 x 10" •7 Dose 2 0.33538 X 1 0 " 2 < 5 x 10" •7 I r r a d i a t i o n Time 1 0.37604 X 1 0 ~ 5 0 . 0 0 0 7 Dose x I r r a d i a t i o n Time 2 0.48470 X 1 0 ~ 5 < 5 x 10" •5 Storage 1 0.43003 X 10 e L < 5 x 10" •7 Temperature 1 0.38189 X -2 10 < 5 x 10" •7 Atmosphere 1 0.60357 X 1 0 ~ 5 0.6306 R a d i a t i o n Treatment x S t o . 6 0.53696 X 1 0 " 4 0 . 0 7 9 9 R a d i a t i o n Treatment x Temp. 6 0.18556 X 1 0 ~ 5 0.0001 R a d i a t i o n Treatment x Atmo. 6 0.18869 X 1 0 ~ 4 0.6071 Storage x Temperature 1 0.14629 X 1 0 " 5 0 . 0 2 1 5 Storage x Atmosphere 1 0.51429 X 1 0 ~ 5 0.6560 Temperature x Atmosphere 1 0.55714 X 1 0 ~ 5 0.7067 E r r o r 25 0.24750 X 1 0 " 4 < 5 x 10" •7 E r r o r between d u p l i c a t e 57 0.23421 X 1 0 ~ 5 T o t a l 115 76 Table D2. A n a l y s i s o f v a r i a n c e f o r c h r o m a t i c i t y c o o r d i n a t e ( y ) . Experiment 4. Source DP Mean Square P r o b a b i l i t y F r e s h vs A l l 1 0 . 5 5 3 6 3 X 1 0 " 5 < 5 x 10' R a d i a t i o n Treatment g 0 . 1 1 7 3 4 X 1 0 " 2 < 5 x 10' C o n t r o l vs Treatment 1 0.2704-0 X 1 0 e L ^ 5 x 10' Dose 2 0 . 1 7 9 7 3 X 1 0 " 2 < 5 x 10' I r r a d i a t i o n Time 1 0 . 3 6 0 3 8 X 1 0 " 5 0 . 0 0 0 3 Dose x I r r a d i a t i o n Time 2 0 . 1 9 0 5 3 X 1 0 " 5 0 . 0 0 0 7 S t o r a g e 1 0.24-014 X 1 0 " 5 0 . 0 0 1 7 Temperature 1 0 . 1 5 7 5 0 X 1 0 " * 0 . 3 7 6 7 Atmosphere 1 0 . 5 3 2 9 1 X 1 0 " ^ 0 . 9 5 0 2 R a d i a t i o n Treatment x S t o . 6 0 . 3 0 8 1 0 X 1 0 ~ * 0 . 1 8 5 9 R a d i a t i o n Treatment x Temp. ' 6 0 . 2 5 8 7 5 X 10""* O . 2 7 2 3 R a d i a t i o n Treatment x Atmo. 6 0 . 3 1 8 3 3 X 1 0 " * 0 . 1 7 1 6 S t o r a g e x Temperature 1 0 . 1 2 8 9 3 X 1 0 " * 0.4248 Storage x Atmosphere 1 0 . 5 1 4 2 9 X 1 0 ~ 5 0.6146 Temperature x Atmosphere 1 0 . 1 5 7 5 0 X 1 0 " * 0 . 3 7 6 7 E r r o r 25 0 . 1 9 1 5 9 X 1 0 " * 0.2081 E r r o r between d u p l i c a t e 57 0 . 1 4 7 9 8 X 1 0 " * T o t a l 113 77 Table D3. Analysis of variance for (^Y). Experiment 4. Source DF Mean Square Probability Fresh vs A l l 1 30.85 0.0012 Radiation Treatment 6 51.81 < 5 x 10' Control vs Treatment 1 5 5 . 9 3 0.0001 Dose 2 90.17 < 5 x 10 Irradiation Time 1 69.77 < 5 x 10 Dose x Irradiation Time 2 2.41 0.3639 Storage 1 48.18 0.0001 Temperature 1 6 .50 0.1002 Atmosphere 1 0.12 0.8061 Radiation Treatment x Sto. 6 4.93 0.0808 Radiation Treatment x Temp. 6 10.02 0.0037 Radiation Treatment x Atmo. 6 0.87 0.8835 Storage x Temperature 1 1 . 2 5 0.4726 Storage x Atmosphere 1 0 . 1 3 x IO"1 0.8991 Temperature x Atmosphere 1 1.52 0.4598 Error 25 2.28 < 5 x 10' Error between duplicates 57 0.14 Total 115 -7 78 Table D4. Analysis of variance for beta-carotene equivalent at 450 nm. Experiment 4. Source DF Mean Square Probability Fresh vs A l l 1 0.33848 <5 x 10" •7 Radiation Treatment 6 0.42518 <5 x 10" •7 Control vs Treatment 1 0 . 15717 X 10" 1 O x 10" •7 Dose 2 0.42426 <£5 x 10" •7 Irradiation Time 1 0.86941 X 10" 1 <5 x 10" •5 Dose x Irradiation Time 2 0.21968 X 10" 1 0.0058 Storage 1 0.11870 X 10" 1 0.0716 Temperature 1 0.12764 <5 x 10" •5 Atmosphere 1 0.13241 <5 x 10" •5 Radiation Treatment'x Sto. 6 0.19887 X 10""2 0.7447 Radiation Treatment x Temp. 6 0 . 5 0 0 7 5 X 10" 2 0.2322 Radiation Treatment x Atmo. 6 0.34273 X 10""2 0.4487 Storage x Temperature 1 0.29251 X 10" 5 0.7648 Storage x Atmosphere 1 0.14401 X 10' 5 0.8205 Temperature x Atmosphere 1 0.80072 X 10" 2 0.1356 Error 25 0.34328 X 10~ 2 0.0543 Error between duplicates 57 0.20463 X 10" 2 Total 113 79 Table D5. Analysis of variance for beta-carotene equivalent at 460 nm. Experiment 4. Source DP Mean Square Probability Fresh vs A l l 1 0.32258 <5 x 1 0 " •7 Radiation Treatment 6 0.39170 O x 1 0 " •7 Control vs Treatment 1 0.14380 X 10 ' -1 <5 x 1 0 " •7 Dose 2 0.40538 <5 x 1 0 " •7 Irradiation Time 1 0.70742 X 10" -1 0 . 0 0 0 2 Dose x Irradiation Time 2 0 . 1 5 3 4 3 X 10' -1 0 . 0 2 7 9 Storage 1 0.11482 X 10' -1 0.0879 Temperature 1 0 .13111 <5 x 1 0 " •5 Atmosphere 1 0.12585 <C5 x 1 0 " •5 Radiation Treatment x Sto. 6 0.29024 X 10' -2 0 . 5 9 5 5 Radiation Treatment x Temp. 6 0.54812 X 10' -2 0 . 2 2 7 9 Radiation Treatment x Atmo. 6 0.32203 X 10' -2 0.5354 Storage x Temperature 1 0.36800 X 10' -2 0.3316 Storage x Atmosphere 1 0.22857 X 10' -5 0 . 9 2 9 4 Temperature x Atmosphere 1 0.68829 X 10' -2 0.1832 Error 25 0 . 5 7 2 5 7 X 10' -2 0.0479 Error between duplicates 57 0 .21772 X 10' -2 Total 113 80 T a b l e D6. C o r r e l a t i o n m a t r i x and mean v a l u e . Experiment 4. V a r i a b l e X y °/o1 450nm 460nm Mean X 1.0000 0.4959 y 0 . 6 3 6 5 1.0000 0 . 3 9 6 0 * * 0.4559 ** 0 .7545 1.0000 2 3 . 0 7 450 nm ** 0.6153 0 . 7 9 0 6 ** 0.6198 1.0000 0 . 7 9 0 2 460 nm 0.6070 * * 0.7810 ** 0.6164 * * 0.9980 1.0000 0.8019 C o r r e l a t i o n c o e f f i c i e n t s a re s i g n i f i c a n t l y d i f f e r e n t a t P <0.01. 

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