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Physical, chemical and rheological studies of the hen's egg Tung, Marvin Arthur 1970

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PHYSICAL, CHEMICAL AND RHEOLOGICAL STUDIES OF THE HEN'S EGG  by  MARVIN ARTHUR TUNG B.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1960 M.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1S67  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Food Science  We accept t h i s t h e s i s as conforming t o the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1970  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  requirements f o r an advanced degree at the U n i v e r s i t y o f 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 reference  and study.  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 copying o f 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 o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood  copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n  permission.  MARVIN A. TUNG  Department o f Food Science The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada  that  ABSTRACT  A t h r e e - p a r t i n v e s t i g a t i o n i s d e s c r i b e d i n which hardness  a t r e g u l a r i n t e r v a l s a c r o s s the t h i c k n e s s o f egg  s h e l l s i s measured and r e l a t e d t o chemical composition a t s i m i l a r p o s i t i o n s i n the same s h e l l s ; e*gg s h e l l membranes are viewed by e l e c t r o n microscopy  t o compare the s t r u c t u r e o f  i n n e r and o u t e r l a y e r s ; and rheology o f egg albumen i s s t u d i e d using a r o t a t i o n a l  viscometer.  M i c r o i n d e n t a t i o n t e s t s o f 27 egg s h e l l s r e v e a l a maximum hardness  at the o u t e r s u r f a c e , i n t e r m e d i a t e hardness a t  the i n n e r s u r f a c e and a minimum hardness t h i c k n e s s o f the s h e l l .  midway a c r o s s the  E l e c t r o n probe microanalyses  indicate  a g r a d u a l l i n e a r i n c r e a s e i n c a l c i u m toward the i n n e r s h e l l surface.  Magnesium and phosphorus are a maximum a t the o u t e r  s u r f a c e and f o l l o w q u a d r a t i c and e x p o n e n t i a l g r a d i e n t s r e s p e c t i v e l y a c r o s s the egg s h e l l t h i c k n e s s .  Hardness g r a d i e n t s ,  composition g r a d i e n t s and t h e hardness-composition change from s h e l l t o s h e l l . average  relationships  For i n d i v i d u a l egg s h e l l s , an  o f 91 percent o f the v a r i a t i o n i n hardness  f o r by v a r i a t i o n s i n chemical composition. 27 s h e l l s , 48 percent o f hardness  i s accounted  In pooled data o f  variance i s associated with  composition changes; whereas 81 p e r c e n t may be a t t r i b u t e d t o v a r i a t i o n s i n composition and p o s i t i o n i n the s h e l l . E l e c t r o n m i c r o s c o p i c a l study and measurement o f f o u r egg s h e l l membranes show t h a t the s t r u c t u r e s c o n s i s t o f an open  network o f f i b e r s b u i l t up i n l a y e r s p a r a l l e l t o the membrane surfaces.  Two r e g i o n s are e v i d e n t i n t r a n s v e r s e s e c t i o n .  The  o u t e r membrane i s three times the t h i c k n e s s o f the i n n e r membrane and the combined dimension i s about 100 microns. Each f i b e r has a c e n t r a l core encased by a g r a n u l a r mantle. For the o u t e r membrane, f i b e r core diameters a r e s i g n i f i c a n t l y g r e a t e r and f i b e r s u s u a l l y occupy a l a r g e r percentage o f membrane volume than i s the case f o r i n n e r membranes.  The  i n s i d e s u r f a c e o f the i n n e r membrane i s l i n e d by a 0.1 micron l a y e r o f m a t e r i a l s i m i l a r t o the f i b e r mantle. membrane s t r u c t u r e i s d i s c u s s e d  Egg s h e l l  i n r e l a t i o n to microbial  penetration. Viscous  behavior  o f egg albumen i s d e s c r i b e d a t 10,  20,  30 and U0°C between shear r a t e s o f 220 and 3140 s e c  Egg  albumen i s a time-dependent p s e u d o p l a s t i c f l u i d .  v i s c o s i t y decay a t constant  shear r a t e i s p a r t i a l l y  - 1  .  Apparentrecoverable  between 32 hour t e s t s and i s a f f e c t e d by the temperature and shear r a t e used.  Flow behavior  power-law and E l l i s models. flow b e h a v i o r  i s a c c u r a t e l y d e s c r i b e d by the  Shear h i s t o r y s t r o n g l y i n f l u e n c e s  w i t h h i g h e r shear r a t e s r e s u l t i n g i n g r e a t e r  p s e u d o p l a s t i c i t y and s e n s i t i v i t y t o temperature  effects.  TABLE  OF C O N T E N T S  Page  LIST  OF T A B L E S  ix  LIST  OF F I G U R E S  x i  ACKNOWLEDGMENTS  xiv  INTRODUCTION CHAPTER  I.  REVIEW  1 HARDNESS  AND C O M P O S I T I O N OF T H E S H E L L  OF T H E L I T E R A T U R E  EXPERIMENTAL  of  4  Shell  Preparation Test  3  METHODS  Measurement  of  Hardness  4  specimens  4  procedure  Measurement Calcium  of  5  Average  and  2  Chemical  magnesium  Composition  determination  6 6  Phosphorus  determination  8  Estimation  of  8  Electron  Probe  Preparation Electron  of  of  9  of  Conversion Relation  9  specimens scans  across  Reduction  content  Microanalyses  beam  Step-scans  organic  of  9  the  shell  microprobe microprobe  Hardness  to  data data  Chemical  - v -  1 0 1 1 to  composition  Composition  1 2 1 7  Page  RESULTS AND DISCUSSION R e s u l t s o f Hardness  17 Tests  17  R e s u l t s o f Chemical Composition by S o l u t i o n Methods  23  R e s u l t s o f E l e c t r o n Probe M i c r o a n a l y s e s  23  E l e c t r o n beam scans  23  Step-scans a c r o s s the s h e l l  27  Comparison o f Chemical Analyses by S o l u t i o n Methods and the E l e c t r o n Microprobe  34  R e l a t i o n o f Hardness  35  t o Chemical Composition  SUMMARY AND CONCLUSIONS CHAPTER I I .  ,  STRUCTURE OF EGG SHELL MEMBRANES  40 42  REVIEW OF THE LITERATURE  42  EXPERIMENTAL METHODS  44  P r e p a r a t i o n o f Specimens  44  Examination by E l e c t r o n Microscopy  45  Measurement o f F i b e r Dimensions  46  A n a l y s i s o f Data  46  RESULTS AND DISCUSSION  49  Q u a l i t a t i v e Observations on' Membrane S t r u c t u r e  52  R e s u l t s o f Measurements o f Membrane S t r u c t u r e  52  Membrane t h i c k n e s s and f i b e r area d e n s i t y  52  Mean v a l u e s o f f i b e r measurements  54  V a r i a t i o n i n f i b e r measurements a c r o s s the membrane  56  - vi -  Page  60  SUMMARY AND CONCLUSIONS CHAPTER I I I .  RHEOLOGY OF EGG ALBUMEN  62  REVIEW OF THE LITERATURE  62  EXPERIMENTAL METHODS  63 63  P r e p a r a t i o n o f Samples  6M-  T e s t Procedures  65  R h e o l o g i c a l Models Models of time-dependent b e h a v i o r • Models o f flow-behavior Temperature E f f e c t s on Flow Behavior  65 67 68  Temperature dependence o f flow parameters  68  Temperature dependence o f v i s c o s i t y  69  S t a t i s t i c a l Methods Used i n Data A n a l y s i s  69 70  RESULTS AND DISCUSSION Sample Q u a l i t y  70  R e s u l t s o f Time Dependence T e s t s  70  Recovery o f apparent  v i s c o s i t y a f t e r storage  70  E f f e c t o f shear r a t e on a p p a r e n t - v i s c o s i t y decay  72  Effect decay  on a p p a r e n t - v i s c o s i t y  72  Tests  76  o f temperature  R e s u l t s o f Flow-Behavior  E f f e c t o f storage on flow behavior  76  Effect  76  o f maximum shear r a t e on flow b e h a v i o r  - v i i-  Page  The power-law model o f flow behavior  78  The  81  E l l i s model o f flow behavior  R e s u l t s f o r Temperature E f f e c t Behavior  on Flow  81  E f f e c t o f temperature parameters  on flow-behavior  81  E f f e c t o f temperature  on apparent  84  SUMMARY AND CONCLUSIONS  viscosity  90  LITERATURE CITED  91  » • •  - v m  -  LIST OF TABLES  Table I  II III IV V  Page Atomic A b s o r p t i o n Spectrophotometer Operating Conditions  7 ' .  2 Diamond Pyramid Hardness (Kg/mm ) a c r o s s the Thickness o f Egg S h e l l s  20  A n a l y s i s o f Variance i n Hardness Data  22  Percentage Composition S o l u t i o n Methods  24  o f Egg S h e l l s by  Average Percentage Composition o f Egg S h e l l s by E l e c t r o n Probe M i c r o a n a l y s i s  28  VI  Average Percentage Composition a t Ten S h e l l L e v e l s i n 27 Egg S h e l l s by E l e c t r o n Probe Microanalysis  29  VII  F-Ratios from Analyses o f the V a r i a n c e i n Composition o f Egg S h e l l s by E l e c t r o n Probe Microanalysis  33  M u l t i p l e C u r v i l i n e a r Regressions o f Hardness on Composition - Pooled B a s i s (n = 270)  36  C o e f f i c i e n t s o f M u l t i p l e Determination f o r Regressions o f Hardness on Composition I n d i v i d u a l S h e l l B a s i s (n = 10)  37  X  M u l t i p l e C u r v i l i n e a r Regressions o f Hardness on Composition and S h e l l L e v e l - Pooled B a s i s (n = 270)  39  XI  Thickness and F i b e r Area D e n s i t y o f Egg S h e l l 'Membranes  53  VIII IX  XII  Mean Values o f Egg S h e l l Membrane Measurements  55  XIII  A p p a r e n t - V i s c o s i t y Decay Curves Spindle  f o r NV  74  XIV  A p p a r e n t - V i s c o s i t y Decay Curves Spindle.  f o r MV1  75  - ix -  XV  Power-Law Flow-Behavior Curves f o r Egg Albumen  80  XVI  E l l i s Model Flow-Behavior Curves f o r Egg Albumen  82  XVII  Temperature Dependence o f Power-Law FlowBehavior Parameters  85  Temperature Dependence o f E l l i s Model FlowBehavior Parameters  85  Temperature Dependence of Apparent V i s c o s i t y U s i n g Shear Rate a s . a Parameter  89  XVIII XIX  - x -  LIST OF FIGURES Figure  Page  1  Hardness i n 27 egg s h e l l s . The mean + one standard d e v i a t i o n i s shown at each shell level.  -  2  Calcium K e l e c t r o n beam scan at the outer edge o f an egg s h e l l . (790X)  25  3  Magnesium K e l e c t r o n beam scan a t the o u t e r edge o f an egg s h e l l . (790X)  25  4  Phosphorus K e l e c t r o n beam scan a t the o u t e r edge o f an egg s h e l l . (79OX)  26  5  Magnesium K e l e c t r o n beam scan a t the i n n e r edge o f an egg s h e l l . (790X)  26  6  CaO content o f 27 egg s h e l l s . The mean +_ one standard d e v i a t i o n i s shown a t each shell level.  30  7  MgO content o f 27 egg s h e l l s . The mean _+ one standard d e v i a t i o n i s shown a t each shell level.  30  8  P2°5 content o f 27 s h e l l s . The mean +_ one standard d e v i a t i o n i s shown a t each s h e l l level.  32  9  Schematic diagram o f an o b l i q u e s e c t i o n through an egg s h e l l membrane f i b e r t o i l l u s t r a t e t h e dimensions measured.  47  10  WL1 o u t e r egg s h e l l membrane (6660X).  50  11  WL2 i n n e r egg s h e l l membrane (6660X).  50  12  NH1 outer egg s h e l l membrane (7050X).  51  13  NH1 i n n e r egg s h e l l membrane (11200X).  51  14  F i b e r core diameters f o r membrane V1L1. The mean and standard d e v i a t i o n a t each l e v e l are shown.  57  a  a  a  a  - xi-  18  F i b e r core diameters f o r membrane WL2. The mean and standard d e v i a t i o n a t each l e v e l are shown. F i b e r core diameters f o r membrane NH1. The mean and standard d e v i a t i o n at each l e v e l are shown. F i b e r core diameters f o r membrane NH2. The mean and standard d e v i a t i o n a t each l e v e l are shown. Average f i b e r mantle t h i c k n e s s f o r membranes WL1 and WL2. Average f i b e r mantle t h i c k n e s s f o r membranes NH1 and NH2. A p p a r e n t - v i s c o s i t y decay i n egg albumen t e s t e d b e f o r e and a f t e r 32 hours s t o r a g e . T e s t s a t 10°C and 3140 s e c shear r a t e . - 1  A p p a r e n t - v i s c o s i t y decay i n egg albumen at 10°C w i t h shear r a t e s of 1570 and 3140 s e c " . 1  A p p a r e n t - v i s c o s i t y decay curves at 10, 30 and 40°C - NV s p i n d l e .  20,  A p p a r e n t - v i s c o s i t y decay curves a t 10, 30 and 40°C - MV1 s p i n d l e .  20,  Flow b e h a v i o r of egg albumen samples t e s t e d b e f o r e and a f t e r 32 hours s t o r a g e . T e s t s a t 10°C w i t h NV s p i n d l e . Flow b e h a v i o r of egg albumen a t 10°;C f o r _ ^ maximum shear r a t e s o f 1570 and 3140 sec" . Flow-behavior curves f o r egg albumen at 20, 30 and 40°C - NV and MV1 s p i n d l e s . Temperature dependence o f the power law parameter m. Temperature dependence of the power law parameter n.  10,  Figure  Page  29  Temperature dependence o f the E l l i s parameter a.  30  Temperature dependence of the E l l i s parameter  31  86 '  86  ^-^/2'  Temperature dependence o f the E l l i s parameter n • o  • • •  - x m  -  87  ACKNOWLEDGEMENTS  The author extends h i s a p p r e c i a t i o n f o r a s s i s t a n c e by Dr. J.F. R i c h a r d s , and  Food Science  Department; P r o f e s s o r L.M. S t a l e y  P r o f e s s o r E.L. Watson, A g r i c u l t u r a l E n g i n e e r i n g  Department;  Dr. J.B. Farmer, Chemistry Department; Dr. J . B i e l y , P o u l t r y Science  Department and Dr. L.C. Brown, M e t a l l u r g y  Department, the  U n i v e r s i t y of B r i t i s h Columbia. The w r i t e r i s g r a t e f u l f o r the use of an e l e c t r o n probe and hardness t e s t e r i n the M e t a l l u r g y  Department, an  e l e c t r o n microscope i n the B i o l o g y Department and a viscometer i n the A g r i c u l t u r a l Engineering  Department.  T h i s r e s e a r c h was f i n a n c e d by the N a t i o n a l Research C o u n c i l o f Canada.  - xiv -  INTRODUCTION  The hen's egg i s an important food i n most p a r t s of the world f o r which the annual p r o d u c t i o n i s i n excess of 300 b i l l i o n  eggs.  P r e s e r v a t i o n o f the e d i b l e p o r t i o n o f  the egg i s a i d e d by a l e a t h e r y membrane and c a l c a r e o u s s h e l l encasement t h a t minimizes microbial invasion.  chemical d e g r a d a t i o n and  The s h e l l a l s o p r o v i d e s a r e l a t i v e l y  d u r a b l e , convenient u n i t f o r h a n d l i n g the product.  Physical  and chemical s t u d i e s o f egg s h e l l s and t h e i r membranes are fundamental  t o e x p l a i n i n g t h e i r r o l e s i n m a i n t a i n i n g egg  quality. Large q u a n t i t i e s o f l i q u i d egg are processed commercially a f t e r removing the s h e l l and membranes. Operations i n c l u d e s e p a r a t i n g , pumping, heat  exchanging,  mixing and spray d r y i n g -- a l l o f which are governed v i s c o u s nature of the f l u i d s .  by the  R h e o l o g i c a l s t u d i e s on egg  c o n s t i t u e n t s would p r o v i d e data u s e f u l i n the design and e v a l u a t i o n o f l i q u i d egg h a n d l i n g equipment. This t h e s i s describes a three-part i n v e s t i g a t i o n of the hen's egg i n which hardness  a t r e g u l a r i n t e r v a l s a c r o s s the  t h i c k n e s s o f egg s h e l l s was measured and r e l a t e d t o chemical composition a t s i m i l a r p o s i t i o n s i n the same s h e l l s ; egg s h e l l membranes were viewed  by e l e c t r o n microscopy  i n order to  compare the s t r u c t u r e o f i n n e r and o u t e r l a y e r s ; and r h e o l o g y of egg albumen was s t u d i e d u s i n g a r o t a t i o n a l v i s c o m e t e r . -  1 -  CHAPTER I.  HARDNESS AND COMPOSITION OF THE SHELL  Most eggs are marketed i n t h e i r s h e l l s ; t h u s , damaged s h e l l s are u n d e s i r a b l e and present a p o t e n t i a l h e a l t h hazard.  public  Losses due to broken and cracked s h e l l s are  estimated a t over f i v e percent of the r e t a i l product v a l u e . Egg s h e l l s t r e n g t h has r e c e i v e d c o n s i d e r a b l e study i n the past decade (W3,  H3, T9)* as a r e s u l t of i t s economic  importance. S h e l l hardness  i s of interest  because hardness  c r y s t a l l i n e materials i s closely related may  t o s t r e n g t h ( T l ) and  be used as a measure of mechanical p r o p e r t i e s  Since m i c r o i n d e n t a t i o n hardness  (M3).  t e s t s permit measurements on  s m a l l amounts o f m a t e r i a l and on b r i t t l e specimens, hardness may  of  egg  shell  be t e s t e d by t h i s method.  Egg s h e l l s are mainly c a l c i u m carbonate i n the c a l c i t e form  (T2) with s m a l l amounts o f i m p u r i t i e s such as  magnesium and phosphorus.  These elements  r e l a t i o n t o hardness because  magnesium s u b s t i t u t i o n  c a l c i u m i n the c a l c i t e l a t t i c e r e s u l t s and f u r t h e r m o r e , c a l c i u m phosphate carbonate.  are of i n t e r e s t i n  i n a harder  for crystal  i s harder than c a l c i u m  The purpose o f t h i s study was  t o measure hardness  and chemical composition p o i n t - b y - p o i n t a c r o s s the t h i c k n e s s of s e v e r a l egg s h e l l s and t o i d e n t i f y the r e l a t i o n between these two *  characteristics.  Alphanumeric c h a r a c t e r s i n parentheses are i n r e f e r e n c e to the l i t e r a t u r e c i t e d .  REVIEW OF'THE LITERATURE Brooks and egg  Hale (B6) measured the hardness of twenty  s h e l l s using r a d i a l s e c t i o n s and r e p o r t e d a l i n e a r  o f hardness d e c r e a s i n g  toward the i n n e r s u r f a c e .  hardness at s h e l l l e v e l s 0.25, and  1,0  correspond  0.50,  to outer and  and  0.75  They t e s t e d  (shell levels  measurements near  shells.  In a p r e l i m i n a r y study by Tung (T3), hardness t e s t e d i n r a d i a l and  t a n g e n t i a l s e c t i o n s of egg  use of t a n g e n t i a l s e c t i o n s permitted  hardness  between s h e l l l e v e l s 0.02  Egg  be hardest  and  0.90.  shell.  be s i m i l a r f o r r a d i a l and  0.20  and  0.70  r e p o r t e d by Brooks (B7) and  solutions.  i n the s h e l l  l a t e r by Itoh and  and  l a y e r s and  analyze  Hatano ( I I ) The  who  method  the  resulting  Magnesium t o c a l c i u m r a t i o s were g r e a t e s t near decreased  t o a minimum near s h e l l  G r a d i e n t s were s e m i - l o g a r i t h m i c l i n e a r f o r Itoh and  r a t i o was  was  to p r o g r e s s i v e l y d i s s o l v e the s h e l l i n  equal  the outer s u r f a c e and 0.70.  Gradients  were found to  a l s o noted an unequal d i s t r i b u t i o n of phosphorus.  five.approximately  hardness  t a n g e n t i a l t e s t s on the same s h e l l s .  V a r i a t i o n i n magnesium content  used i n each case was  The  s h e l l s were found to  s o f t e s t midway between.  of hardness between s h e l l l e v e l s  was  determinations  near the o u t e r s u r f a c e , o f i n t e r m e d i a t e  near the i n n e r s u r f a c e and  0.0  inner surfaces r e s p e c t i v e l y )  because b r i t t l e n e s s of the m a t e r i a l prevented the unsupported edge of the  gradient  Hatano.  The  f o r the data of Brooks  phosphorus to  a maximum i n the outer l a y e r of the s h e l l  e s s e n t i a l l y constant  throughout the remaining -  3 -  level  calcium and  portions.  - 4 -  A r e c e n t advance i n a n a l y t i c a l chemistry i s the e l e c t r o n microprobe  ( B 3 > B 8 , K 2 , 0 1 ) which can be used  to  measure the chemical composition of a few c u b i c microns s o l i d m a t e r i a l and  i s thus well suited to detecting  composition d i f f e r e n c e s i n c r y s t a l l i n e substances. use o f hardness  of  g r a d i e n t measurement techniques  d e s c r i b e d by Tung (T3) i n combination  The  as  with e l e c t r o n probe  a n a l y s i s should p r o v i d e the p o i n t - b y - p o i n t comparison of hardness  and chemical composition r e q u i r e d f o r t h i s  study.  EXPERIMENTAL METHODS Measurement of S h e l l Hardness P r e p a r a t i o n of specimens Three eggs from each o f nine Single-Gomb White Leghorn p u l l e t s p r o v i d e d a t o t a l o f 2 7 s h e l l s f o r hardness tests.  A l i n e was  drawn on the equator of each egg  i t s diameter measured p r i o r t o b r e a k i n g the egg and s h e l l membranes by b o i l i n g i n 0.6 s h e l l was  and removing  M sodium hydroxide.  The  then t h o r o u g h l y r i n s e d i n water and d r i e d i n an  oven at 80°C.  Thickness at the s h e l l equator was  A sample o f the s h e l l was  measured.  mounted i n epoxy (Epon 828  +  10%  d i e t h y l e n e t r i a m i n e by weight) and the convex s u r f a c e ground away t o expose a t a n g e n t i a l s e c t i o n a t the s h e l l The  s u r f a c e was  equator.  p o l i s h e d with a s e r i e s o f emery papers  and  aluminum oxide l a p i d a r y wheels b e f o r e measuring the l e n g t h  - 5 -  of the exposed  s e c t i o n i n the d i r e c t i o n o f the egg equator.  Since the egg i s e s s e n t i a l l y c i r c u l a r i n the plane o f i t s equator, the s h e l l diameter and t h i c k n e s s may be used t o l o c a t e any d e s i r e d s h e l l l e v e l on the exposed t a n g e n t i a l surface Test  (T4) with an estimated  e r r o r of l e s s than 2 p e r c e n t .  procedure Hardness was measured u s i n g a Tukon  microindentation  hardness t e s t e r (M3) equipped with a square-based diamond pyramid i n d e n t e r under 100 g l o a d .  The i n d e n t e r was f o r c e d  i n t o the p o l i s h e d s u r f a c e o f the s h e l l and the d i a g o n a l l e n g t h s " o f the r e s u l t i n g  i n d e n t a t i o n were measured with an  o c u l a r micrometer on the t e s t i n g machine.  Diamond pyramid  hardness was c a l c u l a t e d from the average d i a g o n a l  length  u s i n g the equation „  1.8544 L DP "  r  72  -  d 2 where  H^p = diamond pyramid hardness, kg/mm L  = l o a d on i n d e n t e r , kg  d  = average d i a g o n a l l e n g t h ,  Hardness measurements were s t a n d a r d i z e d  mm. d u r i n g each s e r i e s  of t e s t s by means o f a c a l i b r a t e d r e f e r e n c e  block.  Three t e s t s were made a t s h e l l l e v e l s 0.02, 0.10, 0.20,  0.30, 0.40 and 0.50 s t a r t i n g from one edge o f the  exposed  s e c t i o n along a narrow  n  L 1 ]  s t r i p c o n t a i n i n g the equator.  A d u p l i c a t e s e t o f t e s t s was made s t a r t i n g a t the o p p o s i t e edge o f the s e c t i o n so t h a t the average o f s i x i n d e n t a t i o n s  - 6 -  was used to c a l c u l a t e hax^dness at each s h e l l l e v e l . t e s t s were made from l e v e l s 0.02 b l o c k was  After  t o 0.50, the o r i g i n a l  test  cast i n epoxy and ground away t o expose a  t a n g e n t i a l s e c t i o n f r o m the concave s u r f a c e on which hardness was measured  at l e v e l s 0.50,  s i m i l a r manner.  0.60,  0.70,  0.80  and 0.90  Overlapping t e s t s at s h e l l l e v e l  0.50  p r o v i d e d 12 i n d e n t a t i o n s from which hardness a t t h a t was  in a  level  calculated. Measurement o f Average Chemical Composition  Calcium and magnesium d e t e r m i n a t i o n For  each o f the 27 egg s h e l l s , a sample was  powdered  u s i n g a mortar and p e s t l e and 0.3000 g weighed i n t o a 100 ml beaker. HC1  Three ml o f c o n c e n t r a t e d HNO^,  and 9 ml o f water were added.  3 ml o f c o n c e n t r a t e d  The beaker was covered  w i t h a watch g l a s s and b o i l e d f o r t e n minutes t o d i s s o l v e the  sample.  The s o l u t i o n was  f i l t e r e d i n t o a 500 ml v o l u m e t r i c  f l a s k and the f i l t e r paper was washed with 20 ml o f 0.5 N HC1. Twenty ml o f 5 p e r c e n t lanthanum c h l o r i d e s o l u t i o n was and the f l a s k f i l l e d t o the mark with water. s o l u t i o n was  The  added  resulting  anlyzed f o r magnesium and a p o r t i o n o f i t  d i l u t e d t o s i x times i t s volume and analyzed f o r c a l c i u m . The purpose o f the lanthanum was to overcome  possible  i n t e r f e r e n c e s by o t h e r ions i n the analyzed s o l u t i o n s ( E l ) . Chemicals were o f a n a l y t i c a l grade and d i s t i l l e d water was used i n a l l  preparations.  deionized  - 7  A Unicam SP SO atomic a b s o r p t i o n  spectrophotometer  equipped with a s t r i p c h a r t r e c o r d e r was used i n the analyses f o r magnesium and c a l c i u m .  The instrument was f i t t e d w i t h a  10 cm a c e t y l e n e burner operated t o give a f u e l - r i c h  luminous  flame under the c o n d i t i o n s s p e c i f i e d i n Table I.  TABLE I .  ATOMIC ABSORPTION SPECTROPHOTOMETER OPERATING CONDITIONS  Wavelength, nm S l i t w i d t h , nm  Calcium  Magnesium  422. 7  285. 2  0. 08 12  Lamp c u r r e n t , mA ' 2 A c e t y l e n e p r e s s u r e , kg/cm A c e t y l e n e flow r a t e , ml/min 2 A i r p r e s s u r e , kg/cm A i r flow r a t e , 1/min  0. 7 1500  4 0. 7 1500  2. 1  2. 1  5  5  A s e r i e s o f s o l u t i o n s up t o 4.0 mg/1 and 40.0 mg/1  0. 1  f o r magnesium  f o r c a l c i u m was prepared f o r the s p e c t r o p h o t o -  meter c a l i b r a t i o n .  The standard curves were concave t o the  absorbance  axes;  hence, the l e a s t squares q u a d r a t i c f i t o f  absorbance  t o c o n c e n t r a t i o n formed the c a l i b r a t i o n curve i n  each s e r i e s o f t e s t s .  Percentage  compositions o f c a l c i u m  and magnesium i n the o r i g i n a l samples were based on the  - 8 -  c a l i b r a t i o n c u r v e s , d i l u t i o n f a c t o r s and o r i g i n a l  sample  weights. Phosphorus  determination Powdered  samples o f each s h e l l were p l a c e d i n  weighed c r u c i b l e s , d r i e d o v e r n i g h t at 105°C and reweighed. Specimens were then heated i n an oven a t 1000°C t o remove o r g a n i c matter, and weighed a g a i n .  D u p l i c a t e 0.1200 g  p o r t i o n s o f the r e s i d u e from each egg s h e l l were p l a c e d i n 250 ml Erlenmeyer f l a s k s , 20 ml o f 60% p e r c h l o r i c a c i d v  added  and the mixture b o i l e d f o r 10 minutes t o d i g e s t the sample. The s o l u t i o n s were made up t o 250 ml with water i n v o l u m e t r i c flasks. F i f t y ml p o r t i o n s o f the a c i d s o l u t i o n s were t r a n s f e r r e d t o 100 ml v o l u m e t r i c f l a s k s t o be analyzed f o r phosphorus  (B4).  F i v e ml o f 0.1 M sodium molybdate i n 5.0 M  s u l f u r i c a c i d were added along w i t h 2.0 ml o f 0.012 M h y d r a z i n e s u l f a t e and the f l a s k s f i l l e d The f l a s k s were immersed then c o o l e d r a p i d l y .  t o volume with water.  i n b o i l i n g water f o r 10 minutes,  Absorbance a t 8 30 nm was read i n a  S p e c t r o n i c 20 spectrophotometer f o r the unknown and standard phosphate s o l u t i o n s .  Values o f percentage phosphorus i n the  egg s h e l l samples were c a l c u l a t e d from the c a l i b r a t i o n d i l u t i o n f a c t o r s and i n i t i a l  curves,  sample weights.  E s t i m a t i o n o f o r g a n i c content Egg s h e l l s are known t o possess an o r g a n i c matrix  -  (S2, T2) t h a t extends  9  through  -  the c a l c i t e c r y s t a l s .  An  estimate o f the o r g a n i c content o f the s h e l l may be d e r i v e d from the weight l o s s when heated t o 1000°C.  Calcium and  magnesium, present as carbonates, decompose t o t h e i r  oxides  while phosphorus i s s t a b l e as phosphate; t h u s , weight due t o carbon d i o x i d e p r o d u c t i o n may be c a l c u l a t e d c a l c i u m and magnesium composition.  The remaining  was a t t r i b u t e d t o o x i d a t i o n o f o r g a n i c matter c o n s t i t u e n t s do not exceed  loss  from decrement  s i n c e other  t r a c e amounts (R3). I t should  be p o i n t e d out t h a t t h i s method p r o v i d e d an estimate o f o r g a n i c content s u b j e c t t o i n a c c u r a c y due t o the combined u n c e r t a i n t i e s i n measurement o f magnesium, c a l c i u m and phosphorus and the d i s r e g a r d o f p o s s i b l e t r a c e  elements.  E l e c t r o n Probe M i c r o a n a l y s e s P r e p a r a t i o n o f specimens Samples from the equators o f a l l 27 experimental s h e l l s were c a s t i n epoxy along w i t h standard specimens f o r c a l c i u m , magnesium and phosphorus.  The epoxy b l o c k s were  p o l i s h e d t o expose r a d i a l s e c t i o n s o f s h e l l with a 0.05 micron f i n i s h , then a t h i n c o a t i n g o f carbon was vacuum  evaporated  onto the s u r f a c e of the b l o c k t o render i t c o n d u c t i v e .  The  standards were c a l c i t e f o r c a l c i u m , magnesium carbonate f o r magnesium and f l u o r a p a t i t e  [Ca,. (PO^) ^F] f o r phosphorus.  E l e c t r o n beam scans Composition  o f c r y s t a l l i n e m a t e r i a l s may  differ  - 10 -  from c r y s t a l t o c r y s t a l boundaries, hence p r e l i m i n a r y t e s t s were conducted  t o determine  whether c a l c i u m , magnesium and  phosphorus occur u n i f o r m l y w i t h i n the s h e l l . The U.B.C. Department o f M e t a l l u r g y e l e c t r o n microprobe  (JEOLCo Model JXA-3A w i t h 20° t a k e o f f angle) was  used t o o b t a i n q u a l i t a t i v e  i n f o r m a t i o n on the d i s t r i b u t i o n o f  c a l c i u m , magnesium and phosphorus i n the s h e l l . X - r a d i a t i o n was analyzed with a q u a r t z c r y s t a l whereas magnesium K crystals.  a  and phosphorus K  a  A n a l y s i s o f phosphorus i n the presence  (3.090 A) r a d i a t i o n are d i f f r a c t e d  used  spectrometer  l i n e s used mica  was complicated by i n t e r f e r e n c e o f second  two  Calcium  with P K  a  o r d e r Ca  (6.154 A) because both  at the same spectrometer  l i n e s have d i f f e r e n t  of calcium  setting.  lines  Since the  e n e r g i e s , p u l s e h e i g h t a n a l y s i s was  to exclude the i n t e r f e r i n g c a l c i u m  signal.  The probe was operated a t 15 kV a c c e l e r a t i n g potential  with 0.05 uA sample c u r r e n t normalized on brass and  a minimal  spot s i z e .  E l e c t r o n beam scans were made near  both  edges o f one egg s h e l l i n areas t h a t i n c l u d e d s e v e r a l c r y s t a l boundaries.  Absorbed e l e c t r o n images were recorded t o a i d  in identifying Step-scans  the r e g i o n s scanned.  across the s h e l l F i v e - m i c r o n step-scans a t 10 second  counts were run  on r a d i a l s e c t i o n s o f 27 egg s h e l l s t o measure c a l c i u m , magnesium and phosphorus content p o i n t - b y - p o i n t across the  - 11 -  t h i c k n e s s o f each s h e l l . accelerating potential,  Probe c o n d i t i o n s were 15 kV 0.05 yA sample c u r r e n t normalized on  brass and a spot diameter  o f about 5 microns.  Spots were  t e s t e d on each standard a t one-hour i n t e r v a l s calibration  f o r signal  and d r i f t e v a l u a t i o n .  Reduction o f microprobe The  data  s h e l l l e v e l corresponding t o each analyzed  spot was computed and the raw X-ray  intensity  data d i v i d e d  i n t o t e n p o r t i o n s such t h a t each l e v e l p r e v i o u s l y t e s t e d f o r hardness  was spanned by a s e t o f microprobe  data.  For  example: X = 0.02, 0.0 < X < 0.05; X = 0.10, 0.05 < X < 0.15,..., X = 0.90, 0.85 < X < 0.95 where X i s the s h e l l l e v e l . e v a l u a t e c a l c i u m , magnesium and phosphorus X-ray at the t e n s h e l l l e v e l s  of i n t e r e s t ,  the l e a s t  simple l i n e a r r e g r e s s i o n s o f i n t e n s i t y  To  intensities  squares  on s h e l l l e v e l were  computed f o r the t h r e e elements i n each range and the equations s o l v e d f o r the d e s i r e d s h e l l l e v e l . and phosphorus X-ray 0.20,  intensities  0.90 were o b t a i n e d .  smooth the microprobe  Thus, c a l c i u m , magnesium  at s h e l l l e v e l s T h i s procedure  0.02, 0.10,  serves t o  data and preserve the e f f e c t o f  composition g r a d i e n t s where the data are not symmetrical the s h e l l l e v e l o f i n t e r e s t . reduced  about  The amount o f data i s a l s o  t o correspond w i t h the hardness  information  available.  - 12 -  Conversion  of microprobe data to  composition  D r i f t due to f l u c t u a t i o n s i n the e l e c t r o n i c components was on standards  evaluated from the p e r i o d i c  and a l i n e a r d r i f t  c o r r e c t i o n was  the counting r a t e s o f the standards data o f each egg s h e l l .  instrument  applied to  associated with  Thus, d r i f t was  to  The was  T h i s procedure  be s a t i s f a c t o r y s i n c e the average d r i f t  s h e l l t o s h e l l was  l e s s than 0.5  the  assumed t o be  l i n e a r between t e s t s o f s u c c e s s i v e s h e l l s and was d u r i n g the a n a l y s i s o f each s h e l l .  tests  neglected was  judged  c o r r e c t i o n from  percent.  c a l c i u m weight f r a c t i o n f o r each s h e l l  level  c a l c u l a t e d by assuming d i r e c t p r o p o r t i o n a l i t y between  X-ray i n t e n s i t i e s and c o n c e n t r a t i o n s i n both the and the egg  shell.  calcite  T h i s method i s simple, yet s u i t a b l e f o r  the p r e s e n t a n a l y s i s because o f the c l o s e s i m i l a r i t y between the standard and unknown. Magnesium and phosphorus X-ray i n t e n s i t y data f o r the standards  and unknowns were m o d i f i e d by a number o f  c o r r e c t i o n procedures  (G2, S6).  dead-time on a l l s i g n a l s was N  N  2  where  t  The  corrected using  l  (1-y)  =  influence of detector  C  =  observed  count r a t e  =  t r u e count r a t e and  =  d e t e c t o r dead-time'.  The background i n t e n s i t y was N  3  where B  then c o n s i d e r e d .  =  N  =  c o r r e c t e d count r a t e  =  background count r a t e .  2  That i s ,  - B and  Apparent weight c o n c e n t r a t i o n s o f the specimens were c a l c u l a t e d by d i r e c t p r o p o r t i o n a l i t y between X-ray i n t e n s i t i e s and  c o n c e n t r a t i o n s f o r standards (N ) 3  c  where  C  l  unknowns.  u  STN7J7  =  s  and  C  O S  =  weight c o n c e n t r a t i o n o f  =  apparent weight c o n c e n t r a t i o n  u and  standard  s r e f e r to unknown (egg s h e l l ) and  standard.  The  assumption o f p r o p o r t i o n a l i t y i s i n a c c u r a t e when samples  and  standard are d i s s i m i l a r because o f the d i f f e r i n g  o f atomic number, mass a b s o r p t i o n and r e s p e c t i v e X-ray  composition.  f l u o r e s c e n c e on  procedures  f o r atomic numbe  f l u o r e s c e n c e r e q u i r e an estimate o f specimen However, when the c o r r e c t i o n s have been a p p l i e  a b e t t e r estimate  of composition  i s a v a i l a b l e which may  used t o r e c a l c u l a t e the c o r r e c t i o n f a c t o r s . i s repeated  u n t i l no f u r t h e r improvement r e s u l t s .  [ 4 ] and  c a l c i u m c o n c e n t r a t i o n from the  described e a r l i e r .  Carbon and  oxygen o f the  be  Thus the c y c l e  and phosphorus apparent c o n c e n t r a t i o n s were taken equation  the  intensities.  Microprobe c o r r e c t i o n a b s o r p t i o n and  effects  Magnesium from  calculation  crystalline  - 14 -  phase were computed on the assumption that c a l c i u m c a r b o n a t e , magnesium carbonate and c a l c i u m phosphate were the o n l y i n o r g a n i c substances p r e s e n t .  The remaining weight o f the  s h e l l was a t t r i b u t e d t o the o r g a n i c m a t r i x which i s known to be a p r o t e i n - a c i d mucopolysaccharide complex ( S 2 ) . Composition o f the o r g a n i c m a t e r i a l was approximated by 4 7 percent carbon, 4 3 p e r c e n t oxygen and 10 p e r c e n t n i t r o g e n . With t h i s i n f o r m a t i o n the f r a c t i o n a l weight composition was c a l c u l a t e d f o r each element p r e s e n t i n the s h e l l . The average atomic number o f a t e s t m a t e r i a l has an e f f e c t on e l e c t r o n b a c k s c a t t e r i n g and e l e c t r o n  retarda-  t i o n , hence the c o r r e c t i o n f o r these two phenomena i s termed the atomic number c o r r e c t i o n t r a t i o n was m o d i f i e d  where  C.  =  R  =  ( D I ) . Apparent weight concen-  using  S R" C, ^ S R s u Z  [5]  i i i R  [ J  C  6  R.  =  the b a c k s c a t t e r c o e f f i c i e n t and  C^  =  weight c o n c e n t r a t i o n f o r each element i .  x  !•> i s o b t a i n e d from S . where  =  Z.C.S. i l l i , 1.166 X 1 0 E = -r— l n = A. J Z  S. i  c  [7]  3  ±  r  L8J e  1  - 15 -  Z.  1  = atomic number = atomic weight = mean i o n i z a t i o n p o t e n t i a l E  E  =  E  q  E  C  -E  -2^°  [9]  = a c c e l e r a t i n g p o t e n t i a l used and = e x c i t a t i o n p o t e n t i a l f o r the X-ray l i n e studied  A mass a b s o r p t i o n c o r r e c t i o n i s r e q u i r e d t o compensate f o r p a r t i a l a b s o r p t i o n o f X-rays as they t r a v e l from p o i n t o f o r i g i n i n the sample t o the s u r f a c e . modified  The  P h i l i b e r t method ( P I , D 2 ) 'was used i n the  correction. f<x ) s  A  where  u  = apparent weight c o n c e n t r a t i o n a f t e r atomic number and a b s o r p t i o n c o r r e c t i o n s fTxT  =  +  1.2  N^  [ I ] '  E.N.A. _ [Z.N.Z.] 1  1  concentration  cosec 0  = mass a b s o r p t i o n c o e f f i c i e n t 0  [12]  1  2  = atomic ^  r^TT  [13] (H5)  = t a k e o f f angle 2.39 X 1 0 1.5 _ 1.5 o c 5  E  E  [14]  - 16 -  In equation  [12] weight c o n c e n t r a t i o n , C\ , was used t o  approximate atomic c o n c e n t r a t i o n . C o r r e c t i o n s f o r secondary f l u o r e s c e n c e may be needed when an element i s analyzed c h a r a c t e r i s t i c and continuous than the l i n e o f i n t e r e s t . continuous  i n the presence o f  X - r a d i a t i o n o f h i g h e r energy  In g e n e r a l , enhancement due t o  r a d i a t i o n i s s m a l l and very d i f f i c u l t t o e v a l u -  ate i n comparison with t h a t o f c h a r a c t e r i s t i c (K2).  radiation  Thus, only f l u o r e s c e n c e by c h a r a c t e r i s t i c  l i n e s was c o n s i d e r e d .  emission  C o r r e c t i o n s b f magnesium and  phosphorus i n t e n s i t y r a t i o s f o r f l u o r e s c e n c e by c a l c i u m c h a r a c t e r i s t i c r a d i a t i o n were c a l c u l a t e d with Reed's (RI) s i m p l i f i e d procedure and found  t o be l e s s than 0.1  hence, f l u o r e s c e n c e c o r r e c t i o n s were  percent;  omitted.  Thus C where  4  C^  =  C  [15]  =  apparent weight c o n c e n t r a t i o n a f t e r atomic number, a b s o r p t i o n and f l u o r e s c e n c e corrections.  3  At t h i s p o i n t the apparent weight  concentrations  of magnesium and phosphorus were used t o r e a s s e s s composition  specimen  f o r the r e c a l c u l a t i o n o f atomic number and  absorption c o r r e c t i o n f a c t o r s .  In s e v e r a l separate  tests  o f t h i s procedure with a computer program, t h e r e appeared t o be no advantage i n performing three  times.  the c a l c u l a t i o n s more than  - 17 -  For the purpose o f p r e s e n t i n g the t o t a l  chemical  composition, weight f r a c t i o n s were converted t o percentage composition expressed  as o x i d e s ;  thus egg s h e l l composition  as percent CaO, MgO, 2 ^ 5 ' P  a n c  ^  o  r  g  a  n  i  c  I t should be emphasized t h a t the p o s s i b l e presence amounts o f o t h e r elements and the accumulated  was material. of trace  errors i n  chemical analyses would be r e f l e c t e d i n the o r g a n i c category because o f i t s method o f  computation.  R e l a t i o n o f Hardness t o Chemical  Composition  The e l e c t r o n probe m i c r o a n a l y s e s p r o v i d e d i n f o r m a t i o n c o r r e s p o n d i n g t o hardness ten s h e l l l e v e l s i n 27 egg s h e l l s ; s e t s o f data were a v a i l a b l e .  composition  measurements a t the same  t h e r e f o r e a t o t a l o f 270  R e l a t i o n s among the v a r i a b l e s  were s t u d i e d by means o f a n a l y s i s o f v a r i a n c e , c o r r e l a t i o n and r e g r e s s i o n methods.  Regressions o f hardness  c o n s t i t u e n t s , s i n g l y and i n combination assess t h e i r r e l a t i v e importance the dependent  on chemical  were computed t o  i n explaining variation i n  variable.  RESULTS AND  DISCUSSION  R e s u l t s o f Hardness Tests Diamond pyramid  hardness  numbers a t t e n l e v e l s i n  each o f the 27 egg s h e l l s appear i n Table I I . In polynominal f i t s t o the pooled data (n = 2 7 0 ) , v a r i a t i o n o f hardness a c r o s s the s h e l l t h i c k n e s s ( f i g u r e 1) i s d e s c r i b e d by the curve  19 -  H  D p  = 184.6 - 350.2X + 591.5X  2  - 259.OX  [16]  3  2 where  H^p = diamond pyramid hardness, Kg/mm X  = shell level.  .  TABLE I I .  DIAMOND PYRAMID HARDNESS (Kg/mm ) ACROSS THE THICKNESS OF EGG SHELLS 2  Shell level Shell Number  Bird  0.02  0.10  0.20  0. 30  0.40  0.50  0.60  0.70  0.80  0. 90  1 2 3  1 1 1  178.3 180.9 190.0  155.2 169.1 154.6  145.0 146.2 135.8  133.7 124.1 118.8  124. 1 115. 3 115. 2  116.5 120.1 124.9  130. 3 137. 5 140. 2  14 3. 7 148. 6 132. 4  144. 0 159. 7 150. 7  162. 7 161. 5 168. 8  4 5 6  2 2 2  171.5 170.5 172.6  149.9 154.8 146.7  133.6 137.6 136.9  124. 3 124.6 128.2  123. 2 122. 3 129. 6  129.2 121.8 140.9  149. 2 144. 9 150. 4  141. 0 143. 9 145. 5  141. 7 160. 2 161. 6  162. 3 150. 5 169. 5  7 8 9  3 3 3  184.9 183.7 188.9  165.7 158. 3 168.2  141.0 136.0 155.4  138.6 130.8 145.6  126. 5 126. 6 134. 1  140. 7 132.1 128.8  146. 3 157. 3 135. 7  148. 9 144. 3 177. Q  16 3. 2 152. 4 13 7. 2  160. 0 165. 8 160. 8  10 11 12  4 4 4  176.5 176.5 179.6  155.8 153.1 163.8  137.0 141.7 151.6  121.9 136.0 135.2  114. 1 126. 2 127. 0  108.1 117.0 117.7  113. 6 125. 1 139. 0  ,132. 6 131. 9 135. 7  129. 4 141. 4 155. 5  .15 3. 4 162. 2 153. 4  13 14 15  5 5 5  177.6 183.6 187.8  146.9 133. 6 164.9  131.6 126.0 133. 0  119.8 125.8 127.5  122. 0 122. 4 .124. 3  119.9 128.2 111.9  130. 5 129. 1 117. 2  149. 0 148. 7 140. 9  14 3. 3 14 8. 9 132. 6  164. 8 150. 3 157. 2  16 17 18  6 6 6  173.8 179.1 178.4  156.6 154.2 153.0  130.4 126.0 129. 7  119.9 118.1 125.0  118. 7 116. 1 116. 9  111.7 117.8 119.4  111. 2 121. 7 120. 4  135. 6 141. 5 138. 1  142. 7 145. 1 153. 2  164. 6 163. 1 165. 0  Continued  TABLE I I (Continued)  Shell  Shell Number  -Bird  19 20 21  7 7 7  22 23 24 25 26 27  .0.02  -0.10  0.20  165.6 177.2 166.9  139. 3 154.4 155.6  124. 4 14 3. 0 132. 4  8 8 8  172.4 185. 7 174.6  153. 3 148.5 161.2  9 9 9  174.1 159.6 186.4  144.6 162.2 160.7  0.30  level  0.40  0.50  0.60  0.70  0.80  0.90  126.5 121.0 130.1  129. 0 126.0 124.6  129. 7 118. 8 123. 6  137. 8 128. 3 128. 5  152. 3 135. 7 139. 0  153.5 129. 3 142.6  143. 6 161. 0 155. 3  139. 6 136. 1 134. 2  124.7 124.0 122.5  124.1 120.6 118.7  123. 9 121. 2 121. 4  135. 4 127. 8 133. 2  150. 2 141. 7 132. 4  148.5 153.5 160.9  161. 2 180. 4 170. 4  132. 4 144. 0 140. 7  118.7 120.6 127.8  114.4 121.9 121.1  115. 7 115. 6 124. 9  121. 0 135. 3 134. 9  126. 8 141. 6 134. 6  .130.4 150.8 155.0  142. 7 152. 7 146. 3  - 22 -  TABLE I I I .  ANALYSIS OF VARIANCE IN HARDNESS DATA  Degrees o f Freedom  Source o f V a r i a t i o n  Shells -  Shell  275.9  5.07**  8  627 .1  11.52**  18  119.8  2.20**  i n birds  9  levels  Error  234  Total  269  **  Significant  F-Ratio  (26)  Birds  - Shells  Mean Square  8792.  161.55**  54.4  at P < 0.01  Hardness d i f f e r s s i g n i f i c a n t l y (P < 0.01) i n d i f f e r e n t egg s h e l l s as shown by a n a l y s i s III).  Significant  differences  o f variance  (P < 0.01) were found among  s u c c e s s i v e s h e l l s from the same b i r d and among s h e l l s by d i f f e r e n t  (Table  produced  birds.  The g r a d i e n t o f hardness across t h e t h i c k n e s s o f the  s h e l l i s p a r t i c u l a r l y noteworthy. For the c u b i c  function  f i t t e d t o t h e pooled d a t a , t h e minimum occurs a t s h e l l l e v e l 2 0.40 with a value o f 122.5 kg/mm w h i l e s u r f a c e hardness, by 2 e x t r a p o l a t i o n , i s 184.6 and 166.9 kg/mm  f o r o u t e r and i n n e r  s u r f a c e s which r e p r e s e n t i n c r e a s e s o f 51 and 36 p e r c e n t respectively.  The d i r e c t r e l a t i o n between hardness and  s t r e n g t h o f c r y s t a l l i n e m a t e r i a l s i s w e l l known ( T l ) and t h e  - 23 -  importance  o f s h e l l hardness  has been r e p o r t e d (T3).  to c r u s h i n g s t r e n g t h o f eggs  G r a d i e n t s o f hardness  those i n egg s h e l l s are common i n case-hardened  similar to metals ( s e e ,  f o r example, p. 175 i n r e f e r e n c e M3) which possess  durable  s u r f a c e s , w h i l e the core is. s o f t so t h a t the m a t e r i a l maintains a h i g h shock r e s i s t a n c e .  The g r a d i e n t o f hardness  i n egg s h e l l s may be o f fundamental importance s t r e n g t h o f the egg;  t o the  however, d i r e c t evidence o f t h i s i s  not a v a i l a b l e a t p r e s e n t . R e s u l t s o f Chemical Percentage  Composition  by S o l u t i o n Methods  composition as oxides o f c a l c i u m ,  magnesium and phosphorus along w i t h estimated o r g a n i c content f o r each s h e l l appear i n Table IV.  These r e s u l t s show  r e l a t i v e l y c l o s e agreement with egg s h e l l r e p o r t e d elsewhere  (R2, S8).  composition  I t should be noted t h a t s m a l l  amounts o f o t h e r elements may o c c u r i n the s h e l l , t h e i r presence  although  was not determined.  R e s u l t s o f E l e c t r o n Probe M i c r o a n a l y s e s E l e c t r o n beam scans Large through  amounts o f c a l c i u m , u n i f o r m l y d i s t r i b u t e d  the s h e l l , are e v i d e n t i n the e l e c t r o n beam scan o f  f i g u r e 2.  F i g u r e s 3 and 5 show t h a t magnesium occurs i n s o l i d  s o l u t i o n w i t h the c a l c i u m carbonate  and t h a t s l i g h t l y h i g h e r  c o n c e n t r a t i o n s may be present i n a zone near the o u t e r edge o f the s h e l l .  The phosphorus content  ( f i g u r e H) i s  - 24 TABLE IV  PERCENTAGE COMPOSITION OF EGG SHELLS BY SOLUTION METHODS  2°5  Organic Matter '  0.713  0.588  2.97  52.58  0.705  0.515  3.04  3  52.49  0.656  0.611  3.23  4  52.86  0.476  0.485  3.14  5  51.67  0.584  0.487  4.00  6  52.19  0.618  0.517  3.25  7  52.26  0.607  0.462  3.37  8  51.61  0.561  0.694  3.75  9  51. 88  0.59 7  0.586  3.11  10  51.74  0. 552  0.532  3. 86  11 12  51.99  0.630  0.587  3.43  51.92  0.677  0.610  3.65  13  51.26  0.642  0.473  4.14  14  51.70  0.561  0.422  3. 39  15  51.50  0.605  0.617  2.96  16  51.72  0.754  0.629  5.38  17  51.57  0.648  0.468  3.57  18  51.57  0.638  0.428  3.62  19  52.18  0.547  0.499  3.12  20  51.49  0.633  - 0.541  3.81  21  51.94  0.579  0.479  3.22  22  51.9.9  0.767  0.426  3. 30  23  52.07  0.686  0.541  3.15  24  51.28  0.614  0.559  4.25  25  52. 30  0.586  0.494  3.49  26  52.00  0.665  0.595  3.08  27  52. 80  0.586  0.507  2.47  Average  •51.98  0.625  0.532  3.47  Shell  *  CaO  MgO  1  52.87  2  P  Organic matter estimated by d i f f e r e n c e .  Figure 2.  Calcium K e l e c t r o n beam scan at the a o u t e r edge o f an egg s h e l l . (790x)  Figure 3.  Magnesium  e l e c t r o n beam scan  o u t e r edge o f an egg s h e l l .  a t the  (790X)  26  Figure  5.  Magnesium K  A  -  e l e c t r o n beam scan a t the  i n n e r edge o f an egg s h e l l .  (790X)  - 27 -  relatively decreases  h i g h near the o u t e r s u r f a c e of the s h e l l r a p i d l y ' t o w a r d the s h e l l i n t e r i o r .  boundaries  and  Several  crystal  are i n c l u d e d i n the e l e c t r o n beam scans but  no  d i f f e r e n c e s are evident i n c a l c i u m , magnesium o r phosphorus content between c r y s t a l s scans on r a d i a l tative  and t h e i r boundaries.  Thus, step-  s e c t i o n s of the s h e l l should be  of composition  Step-scans a c r o s s the  a c r o s s the egg  represen-  shell thickness.  shell  To t e s t whether a s i n g l e step-scan a c r o s s t h i c k n e s s of a s h e l l adequately  r e p r e s e n t s i t s composition,  t h r e e separate step-scans were made i n d i f f e r e n t along the equator of one i n composition difference  shell.  showed no  among the mean MgO  the t h r e e separate step-scans  of one  Mean v a l u e s of percentage ten  analyzed  shell levels  average composition  positions  A n a l y s i s of the v a r i a n c e  a t ten s h e l l l e v e l s  (P > 0.05)  the  significant  and P2^5  ' ent  of  t  shell. CaO,  MgO  and  ?2®5  were taken t o r e p r e s e n t  o f each s h e l l  c o n  (Table V).  Egg  a  t  t  ^  i e  the shell  composition v a r i e s c o n s i d e r a b l y a c r o s s the t h i c k n e s s of the s h e l l as shown by average r e s u l t s (Table V I ) ,  levels  T e s t s o f the c o m p o s i t i o n - s h e l l l e v e l  show t h a t c a l c i u m oxide content s u r f a c e of the s h e l l  where  at ten s h e l l  (figure  i n c r e a s e s toward the i n n e r  6) as d e s c r i b e d by the  CaO  = 51.21+ + 3.13X  CaO  = percent c a l c i u m oxide by weight .  For equation  relation  [17] the standard e r r o r of estimate  equation [17]  is  1.27.  - 28 -  TABLE V.  AVERAGE PERCENTAGE COMPOSITION OF EGG SHELLS BY ELECTRON PROBE.MICROANALYSIS  .Organic Matter  CaO  MgO  1  51.38  0.645  1.138  6.82  2  52.51  0.608  0.779  4.90  3  52.30  0.474  0.816  5.56  4  53.52  0.266  0.43 2  3.84  5  53.73  0.399  0.402  3.19  6  52.90  0.428  1.108  4.56  7  53.34  0.432  0.455  3.81  8  53.35  0.523  0.716  3.59  9  53.06  0.447  0.49 0  4.28  10  53.01  0 .744  0.617  3.74  11  52.73  0 .510  0 .481  4.73  12  52.54  0 .498  0 .566  5.11  13  52.65  0.471  0.363  4.98  14  52 . 82  0 . 354  0.410  4.91  15  52 .15  0 .541  0 .496  5.72  16  52.42  0.569  0.507  5.17  17  53 .20  0.465  0.400  4.00  18  53.02  0.472  0.345  4.31  19  52.07  0.318  0.507  6 . 32  20  52.97  0.302  0.428  4.75  21  52.43  0.389  0.499  5.53  22  52.35  0.579 '  0 .410  5.28  23  52.56  0 .496  5.16  24  51.77  0.355  0.869  6.75  25  50.42  0.425  ,0 .634  9.04  26  52 .89  0.590  0.611  4.28  27  53.61  0.322  0.305  3.57  Average  52.65  0.466  0.566  4.96  Shell  *  . 0.449  P  2°5  Organic matter estimated by d i f f e r e n c e .  - 29 TABLE V I .  AVERAGE PERCENTAGE COMPOSITION AT TEN SHELL LEVELS IN 27 EGG SHELLS BY ELECTRON PROBE MICROANALYSIS  Shell level  CaO  MgO  0.02  50.57  1.166  2.657  7.06  0.10  52.68  0.938  0.888  3.90  0.20  52.11  0.70 2  0.462  5.45  0.30  52.02  0.537  0.363  5.96  0.40  51.89  0 .401  0.308  6.48  0.50  52.33  0.315  0.271  5.87  0.60  53.37  0.205  0.249  4.25  0.70  53.67  0.077  0.180  3.99  0.80  54.06  0.062  0.134  3.33  0.90  53. 85  0.255  0.147  3.30  Average  52.66  0.466  0.566  4.96  P  2°5  Organic matter estimated by d i f f e r e n c e  Organic Matter*  - 30 -  50  48  0  0.2  0.4 Shel  Figure  6.  0.6  0.8  1.0  I eve  CaO content o f 2 7 egg s h e l l s . The mean +_ one standard d e v i a t i o n i s shown a t each s h e l l l e v e l  2  0  0.2  0.4 Shell  Figure  7.  0.6  0.8  1.0  level  MgO content o f 27 egg s h e l l s . The mean + one standard d e v i a t i o n i s shown a t each s h e l l l e v e l .  - 31 -  Magnesium oxide content i s a maximum a t the o u t e r s u r f a c e , f a l l s t o a minimum a t s h e l l l e v e l the i n n e r s h e l l s u r f a c e .  0.75, then i n c r e a s e s toward  The g r a d i e n t ( f i g u r e  7, equation  18)  i s q u a d r a t i c w i t h a standard e r r o r o f estimate o f 0.23.  where  MgO  = 1.22 - 2.86X + 1.89X  [18]  MgO  = p e r c e n t magnesium oxide by weight.  2  Phosphorus c o n c e n t r a t i o n s are h i g h near the outer s u r f a c e of the egg s h e l l and f o l l o w an e x p o n e n t i a l decrease  (figure  8)  g i v e n by P 0 2  where  P  2^5 X  = 0 .1U3X  5  =  P  e r c e n - t  = shell  u  '  ,  £  [19]  ±  phosphorus pentoxide by weight level  A n a l y s i s o f the v a r i a n c e (Table V I I ) i n the composition data shows t h a t c a l c i u m and magnesium contents are s i g n i f i c a n t l y d i f f e r e n t different  (P < 0.01) f o r the s h e l l s o f  b i r d s and f o r s u c c e s s i v e s h e l l s  from a  hen; however, the phosphorus data do not show differences.  Highly s i g n i f i c a n t  single  significant  d i f f e r e n c e s i n chemical  data among t e n p o s i t i o n s a c r o s s the t h i c k n e s s o f egg  shells  r e f l e c t the presence o f c a l c i u m , magnesium and phosphorus composition  gradients discussed e a r l i e r .  - 32 -  F i g u r e 8.  p 2  °5  content o f 2 7 s h e l l s .  The mean + one  d e v i a t i o n i s shown at each s h e l l  level.  standard  -  TABLE V I I .  33 -  F-RATIOS FROM ANALYSES OF THE VARIANCE IN COMPOSITION OF EGG SHELLS BY ELECTRON PROBE MICROANALYSIS  Degrees o f Freedom  CaO  Shells  (26).  5 .17**  2.9 8**  1.05ns  -  8  6 .32**  4.84**  1.53ns  18  4 .67**  2.16**  0.8 3ns  9  32 .73**  86.69**  36.13**  Source o f V a r i a t i o n  Birds  - Shells i n birds Shell levels Error  2 34  Total  269  **  S i g n i f i c a n t a t P < 0.01  ns  Not s i g n i f i c a n t a t P < 0.0 5  MgO  P  2°5  - 34 -  Comparison o f Chemical Analyses by S o l u t i o n Methods and the E l e c t r o n  Microprobe  The 27 s e t s o f composition d a t a , o b t a i n e d by a n a l y z i n g egg s h e l l s o l u t i o n s  (Table I V ) , were compared  w i t h c o r r e s p o n d i n g e l e c t r o n probe r e s u l t s t - t e s t s o f t h e i r meaas. significant differences whereas F^^s MgO  c o m  P  o s  itions  CaO and MgO  (Table V) u s i n g  d e t e r m i n a t i o n s show  (P < 0.01) f o r the two methods, are e s s e n t i a l l y the same.  Lower  values by the e l e c t r o n probe method are expected  because the average  i s based  on composition between s h e l l  l e v e l s 0.0 and 0.95 and magnesium content i s known t o i n c r e a s e a t the i n n e r s h e l l s u r f a c e (H7, T 6 ) .  Furthermore,  the s o l u t i o n methods i n c l u d e d egg s h e l l samples from the p o l e s o f the s h e l l whereas e l e c t r o n probe a n a l y s e s were r e s t r i c t e d t o the equator.  Since the p h y s i c a l s t r u c t u r e  o f the s h e l l v a r i e s . a l o n g i t s l e n g t h [ f o r example, thickness  (T8) a n d ^ p o r o s i t y ( W l ) ] , the chemical  may a l s o change.  The s i g n i f i c a n t c o r r e l a t i o n  o f 0.525 (P < 0.01) between the p a i r s o f MgO  nature  coefficient  compositions  i n d i c a t e s a l i n e a r r e l a t i o n between the two methods.  - 35 -  R e l a t i o n o f Hardness t o Chemical  Composition  Hardness proved t o be a c u r v i l i n e a r f u n c t i o n o f c a l c i u m , magnesium and phosphorus content, thus m u l t i p l e c u r v i l i n e a r r e g r e s s i o n s were used t o determine the r e l a t i o n of hardness t o composition l e v e l s i n 27 egg s h e l l s .  i n the pooled data f o r t e n s h e l l Of the three chemical  constituents  i n the r e g r e s s i o n s (Table V I I I ) , magnesium c o n t r i b u t e s l e a s t t o the e x p l a n a t i o n o f v a r i a n c e i n hardness, while v a r i a t i o n i n c a l c i u m i s the most important  contributor.  When the data  are c o n s i d e r e d on a pooled b a s i s , the c o e f f i c i e n t o f multiple determination  i s 0.482; t h u s , there i s an a p p r e c i a b l e  r e s i d u a l v a r i a n c e i n hardness a f t e r chemical considered.  composition i s  '  Examination o f the hardness-composition  relation  i n the data o f i n d i v i d u a l s h e l l s r e v e a l d i f f e r e n t r e g r e s s i o n s from s h e l l t o s h e l l .  Coefficients of multiple  determination  (Table IX) i n d i c a t e t h a t , on the average, 9 0.9 percent o f the v a r i a t i o n i n hardness can be e x p l a i n e d by v a r i a t i o n s i n c a l c i u m , magnesium and phosphorus i n the same s h e l l . When two o f the chemical  c o n s t i t u e n t s a r e c o n s i d e r e d the  average i n c r e a s e i n e x p l a n a t i o n o f r e s i d u a l v a r i a n c e by i n c l u d i n g the t h i r d component i n the r e g r e s s i o n i s 51.3, 49.5 and 63.0 percent  f o r c a l c i u m , magnesium and phosphorus  r e s p e c t i v e l y ; t h e r e f o r e , each o f the t h r e e i s important t o the r e g r e s s i o n .  TABLE V I I I .  MULTIPLE CURVILINEAR REGRESSIONS OF HARDNESS ON COMPOSITION - POOLED BASIS (n = 270)  Independent Variable CaOt (CaO)  2  -  (MgO)  2  2°5  -  ) 2  Constant Sytt R ttt 2  -19 0.6**  4  -  -134.5**  1.312**  1.848**  3.448ns  8.704ns  -  - 19.9 8**  4.115ns  8.868**  -  10. 7 5**  25.91**  2°5  ( P  Regression c o e f f i c i e n t 2 3  -133.14**  MgO  P  1  2.614**  3500. 13.96 0.482  1.326**  -  5046. 15.33 0. 371  28.21** -  2.816**  3532. 13.96 0.478  25. 30** -  2. 597**  136. 6 15. 94 0. 320  **  S i g n i f i c a n t a t P < 0.01  ns  Not s i g n i f i c a n t a t P < 0.05  t  Percentage compositions by weight are used i n the r e g r e s s i o n f o r the i n d i c a t e d compounds  tt Standard e r r o r o f estimate ttt C o e f f i c i e n t of multiple determination  - 37 -  TABLE I X .  C O E F F I C I E N T S OF M U L T I P L E D E T E R M I N A T I O N FOR REGRESSIONS  OF HARDNESS ON  INDIVIDUAL SHELL BASIS  Independent Variables  COMPOSITION-  (n = 10)  Range  Mean  Ca, Mg, P t  0.771 - 0.993  0.909  Ca, Mg  0.419 - 0.968  0.754  Ca, P  0.490 - 0.940  0.820  Mg, P  0.564 - 0.974  0 . 813  t  Ca r e p r e s e n t s CaO and (CaO) and P r e p r e s e n t s  ?2®5  2  2 , Mg r e p r e s e n t s MgO and (MgO)  2 percentage and (P2O,-) where  compositions by weight are used i n the r e g r e s s i o n s f o r the i n d i c a t e d  compounds.  - 38 -  Hardness i s known t o be a c u b i c f u n c t i o n o f s h e l l l e v e l as g i v e n i n equation  [16] f o r which the c o e f f i c i e n t  o f m u l t i p l e d e t e r m i n a t i o n i s 0.79 5 on the pooled-data b a s i s . Since chemical composition  a l s o v a r i e s a c r o s s the t h i c k n e s s  o f the s h e l l , r e g r e s s i o n s o f hardness on composition and s h e l l l e v e l were c o n s i d e r e d  (Table X ) .  The c o e f f i c i e n t o f  m u l t i p l e d e t e r m i n a t i o n o f 0.814 r e p r e s e n t s a 9.3 percent i n c r e a s e i n e x p l a n a t i o n of r e s i d u a l v a r i a n c e due t o i n c l u s i o n of composition  data.  Another e q u a l l y v a l i d  interpretation  i s t h a t i n c l u s i o n o f s h e l l l e v e l i n the r e g r e s s i o n o f hardness on composition  e x p l a i n s an a d d i t i o n a l 64.1 percent o f the  r e s i d u a l v a r i a n c e i n hardness.  I t i s evident, therefore,  t h a t v a r i a t i o n s i n hardness o f egg s h e l l s are not e n t i r e l y e x p l a i n e d by v a r i a t i o n s i n c a l c i u m , magnesium and phosphorus compositions  a c r o s s the t h i c k n e s s o f egg s h e l l s .  Large  improvements i n r e g r e s s i o n s by i n c l u d i n g  s h e l l l e v e l suggest  the presence  o f f a c t o r s important t o  s h e l l hardness t h a t a r e a s s o c i a t e d with s h e l l l e v e l .  Two  a d d i t i o n a l f a c t o r s worthy o f c o n s i d e r a t i o n are c r y s t a l l i t e s i z e and the o r g a n i c matrix t h a t extends through shell.  the egg  C r y s t a l l i t e s i z e i s r e p o r t e d t o be much s m a l l e r  toward the i n n e r s u r f a c e o f the s h e l l than near the o u t e r surface  (S2) and, f o r metals, hardness i n c r e a s e s as the  g r a i n s i z e decreases  (H2). Organic matter i s known t o be  - 39 -  TABLE X.  MULTIPLE CURVILINEAR REGRESSIONS OF HARDNESS' ON COMPOSITION AND SHELL LEVEL - POOLED BASIS (n = 270).  Independent variable - 27 .21ns  CaOt (CaO)  2  MgO (MgO) P  2  2°5>  0 .279ns  0 .290ns  1 .410ns -  1 .429ns  1 . 500ns  1 .785ns  - o .167ns  2  -324  Xtt 2  - 26 . 65ns 0 .273ns -  O  A  A  -224 .4**  3  Constant  842 .7  546  2 •620ns  -  0 .181ns  0 .261ns  -  0 .029ns  -324 531 . 3**  -234 . 3A "A " •3  877 .1  1 .507ns 1 .559ns  -333 .4**  529 . 3 " "  X X  - 28 . 41ns  1 .617ns  2°5  ( P  Regression c o e f f i c i e n t 2 3  -225 .9** 827 .1  -347 584 .3** -254 . rt/ "A "A 184 .6  Syttt  8 .42  8 .39  8 .40  8 .78  R tttt  0 .814  0 .814  0 .814  0 .796  2  Percentage compositions by weight are used i n the r e g r e s s i o n f o r the i n d i c a t e d compounds tt  Shell  level  ttt  Standard e r r o r o f estimate  tttt  Coefficient  ns  Not s i g n i f i c a n t a t P <  **  S i g n i f i c a n t a t P < 0.01  of multiple  correlation 0.05  - UO  -  present throughout the egg s h e l l  (SU) i n the form of  fibrils  and v e s i c l e s , and t h e r e i s evidence of h i s t o c h e m i c a l d i f f e r e n c e s a c r o s s the t h i c k n e s s o f the s h e l l  (S2).  Mean  v a l u e s o f estimated o r g a n i c content obtained i n t h i s research  (Table VI) show v a r i a t i o n s w i t h s h e l l l e v e l .  o b s e r v a t i o n s are s u g g e s t i v e ; however, t h e r e i s no evidence  linking crystallite  direct  s i z e and the nature o f the  o r g a n i c matrix w i t h hardness i n the hen's egg SUMMARY AND  These  shell.  CONCLUSIONS  Hardness and chemical composition were measured at ten p o s i t i o n s a c r o s s the t h i c k n e s s of 27 egg  shells.  Hardness v a r i e s i n a c u r v i l i n e a r manner w i t h maximum v a l u e s at the o u t e r s u r f a c e , i n t e r m e d i a t e v a l u e s a t the i n n e r s u r f a c e and minimum v a l u e s midway between.  Calcium  content  i n c r e a s e s toward the i n s i d e o f the egg s h e l l whereas magnesium and phosphorus are a maximum at the o u t e r s u r f a c e and  f o l l o w q u a d r a t i c and e x p o n e n t i a l g r a d i e n t s r e s p e c t i v e l y . For the data o f i n d i v i d u a l s h e l l s , v a r i a t i o n i n  chemical composition e x p l a i n s an average of 9 0.9  percent  o f the v a r i a t i o n i n hardness; however the r e l a t i o n s h i p between hardness and composition d i f f e r s from s h e l l t o From the a n a l y s i s of pooled data f o r a l l s h e l l s , i n composition account f o r U8.2 hardness.  shell.  variations  p e r c e n t of the v a r i a t i o n i n  I n c l u s i o n o f s h e l l l e v e l i n the r e g r e s s i o n i n c r e a s e s  the c o e f f i c i e n t of m u l t i p l e d e t e r m i n a t i o n t o 81.U  percent;  - Ul -  thus, may  a d d i t i o n a l unknown f a c t o r s a s s o c i a t e d w i t h s h e l l  have an a p p r e c i a b l e  /  i n f l u e n c e on egg  level,  s h e l l hardness.  CHAPTER I I .  Egg  STRUCTURE OF EGG  SHELL MEMBRANES  s h e l l membranes form a f l e x i b l e  p a r t i t i o n between the egg  albumen and  the  leathery  shell.  This  s t r u c t u r e c o n s i s t s o f matted f i b e r s with a combined thickness at the  of about 65 microns (R3).  a i r c e l l region  of the egg  Two  l a y e r s are  evident  where the f i b e r s separate  t o form the  i n n e r and  i n the egg,  the membranes appear as a s i n g l e l a y e r ; however,  they may  o u t e r membranes.  e a s i l y be p u l l e d Although the  o u t e r membrane (R3), preventing  locations  apart.  i n n e r membrane i s t h i n n e r than  the  b a c t e r i a l i n v a s i o n of the egg  of t h i s i n v e s t i g a t i o n was  o f the  egg  (Gl, LI).  t o e l u c i d a t e the  The structure  s h e l l membranes w i t h p a r t i c u l a r emphasis  d i f f e r e n c e s between the  i n n e r and  REVIEW OF THE The  LITERATURE s h e l l membranes  been known f o r over a century a c c o r d i n g  to 1.5  microns were r e p o r t e d ;  however, d e s c r i p t i o n s  l i m i t a t i o n s imposed by the a v a i l a b l e microscopes.  -  of  F i b e r diameters  o f f i n e s t r u c t u r e were not p o s s i b l e at t h a t time due  42  has  to d e s c r i p t i o n s  l i g h t microscopy ( N l , S8).  -  on  outer l a y e r s .  f i b r o u s nature of egg  e a r l y s t u d i e s by  the  i n n e r l a y e r i s more e f f e c t i v e i n  object  o f 0.5  At o t h e r  to  - 43 -  Recent  s t u d i e s ( M l , S4) o f egg s h e l l membranes by  e l e c t r o n microscopy  r e v e a l e d two d i s t i n c t r e g i o n s i n the c r o s s  s e c t i o n o f i n d i v i d u a l f i b e r s - an i n n e r e l e c t r o n dense core and a l i g h t e r g r a n u l a r mantle surrounding the c o r e .  The core  o  consists of a-keratin f i b r i l s  30 t o 40 A i n diameter and the  mantle i s thought t o be a mucopolysaccharide two it  material.  The  r e g i o n s a r e separated by an o p t i c a l l y empty gap; however, i s not known whether the gap i s p r e s e n t i n the normal  t i s s u e o r i s an a r t i f a c t r e s u l t i n g from the p r e p a r a t i o n s f o r electron  microscopy. Observations on a t r a n s v e r s e s e c t i o n through the  membranes (S4) i n d i c a t e t h a t f i b e r diameters are somewhat l a r g e r i n the outer membrane than i n the i n n e r .  In a s i n g l e  plane s e c t i o n through the membrane, f i b e r s are c u t i n c r o s s s e c t i o n and l o n g i t u d i n a l l y ; t h u s , the f i b e r s must r u n i n v a r y i n g d i r e c t i o n s throughout  the membrane.  At the i n n e r  s u r f a c e o f the i n s i d e membrane t h e r e i s a t h i n l a y e r o f m a t e r i a l t h a t appears Experiments  t o be continuous over the s u r f a c e .  w i t h f r e s h eggs have shown t h a t t h i s l a y e r i s  impermeable t o egg albumen (B9, N I ) ; t h e r e f o r e , i t may serve t o exclude egg albumen from the spaces between f i b e r s i n the membrane. In g e n e r a l , s t u d i e s on egg s h e l l membranes have been q u a l i t a t i v e i n nature w i t h l i t t l e  i n f o r m a t i o n on f i b e r  and o r i e n t a t i o n s along w i t h the v a r i a t i o n s i n these  sizes  - 44 -  c h a r a c t e r i s t i c s at d i f f e r e n t p o s i t i o n s i n the membrane. present  experiment was  The  i n i t i a t e d to provide q u a n t i t a t i v e  measurements o f f i b e r s i z e and o r i e n t a t i o n i n egg  shell  membranes. EXPERIMENTAL METHODS P r e p a r a t i o n o f Specimens Two  f r e s h eggs were randomly s e l e c t e d from the  p r o d u c t i o n of Single-Comb White Leghorn p u l l e t s  (WL1  and  WL2)  (NH1  and  and another two  from New  Hampshire p u l l e t s  The  eggs were opened, contents were d i s c a r d e d and  was  washed from the egg  solution  (pH  NH2).  albumen  i n t e r i o r u s i n g phosphate b u f f e r  =7.4).  Membrane samples were taken from the a i r c e l l r e g i o n o f each egg  i n such a manner t h a t o b s e r v a t i o n s  be made on t r a n s v e r s e  s e c t i o n s t h a t would be  l a t i t u d i n a l with r e s p e c t t o the egg almost p a r a l l e l to the egg  could  approximately  geometry, t h a t i s ,  equator.  A p i e c e o f the i n n e r membrane was i n 3% gluteraldehyde-phosphate  e x c i s e d and  placed  b u f f e r s o l u t i o n f o r a 30 minute  primary f i x a t i o n .  A s m a l l p a r t o f the s h e l l with the  adhering  o u t e r membrane was  t r e a t e d w i t h the f i x a t i v e f o r 90 minutes.  F o l l o w i n g f i x a t i o n , membrane samples were washed i n phosphate buffer.  The  egg  s h e l l was  d e c a l c i f i e d using a 6% s o l u t i o n o f  the disodium s a l t of ethylene  diamine t e t r a - a c e t i c  (EDTA)  c o n t a i n i n g 6% paraformaldehyde with the pH a d j u s t e d t o  7.4  - 45 -  by sodium h y d r o x i d e .  Membrane samples were then c u t i n t o  small p i e c e s and f u r t h e r f i x e d and s t a i n e d f o r 2 hours w i t h 1% osmium t e t r o x i d e i n phosphate b u f f e r . dehydrated  with a s e r i e s of i n c r e a s i n g l y  The t i s s u e was concentrated  e t h a n o l s o l u t i o n s , f o l l o w e d by replacement o x i d e , then i n f i l t r a t e d and embedded  with  propylene  i n Epon 812 ( L 3 ) .  Transverse s e c t i o n s o f the membranes were c u t a t o 900-1500 A w i t h a P o r t e r Blum ultramicrotome.  The s e c t i o n s  were p i c k e d up on carbon coated g r i d s and s t a i n e d f o r 15 minutes w i t h u r a n y l a c e t a t e s a t u r a t e d i n 70 percent methanol, then washed and s t a i n e d f o r 10 minutes i n l e a d c i t r a t e  solution  (R2). Examination The microscope  by E l e c t r o n  Microscopy  s e c t i o n s were examined i n a P h i l i p s 75C e l e c t r o n  a t 60kV and images were recorded u s i n g an e l e c t r o n  o p t i c a l m a g n i f i c a t i o n o f 830 diameters.  Micrographs  were  p r i n t e d a t t o t a l m a g n i f i c a t i o n s o f 6000 t o 7000 t i m e s .  Some  h i g h e r m a g n i f i c a t i o n s up to 40,000 times were used t o examine f i n e s t r u c t u r e o f the membranes.  Transverse s e c t i o n s o f the  o u t e r membranes were recorded by choosing s e v e r a l adjacent f i e l d s and p r e p a r i n g composites  o f the r e s u l t i n g  micrographs.  These methods p r o v i d e d views o f complete t r a n s v e r s e s e c t i o n s through both the i n n e r and o u t e r s h e l l membranes o f the f o u r eggs.  - 46 -  Measurement o f Membrane  Dimensions  A c o o r d i n a t e system was a p p l i e d t o each t r a n s v e r s e s e c t i o n by marking a r e c t a n g u l a r area on the micrographs such t h a t one p a i r o f o p p o s i t e s i d e s c o i n c i d e d w i t h the o u t e r and i n n e r boundaries o f the membrane.  F i b e r dimensions  ( f i g u r e 9 ) were measured from micrographs o f t r a n s v e r s e s e c t i o n s f o r each o f the f o u r o u t e r and i n n e r membranes. Many f i b e r s were c u t o b l i q u e l y and appeared t o be elliptical  i n the s e c t i o n .  The mantle  surrounding the f i b e r  core was not o f uniform t h i c k n e s s even i n f i b e r s c u t a t r i g h t angles t o t h e i r major axes; t h u s , the l e a s t of  the e l l i p t i c a l  diameter  s e c t i o n was assumed t o be the t r u e  fiber  diameter and f i b e r core dimensions were measured i n p r e f e r e n c e to  the combined  core and mantle dimensions.  Average  mantle  t h i c k n e s s was e v a l u a t e d i n the d i r e c t i o n o f the l e a s t diameter of  the f i b e r s e c t i o n .  Each f i b e r i n the membrane s e c t i o n  p r o v i d e d f i v e measurements:  f i b e r core diameter ( d ) , c  maximum core diameter (d ), average mantle t h i c k n e s s m  ( t ), m '  the  d i s t a n c e from the f i b e r c e n t e r t o the o u t s i d e edge o f  the  membrane s e c t i o n  (x) and the angle between the o u t s i d e  edge o f the membrane s e c t i o n and the major a x i s o f the f i b e r c r o s s s e c t i o n (<}>). A n a l y s i s o f Data L i n e a r measurements from the micrographs were converted t o a c t u a l dimensions i n the t i s s u e u s i n g the appropriate magnification factors.  The p o s i t i o n o f the f i b e r  -  47  -  F i g u r e 9 . Schematic diagram o f an o b l i q u e s e c t i o n through an egg s h e l l membrane f i b e r to i l l u s t r a t e the dimensions measured.  -  in relation  H8 -  t o the membrane s u r f a c e s was d e r i v e d from the  d i s t a n c e between the f i b e r c e n t e r and the o u t s i d e edge o f the membrane.  T h i s dimension was d i v i d e d by the t o t a l  membrane t h i c k n e s s t o g i v e a number between 0 and 1.0 which was c a l l e d  membrane l e v e l . The  p r o p o r t i o n o f the t r a n s v e r s e s e c t i o n occupied  by f i b e r s was computed f o r each membrane. assumed t o be c i r c u l a r  F i b e r s were  i n c r o s s s e c t i o n with the mantle  u n i f o r m l y d i s t r i b u t e d around the f i b e r c o r e . fibers  t h a t passed through the plane  Thus, f o r  o f the s e c t i o n i n a  d i r e c t i o n other than normal t o the s e c t i o n , the exposed o b l i q u e s e c t i o n s were e l l i p t i c a l and the areas c a l c u l a t e d accordingly.  The r a t i o o f t o t a l exposed f i b e r a r e a and  membrane s e c t i o n area was termed f i b e r area d e n s i t y . Mean v a l u e s o f f i b e r core diameter and mantle t h i c k n e s s were computed f o r the outer and i n n e r membranes o f each egg.  T - t e s t s were used t o determine whether the mean  f i b e r core diameter and mantle t h i c k n e s s d i f f e r between o u t e r and i n n e r membranes o f the same egg.  S i m i l a r comparisons  were a l s o made among o u t e r membrane dimensions and among i n n e r membrane dimensions o f the f o u r eggs by means o f t - t e s t s . The  data f o r each membrane were then  separated  i n t o p o r t i o n s a c c o r d i n g t o membrane l e v e l i n o r d e r t o examine f i b e r dimensions a t d i f f e r e n t t h i c k n e s s o f the s t r u c t u r e s .  l o c a t i o n s a c r o s s the  On the b a s i s o f equal  membrane  - 49 -  l e v e l i n t e r v a l s , data were d i v i d e d  i n t o t e n and f o u r  f o r o u t e r and i n n e r membranes r e s p e c t i v e l y . standard d e v i a t i o n s  groups  Means and  were then computed f o r each group o f  data. RESULTS AND DISCUSSION Q u a l i t a t i v e Observations on Membrane E a r l i e r descriptions structure  Structure  o f egg s h e l l membrane  ( M l , S4) a r e confirmed by t h i s study.  Individual  f i b e r s possess a c e n t r a l core surrounded by a mantle w i t h a distinct 13).  separation  between the two r e g i o n s  Higher m a g n i f i c a t i o n s  ( f i g u r e s 10 t o  r e v e a l the o r d e r l y f i b r o u s  nature  o f the core i n contrast, t o the g r a n u l a r appearance o f the mantle. cross  Mantle t h i c k n e s s  section.  i s v a r i a b l e even f o r f i b e r s c u t i n  The mantle l a y e r i s o f t e n continuous around  a d j a c e n t f i b e r s and appears t o cement f i b e r s t o g e t h e r where they c r o s s .  In a s i n g l e s e c t i o n , f i b e r s are cut i n c r o s s  s e c t i o n and l o n g i t u d i n a l l y as w e l l as a t angles i n t e r m e d i a t e t o the two extremes; t h u s , the f i b e r s t r a v e l i n many d i r e c t i o n s to form a matted network. At s i m i l a r m a g n i f i c a t i o n s ,  f i b e r s i n the o u t e r  membrane appear l a r g e r than those o f the inner membrane. For some p a i r s o f membranes, p a r t i c u l a r l y from the White Leghorn b i r d s , t h e r e appears t o be l a r g e r i n t e r f i b e r spaces i n the i n n e r membrane.  Inside  boundaries o f i n n e r membranes  have a 0.1 micron t h i c k l a y e r of m a t e r i a l  similar i n electron  - 50 -  Figure 10.  WL1 outer egg s h e l l membrane (66 60X).  Figure 11.  WL2 i n n e r egg s h e l l membrane  (6660X).  - 51 -  Figure 12.  NH1 outer egg s h e l l membrane (70 50X).  Figure 13.  NH1 i n n e r egg s h e l l membrane  (11200X).  - 52 -  d e n s i t y t o the mantle substance ( f i g u r e s 11 and 13). i n n e r membrane has two l a y e r s separated  The WL2  by a narrow gap.  Since the o t h e r membrane s u r f a c e s a r e o f open s t r u c t u r e and i n t e r f i b e r spacings t h i s continuous  are l a r g e r than b a c t e r i a l  dimensions,  l a y e r i s the o n l y v i s i b l e p h y s i c a l b a r r i e r  to m i c r o b i a l p e n e t r a t i o n o f egg s h e l l membranes. R e s u l t s o f Measurements o f Membrane S t r u c t u r e Membrane t h i c k n e s s and f i b e r area  density  Outer egg s h e l l membranes a r e approximately times as t h i c k as i n n e r membranes (Table X I ) .  three  The combined  s t r u c t u r e s range from 7 3 t o 114 y i n t h i c k n e s s . F i b e r area d e n s i t y p r o v i d e s  a measure o f combined  f i b e r s i z e and spacing c h a r a c t e r i s t i c s i n the plane o f the membrane s e c t i o n .  F o r the membranes o f the White Leghorn  eggs, o u t e r membranes have a f i b e r area d e n s i t y twice o f i n n e r membranes.  T h i s r e s u l t confirms  appearance o f the i n n e r membrane outer  that  the more open  ( f i g u r e 11) r e l a t i v e t o the  ( f i g u r e 10). The c l o s e n e s s o f the f i b e r area d e n s i t i e s  f o r the two NH1 membranes i s r e f l e c t e d by t h e i r s i m i l a r i t i e s e v i d e n t i n f i g u r e s 12 and 13.  qualitative  - 53 -  TABLE X I .  THICKNESS AND FIBER AREA DENSITY OF EGG SHELL MEMBRANES  Membrane WL1  WL2  NH1  NH2  t  T h i c k n e s s , JJ  F i b e r area d e n s i t y t  - outer  53.2  0.554  - inner  19.5  0.272  - outer  6 5.5  0.490  - inner  24.3  0.238  - outer  91. 8  0. 369  - inner  22.4  0.388  - outer  80.9  0.456  - inner  27.7  0 .331  F i b e r area d e n s i t y i s d e f i n e d as the r a t i o o f t o t a l exposed f i b e r area and membrane s e c t i o n a r e a .  - 54 Mean v a l u e s o f f i b e r measurements The mean f i b e r core diameter o u t e r membrane i s s i g n i f i c a n t l y mean f i b e r core diameter the f o u r eggs.  (Table XII) o f the  g r e a t e r (P < 0.01) than t h e  o f the i n n e r membrane f o r each o f  Comparisons among corresponding WL1 and WL2  membranes i n d i c a t e t h a t mean f i b e r core diameters  differ  (P < 0.01) between the o u t e r membranes whereas the i n n e r • membrane f i b e r core diameters (P > 0.05). significant diameters  are e s s e n t i a l l y  the same  T - t e s t s o f the NH1 and NH2 membranes r e v e a l no differences  (P > 0.05) o f the mean f i b e r  core  i n the p a i r o f o u t e r membranes and i n the p a i r o f  i n n e r membranes.  In a l l comparisons between corresponding  'membranes o f the White Leghorn and New Hampshire eggs, mean f i b e r core diameters were s i g n i f i c a n t l y  g r e a t e r (P < 0.01)  f o r the New Hampshire eggs. The mean f i b e r mantle t h i c k n e s s i n the White Leghorn eggs does not d i f f e r  (P > 0.05) between o u t e r and i n n e r  membranes nor between membranes o f d i f f e r e n t  eggs.  Mantle  t h i c k n e s s i s g r e a t e r i n the i n n e r NH1 membrane than i n the o u t e r membrane whereas the r e v e r s e i s t r u e f o r the NH2 membranes. In g e n e r a l , f i b e r s  i n the New Hampshire membranes possess  t h i c k e r mantles (P < 0.01) than those i n White Leghorn membranes. The  average  coefficient  of v a r i a t i o n o f 57.8 percent  indicates  t h a t mantle t h i c k n e s s i s h i g h l y v a r i a b l e i n egg s h e l l membranes.  TABLE X I I .  MEAN VALUES OF EGG SHELL MEMBRANE MEASUREMENTS  Number o f fibers measured  microns  WL1 - o u t e r  459  - inner  Membrane  • ttt, microns  degrees  0.681  0.105  -0.34  145  0.481  0.102  2.04  WL2 - outer  571  0.789  0.102  -0.76  - inner  222  0.475  0.096  -2.07  NH1 - o u t e r  443  0.871  0.149  -3.22  inner  152  0.574  0.172  0.53  NH2 - o u t e r  433  0.842  0.151  1.57  - inner  128  0.592  0.120  1.37  -  t  Diameter o f f i b e r  tt  Thickness  ttt  Angle between o u t e r edge o f membrane s e c t i o n and major axis of f i b e r cross section  of fiber  core mantle  - 56 -  I t should be noted t h a t f i b e r s i z e  i s r e p o r t e d as  f i b e r core diameter and average mantle t h i c k n e s s f o r reasons discussed e a r l i e r .  O v e r a l l f i b e r diameter may r e a d i l y  be  computed as the sum o f f i b e r core diameter and twice the mantle t h i c k n e s s . The  angle  < f > (Table X I I ) , between the o u t e r edge o f  the membrane s e c t i o n and the major a x i s o f an exposed s e c t i o n through a f i b e r , r e f l e c t s  the f i b e r d i r e c t i o n  i n relation to  the s u r f a c e s o f the membrane.  If fibers  are assumed t o be  cylindrical, < f > = 0° r e p r e s e n t s  a f i b e r i n a surface  parallel  t o the membrane s u r f a c e s , whereas < f > = + 90° d e s c r i b e s a f i b e r p a s s i n g d i a g o n a l l y across the t h i c k n e s s o f the membrane. Since the mean value o f c}> f o r the f i b e r s approaches z e r o , f i b e r s  i n each membrane  are l o c a t e d i n s u r f a c e s  approximately  p a r a l l e l t o the membrane s u r f a c e s . V a r i a t i o n i n f i b e r measurements a c r o s s the membrane Average f i b e r core diameters a t t e n o u t e r membrane levels  and f o u r i n n e r membrane l e v e l s  14 t o 17.  are shown i n f i g u r e s  No c o n s i s t e n t t r e n d i s e v i d e n t i n mean f i b e r  core  diameter across the t h i c k n e s s o f the f o u r o u t e r and the f o u r i n n e r membranes; however, f i b e r diameters are d i s t i n c t l y f o r the i n n e r membranes o f each p a i r .  smaller  For each o f the i n t e r v a l s  a c r o s s the membrane, f i b e r core diameters show c o n s i d e r a b l e variation  as i s i l l u s t r a t e d i n the f i g u r e s by the v e r t i c a l  bars which r e p r e s e n t  the standard  deviation.  - 57 -  0.2  Figure  14.  0.4  0.6 Membrane  0.5  1.0  level  0.6 Membrane  15.  0  F i b e r core diameters f o r membrane WL1. The mean and standard d e v i a t i o n a t each l e v e l a r e shown.  0.4  Figure  0.8  0.8 level  F i b e r core diameters f o r membrane WL2. The mean and standard d e v i a t i o n a t each l e v e l a r e shown.  - 58 -  0  0.2  F i g u r e 16  0.4  0.6  0  0.5  1.0  • M e m b r a n e level F i b e r core diameters f o r membrane NH1. The mean and standard d e v i a t i o n a t each l e v e l a r e shown.  0.4  0.6 Membrane  F i g u r e 17.  0.8  0-8 level  F i b e r core diameters f o r membrane NH2. The mean and standard d e v i a t i o n a t each l e v e l are shown.  - 59 -  0.4  F i g u r e 18.  0.6 0.8 0.5 1 0 M e m b r a n e level Average f i b e r mantle t h i c k n e s s f o r membranes WL1 and WL2.  Outer .0.3  Inner  • = NH1 NH 2 A  r  tsi  C  0)  c o 0.1  0.2 F i g u r e 19.  0.4  0.6 Membrane  ±  0 8 level  0  0.5  Average f i b e r mantle t h i c k n e s s f o r membranes NH1 and NH2.  1.0  - 60 -  The  average mantle t h i c k n e s s f l u c t u a t e s a c r o s s the  membrane ( f i g u r e s 18 and 19); however, the v a r i a t i o n s appear t o be random. SUMMARY AND CONCLUSIONS The  s h e l l membranes o f f o u r eggs were s u b j e c t e d t o  e l e c t r o n m i c r o s c o p i c a l examination a t r e l a t i v e l y low magnifications.  Micrographs were assembled t o permit  measure-  ments o f p o s i t i o n and dimensions f o r each f i b e r i n t r a n s v e r s e s e c t i o n s through the membranes. Egg  s h e l l membranes c o n s i s t o f a f i b r o u s network  t h a t i s separated  i n t o two d i s t i n c t  layers.  The outer membrane  i s t h r e e times as t h i c k as the i n n e r membrane and the combined s t r u c t u r e i s approximately  100 microns i n t h i c k n e s s .  F i b e r s are o r i e n t e d i n many d i r e c t i o n s i n s u r f a c e s t h a t p a r a l l e l the membrane s u r f a c e s . membranes are l a r g e r than those spacings  The f i b e r s o f outer  o f i n n e r membranes and  among the f i b e r s may d i f f e r from membrane t o membrane. A 0.1 micron continuous  l a y e r o f t i s s u e i s present  at the i n s i d e s u r f a c e o f the i n n e r s h e l l membrane. remaining  Since the  s t r u c t u r e s are an open network, t h i s l a y e r may be  o f fundamental importance i n r e t a r d i n g b a c t e r i a l  penetration  o f egg s h e l l membranes. I n d i v i d u a l f i b e r s w i t h i n the membranes possess a c e n t r a l core surrounded by a g r a n u l a r mantle o f v a r y i n g t h i c k n e s s . F i b e r core diameters are s i g n i f i c a n t l y l a r g e r (P < 0.01) i n the o u t e r membrane than i n the i n n e r membrane o f an egg.  - 61 -  V a r i a t i o n s i n f i b e r core diameter  and mantle t h i c k n e s s occur  a c r o s s the t h i c k n e s s o f egg s h e l l membranes; however, these v a r i a t i o n s appear t o be random.  CHAPTER I I I .  RHEOLOGY OF EGG ALBUMEN  Rheological properties of f l u i d  foods i n f l u e n c e  the design o f m a t e r i a l s h a n d l i n g and p r o c e s s i n g Since most foods have a complex v i s c o u s nature SI, W2) t h a t changes with composition,  equipment. ( C l , H4, L2,  temperature, shear  r a t e and d u r a t i o n o f s h e a r i n g ; s t u d i e s o f food rheology are largely empirical. Three hundred m i l l i o n pounds o f egg albumen a r e processed operations  a n n u a l l y on t h i s c o n t i n e n t  ( F l ) by means o f  t h a t i n c l u d e s e p a r a t i n g , pumping, m i x i n g , h e a t i n g ,  c o o l i n g and spray d r y i n g ; however, very l i t t l e i s a v a i l a b l e on the p h y s i c a l p r o p e r t i e s o f t h i s  information fluid.  Lack o f adequate r h e o l o g i c a l c h a r a c t e r i z a t i o n o f egg albumen has  undoubtedly been an o b s t a c l e i n the design o f p r o c e s s i n g  equipment.  The purpose o f t h i s r e s e a r c h was t o supplement  a v a i l a b l e data on the v i s c o u s behavior  o f egg albumen.  REVIEW OF THE LITERATURE Flow o f egg albumen through c a p i l l a r y under constant  pressure  has been used t o compare c o n s i s t e n c i e s  of samples s u b j e c t e d t o d i f f e r e n t experimental (Bl,  tubes  conditions  S5). Such i n f o r m a t i o n i s u s e f u l i n d e t e c t i n g changes  i n v i s c o s i t y ; however, s i n g l e p o i n t v i s c o s i t y measurements do not c h a r a c t e r i z e flow behavior  over a range o f shear r a t e s  as r e q u i r e d f o r d e s i g n purposes.  Damping p r o p e r t i e s o f  i n t a c t eggs have been s t u d i e d u s i n g a t o r s i o n pendulum - 62 -  - 63 ( A l , RH);  however, the d a t a show s m a l l c o r r e l a t i o n s with the  v i s c o u s nature o f the egg components.  The modulus o f  r i g i d i t y o f t h i c k albumen has been e v a l u a t e d by means o f the displacement o f a s m a l l n i c k e l sphere  i n a magnetic  and by t o r s i o n between c o n c e n t r i c c y l i n d e r s  field  (B7).  Kaufman ejt a l . ( K l ) c i t e d unpublished d a t a i n which v i s c o s i t y o f commercial p a s t e u r i z a t i o n temperatures range o f shear r a t e s .  unfrozen egg products a t  d i d not change g r e a t l y over a  P r e l i m i n a r y t e s t s on t h i c k and  thin  egg albumen, s e p a r a t e l y and combined, at temperatures U0°C, showed d e c r e a s i n g apparent shear r a t e s .  v i s c o s i t i e s with  up to  increasing  Shearing a l s o causes a breakdown o f s t r u c t u r e  d u r i n g t e s t s i n a p r e s s u r i z e d c a p i l l a r y viscometer  (S5)  and i n a r o t a t i o n a l v i s c o m e t e r at a constant r a t e o f shear (T5).  Thus egg albumen e x h i b i t s p s e u d o p l a s t i c , time-dependent  flow b e h a v i o r at moderate temperatures. experiments  The  following  were designed t o e v a l u a t e the r h e o l o g i c a l  b e h a v i o r o f egg albumen over a moderate range o f shear r a t e s and  temperatures. EXPERIMENTAL METHODS P r e p a r a t i o n o f Samples Two  experiment.  l o t s of s i x t y f r e s h eggs were used i n t h i s Twenty randomly s e l e c t e d eggs from each l o t were  weighed, albumen h e i g h t measured and Haugh score c a l c u l a t e d (B5) t o serve as a g e n e r a l i n d i c a t i o n o f egg q u a l i t y . albumen from each l o t of. s i x t y eggs was  combined and  The forced  - 64 -  s i x times through a number 3 Buchner f u n n e l .  T h i s procedure  homogenized the t h i c k and t h i n p o r t i o n s o f the albumen and was  intended t o simulate the commercial  p r a c t i c e i n which  albumen i s passed through a f i n e mesh screen t o remove suspended  materials.  Samples were taken f o r measurements  o f pH and percentage s o l i d s Test  (H6).  Procedures  V i s c o u s p r o p e r t i e s were measured with a Haake r o t a t i o n a l viscometer (VI) equipped with MVI  and NV  gapped c o n c e n t r i c c y l i n d e r s p i n d l e s and cups. s p i n d l e with a gap width of 0.96 r a t e o f 1320  sec  r a t e o f 3140  sec  and 0.80  mm.  1  - 1  whereas the NV and was  mm  The  narrowMVI  p r o v i d e d a maximum shear  s p i n d l e had a maximum shear  double-gapped  with widths o f  0.70  C a l i b r a t i o n c o n s t a n t s f o r the s p i n d l e s were  e s t a b l i s h e d by means of o i l v i s c o s i t y s t a n d a r d s . from the dual-range t o r s i o n dynamometer was  Output  f e d t o a 10-inch,  two c h a n n e l , s t r i p c h a r t r e c o r d e r through a switch t h a t p e r m i t t e d s e l e c t i o n of e i t h e r recorder channel.  One  channel was  calibrated  at twice the s e n s i t i v i t y o f the o t h e r t o i n c r e a s e p r e c i s i o n of measurement f o r s m a l l e r s i g n a l s .  Sample temperatures were  maintained w i t h i n +_ 0.5°C by a t h e r m o s t a t i c a l l y water  j a c k e t surrounding the measuring Each sample was  controlled  head.  f i r s t t e s t e d f o r time dependence  by r e c o r d i n g the- s h e a r - s t r e s s decay curve at a c o n s t a n t r a t e o f shear.  The  s h e a r - s t r e s s curve reached a maximum a f t e r  one or two  seconds due t o r e c o r d e r response and  inertial  e f f e c t s , then appeared t o f o l l o w a l o g a r i t h m i c decay to an  - 65 -  e q u i l i b r i u m value i n f i v e t o t e n minutes.  When the shear  s t r e s s reached a constant v a l u e , f l o w - b e h a v i o r curves were obtained by d e c r e a s i n g the shear r a t e over a s e r i e s o f steps and then i n c r e a s i n g the shear r a t e stepwise t o the o r i g i n a l value w h i l e r e c o r d i n g the corresponding shear s t r e s s e s .  The  maximum shear r a t e used i n o b t a i n i n g flow curves d i d not exceed the shear r a t e o f the time-dependence t e s t s . The f o l l o w i n g t e s t s were performed o f egg albumen.  Recovery  u s i n g one l o t  o f shear s t r e n g t h was i n v e s t i g a t e d  at 10°C i n t h r e e samples sheared a t a r a t e o f 3140 sec " ", -  a f t e r which the samples were s t o r e d i n a i r t i g h t  1  plastic  c o n t a i n e r s a t 4°C f o r 32 hours and then r e t e s t e d under identical conditions.  Shear r a t e s o f 1570 and 3140 sec"''"  were used on t r i p l i c a t e samples t o determine of shear r a t e on the time-dependent The  second  the i n f l u e n c e  characteristic.  l o t o f albumen was t e s t e d a t temperatures  of 10, 20, 30 and 40°C u s i n g each o f the two s p i n d l e s .  Shear-  s t r e s s decay curves and e q u i l i b r i u m f l o w - b e h a v i o r curves were o b t a i n e d f o r a l l t e s t s and a t o t a l o f t h r e e runs were made at each e x p e r i m e n t a l c o n d i t i o n . R h e o l o g i c a l Models Models o f time-dependent  behavior  Data were sampled from the c h a r t paper by a method (C2) designed t o p r e s e r v e data e q u a l l y throughout Since shear s t r e s s appeared  i t s range.  t o decrease l o g a r i t h m i c a l l y w i t h  time, the range between the i n i t i a l usable maximum and the  - 66 -  equilibrium  shear-stress  logarithmic  intervals  times n  were were  -n  viscosity  recorded. calculated i n  equilibrium  poise for  experimental  a  was  divided  into  ten  and  the  corresponding  For  the  purpose  for  and  each  n  run.  condition  of  datum.,  where  the  apparent  Data  for  the  pooled  n  is  and  n  and  apparent  viscosity  three  and  stresses  analysis,  is  were  equal  runs  treated  at as  at each  a  single  curve. Using as  the  n  and  independent  n  ~  n  as  e  variable;  dependent l i n e a r ,  hyperbolic  functions  were  tested  the  Functions  that  suitably  data.  n  and where are  t  is  relaxation  and  accuracy  f i t  f i t t e d  i  b  logarithmic  l  o  g  of  the  data  time  to were:  t -*  1  2 0  (n-n >  = a  2  -  b  2  t  [21]  log  (n-n )  = a  3  -  b  3  log t  [22]  e  e  time  by i n  subsequent  accurately  l "  a  and  log  constants.  suggested  in  =  for  variables  respectively.  seconds  Equations  Weltmann  analyses an  with  and  a^,  a  [20]  and  [21]  and  (W4)  thixotropic  with  0.8 3 c o m p a r e d  i n  Hahn  systems.  because  average 0.7 2 a n d  i t  ,  2  et  a  3  ,  b^,  are a l .  Equation  coefficient 0.7 7 f o r  of  2  ,  b  similar (HI)  described  b  the  to  for  [22]  models  stress  was  data  3  used most  determination  Equations  [20]  and  of [21]  - 67 -  Models o f f l o w - b e h a v i o r E q u i l i b r i u m f l o w - b e h a v i o r data were f i t t e d by the w e l l known power-law equation n  » where  = my ' 11  [23]  1  h • \k] * 1/n  n  = apparent  <l/B) 1  v i s c o s i t y , poise -1  Y  = r a t e o f shear, sec  T  = shear s t r e s s , dynes cm~^  m  = the c o n s i s t e n c y index, and  n  = the f l o w - b e h a v i o r  index  Values f o r m and n were d e r i v e d from the i n t e r c e p t and  slope o f the l e a s t - s q u a r e s l i n e a r f i t o f l o g n t o l o g y  for  each experimental c o n d i t i o n . The  power-law i s the s i m p l e s t and most w i d e l y  e m p i r i c a l model f o r non-Newtonian behavior  (B2).  used  Newtonian  flow i s c h a r a c t e r i z e d by a f l o w - b e h a v i o r index o f u n i t y , whereas v a l u e s l e s s than one apply t o p s e u d o p l a s t i c m a t e r i a l s . T h i s model f i t s the flow curves o f many p s e u d o p l a s t i c s over one o r two decades o f shear r a t e but does not apply t o the extremes i n shear r a t e because the equation p r e d i c t s an i n f i n i t e v i s c o s i t y as the shear r a t e approaches zero and a zero v i s c o s i t y f o r i n f i n i t e The  shear  rates.  E l l i s model was a l s o used t o d e s c r i b e the  f l o w - b e h a v i o r data i n the form  n  where  o  T,  = apparent v i s c o s i t y a t zero shear r a t e = shear s t r e s s c o r r e s p o n d i n g with 1/2  /0  a = a  n  parameter  T h i s three-parameter model i s more g e n e r a l l y  applicable  t o r e a l f l u i d s p a r t i c u l a r l y a t low shear r a t e s because i t d e s c r i b e s a zero-shear v i s c o s i t y , n . o  Since e q u a t i o n  [25] i s not s u i t e d to a d i r e c t s o l u t i o n , the  V  T  l/2  parameters  and a were e v a l u a t e d u s i n g a t r i a l - a n d - e r r o r  computer method. Temperature Temperature  E f f e c t s on Flow  dependence o f flow  Behavior  parameters  Flow-behavior parameters have been shown t o be dependent  of polymer  on temperature  solutions (T7) , thus  the power law and E l l i s model parameters were examined as f u n c t i o n s o f temperature.  The data were t e s t e d w i t h  s e v e r a l d i f f e r e n t f u n c t i o n s and were found t o be  suitably  d e s c r i b e d by equations s i m i l a r t o those of T u r i a n (T7). l o g m = l o g m° + A 0  [26]  n = n° + B 0  [27]  log n log T  1/2  Q  = l o g n° + C 0  [28]  TJ  [29]  = log  /2  + D 0  (1/a) = (l/a)° + E 0  [30]  -  rn  where  0 =  69  -  rpo  —  [31]  T° = a r e f e r e n c e temperature, T = temperature,  10°C  °C  A, B, C, D, and E are constants and the s u p e r s c r i p t e d terms are v a l u e s o f the parameters at the r e f e r e n c e temperature  T°.  Temperature dependence of v i s c o s i t y Over a moderate range of temperatures  the  of most f l u i d s changes a c c o r d i n g t o the A r r h e n i u s n = Ae where  viscosity  equation [32]  A E / R T  A = a constant AE = a c t i v a t i o n energy R = gas  constant  T = temperature, Flow-behavior  °K  data were grouped a c c o r d i n g t o shear r a t e  and each s e t of data was e v a l u a t e A and  f o r v i s c o u s flow  f i t t e d by equation [32] to  AE.  Statistical  Methods Used i n Data A n a l y s i s  A l l r h e o l o g i c a l models were f i t t e d l e a s t - s q u a r e s r e g r e s s i o n methods.  t o data by  Time-dependence t e s t s  each p r o v i d e d 30 o b s e r v a t i o n s whereas f l o w - b e h a v i o r u s u a l l y c o n s i s t e d o f 24 separate measurements.  tests  In o r d e r t o  e v a l u a t e the e f f e c t s o f c e r t a i n experimental c o n d i t i o n s on v i s c o u s b e h a v i o r , p a i r s o f r e g r e s s i o n s were t e s t e d f o r d i f f e r e n c e s o f s l o p e and  l e v e l with a covariance a n a l y s i s  o u t l i n e d by Snedecor (S7).  RESULTS AND  DISCUSSION  Sample Q u a l i t y Average Haugh scores of 89.8 for  twenty eggs from l o t s 1 and  corresponding  percentage  and  85.0  were o b t a i n e d  2 respectively.  s o l i d s were 12.92  and  The 11.36  and  pH v a l u e s d u r i n g the t e s t s were e s s e n t i a l l y constant at 8.4  +_ 0.2.  These data i n d i c a t e t h a t the albumen was  of high  q u a l i t y ; hence, some c a u t i o n i s recommended when e s t i m a t i n g r h e o l o g i c a l p r o p e r t i e s of low q u a l i t y albumen on the b a s i s of the data r e p o r t e d h e r e i n .  From the r e s u l t s of p r e v i o u s  experiments a t 2°C on egg albumen of h i g h and (T5),  low  quality  the e f f e c t o f lower q u a l i t y albumen would be more  pronounced i n the time-dependent c h a r a c t e r i s t i c than i n the equilibrium  flow-behavior. R e s u l t s o f Time-Dependence T e s t s  Recovery o f apparent  viscosity after  storage  In f i g u r e 20, the a p p a r e n t - v i s c o s i t y decay for  t h r e e samples a t 10°C  and a shear r a t e of 3140  curve  sec  is  compared with the curve f o r t e s t s repeated on the same samples 32 hours l a t e r . level  Since the two  curves d i f f e r i n slope  and  (P < 0.01), albumen does not r e c o v e r i t s o r i g i n a l  dependent c h a r a c t e r i s t i c .  time-  A c r i t e r i o n of thixotropy i s that  the m a t e r i a l r e t u r n s t o i t s o r i g i n a l s t r u c t u r e when l e f t u n d i s t u r b e d f o r a p e r i o d of time f o l l o w i n g mechanical If  s t r u c t u r a l breakdown i s n o n - r e v e r s i b l e , the f l u i d  - 70 -  agitation. i s s a i d to  - 71 -  0.5  Figure  20.  , Log  1.0  1.5  2.0  2.5  t  A p p a r e n t - v i s c o s i t y decay i n egg albumen t e s t e d before and a f t e r 32 hours s t o r a g e . T e s t s at 10°C and 3140 s e c " shear r a t e . 1  -0.8  P  ^1.4  h  cn o -2.0  h  -2.6  Log  Figure  21  t  A p p a r e n t - v i s c o s i t y decay i n egg albumen at with shear r a t e s o f 1570 and 3140 sec" . 1  10°C  - 72  be r h e o d e s t r u c t i v e recovery  (M2).  -  There i s , however, an  of s t r u c t u r e because the apparent v i s c o s i t y near,  the b e g i n n i n g of the  second curve i s much h i g h e r  apparent v i s c o s i t y o f the i n i t i a l the b a s i s of these r e s u l t s , the i n egg  albumen at constant  and  3140  process.  sec \  Both the  extensive  f o r operations  slope and  (P < 0.01)  low  i n which the v i s c o u s  the  shear r a t e .  As  a  s t r u c t u r e o f albumen i s  preserved.  Apparent-viscosity  f o r the MV1  spindle  because o f t h e i r d i f f e r i n g sec  -1  In covariance  spindle  shear r a t e s  30  and  I t should  s p i n d l e s are c o n s i d e r e d  w h i l e f o r the MV1 analyses  20,  ( f i g u r e 22, Table X I I I )  ( f i g u r e 23, Table XIV).  noted t h a t data f o r the two  Y = 3140  decay  decay curves at 10,  40°C are presented f o r the NV  the two  l e v e l of  shear r a t e s are recommended  E f f e c t o f temperature on a p p a r e n t - v i s c o s i t y  and  apparent-  with the more r a p i d  breakdown at the h i g h e r  r e s u l t of t h i s observation,  t o be  decay  i s shown i n f i g u r e 21 f o r shear r a t e s  curves d i f f e r s i g n i f i c a n t l y decay and  On  s t r u c t u r a l breakdown mechanism  e f f e c t o f d i f f e r e n t shear r a t e s on  v i s c o s i t y decay a t 10°C  the  curve at e q u i l i b r i u m .  E f f e c t o f shear r a t e on a p p a r e n t - v i s c o s i t y The  than  shear r a t e appears to be a combined  thixotropic-rheodestructive  o f 1570  appreciable  ( f o r the NV  spindle, y  = 1320  separately spindle, sec  -1  ).  of a l l p o s s i b l e p a i r s o f l i n e s from  s e t s of data, the  be  slopes are e s s e n t i a l l y the same  - 73 -1.0  0 F i g u r e 22.  0.5  1.0  1.5  2.5  Log t A p p a r e n t - v i s c o s i t y decay curves a t 10, 20, 30 and U0°C - NV s p i n d l e .  0.5  1.0  1.5 Log  F i g u r e 23.  2.0  2.0  2.5  t  A p p a r e n t - v i s c o s i t y decay curves a t 10, 20, 30 and 40°C - MVI s p i n d l e .  TABLE X I I I .  APPARENT-VISCOSITY DECAY CURVES FOR NV SPINDLE  Temperature, °C  Sytt  V  r ttt 2  10  1.138  -0.637  0.086  0.943  20  1.075  -0.676  0.089  0.939  30  0.827  -0.658  0.095  0.925  UO  0.695  -0.715  0.082  0.949  t  a^, bg are constants d e f i n e d by equation [22]  tt  Standard  ttt  C o e f f i c i e n t o f d e t e r m i n a t i o n (n = 30)  e r r o r of estimate  - 75 -  TABLE XIV.  APPARENT-VISCOSITY DECAY CURVES FOR MVI SPINDLE  Temperature, °C  a  3  +  V  Sytt  r ttt 2  10  1.272  -0.4 39  0.166  0.790  20  1.027  -0.529  0.145  0. 821  30  0.678  -0 . 399  0.232  0.531  40  0.457  -0.514  0.148  0.792  t  a^,  are constants d e f i n e d by equation [22]  tt  Standard  ttt  C o e f f i c i e n t of determination  e r r o r o f estimate (n = 30)  - 76 -  (P > 0.05) over the temperature are s i g n i f i c a n t l y d i f f e r e n t of  (P < 0.01). That  s t r u c t u r a l breakdown appears  although v i s c o s i t y  range; however, the l e v e l s i s , the r a t e  u n a f f e c t e d by  i s generally lower a t h i g h e r  R e s u l t s o f Flow-Behavior  temperature temperatures.  Tests  E f f e c t o f storage on f l o w - b e h a v i o r Power-law e q u a t i o n s . f o r t h e flow curves of  the samples t e s t e d a t 10°C b e f o r e and a f t e r  refrigerated  0.32  Y ~ ° '  n  =  n  = o.i8 Y "  0  ,  2  1  [33]  4  [34]  8  flow curves d i f f e r s i g n i f i c a n t l y  and  l e v e l , thus egg albumen appears  (P < 0.01) i n both s l o p e t o undergo a d e t e r i o r a t i o n  v i s c o u s s t r u c t u r e when t e s t e d and s t o r e d .  indices  32 hours o f  storage a r e , r e s p e c t i v e l y  The  in  ( f i g u r e 24)  Flow b e h a v i o r  (n) i n c r e a s e from 0.76 t o 0.82 i n d i c a t i n g t h a t egg  albumen becomes more Newtonian as a r e s u l t o f t h i s  treatment.  E f f e c t o f maximum shear r a t e on f l o w - b e h a v i o r Flow curves f o r samples t e s t e d w i t h the NV s p i n d l e a t 10°C, a t maximum shear r a t e s o f 1570 and 3140 s e c \ in  figure  and  for  25.  a r e shown  The power-law equations f o r the data a r e  r)  = 0.32 Y "  n  = 0.32  y "  0  0  ,  2  3  ,  2  4  [35] [36]  maximum shear r a t e s o f 1570 and 3140 sec  respectively.  Slopes o f the two curves do not d i f f e r  (P > 0.05); however,  the l e v e l s are s i g n i f i c a n t l y d i f f e r e n t  (P < 0.01).  be noted t h a t the flow curves are based apparent  I t should  on e q u i l i b r i u m  v i s c o s i t i e s over a c e r t a i n range o f shear r a t e s and  - 77 -1.12  Log  F i g u r e 24.  2.5  Flow behavior o f egg albumen samples t e s t e d b e f o r e and a f t e r 3 2 hours s t o r a g e . T e s t s a t 10°C w i t h NV s p i n d l e .  2.7  2.9 Log  F i g u r e 25.  y  3.1  3.3  3.5  J  Flow b e h a v i o r o f egg albumen a t 10°C f o r maximum shear r a t e s o f 1570 and 3140 s e c . - 1  - 78 -  t h a t the extent o f s t r u c t u r a l breakdown i n the f l u i d a t a given temperature  i s determined  by the maximum shear r a t e .  Although  most o f the data f o r the two curves a r e w i t h i n the same range of shear r a t e s , the flow p r o p e r t i e s are q u i t e d i f f e r e n t (P < 0.01).  These r e s u l t s i n d i c a t e t h a t egg albumen rheology  i s s t r o n g l y dependent upon the shear h i s t o r y o f the f l u i d . The  power-law model o f flow b e h a v i o r Power-law parameters f o r the albumen flow curves  . ( f i g u r e 26) at 10, 20, 30 and 4 0°C are shown i n Table XV f o r NV and MVI s p i n d l e s .  For each o f the f o u r temperatures,  curves o b t a i n e d by the two s p i n d l e s are s i g n i f i c a n t l y  flow  different  (P < 0.01) t h u s , dependence o f albumen flow behavior on shear h i s t o r y i s confirmed.  The average  c o e f f i c i e n t of determination  o f 0.762 f o r the e i g h t curves i n d i c a t e s t h a t the power-law a c c u r a t e l y d e s c r i b e s the flow b e h a v i o r o f egg albumen over the range o f shear r a t e s t e s t e d . Covariance analyses o f the s i x p o s s i b l e p a i r s o f flow curves o b t a i n e d from the NV-spindle  show s i g n i f i c a n t  differences  (P < 0.05) i n s l o p e s f o r a l l p a i r s except the 20-30°C and 30-40°C •comparisons.  A l l p a i r s o f curves a r e s i g n i f i c a n t l y  (P < 0.01) i n l e v e l .  different  Thus, the flow curves f o r egg albumen  between shear r a t e s o f 520 and 3140 sec  1  show some d i f f e r e n c e s  among f l o w - b e h a v i o r i n d i c e s and s i g n i f i c a n t d i f f e r e n c e s among apparent  v i s c o s i t i e s a t t e n degree i n t e r v a l s from 10 t o U0°C.  M V l - s p i n d l e r e s u l t s do not d i f f e r i n slope (P > 0.05); however, a l l p a i r s o f curves d i f f e r i n l e v e l 20-30°C  lines.  (P < 0.01) except f o r the  - 79 -  F i g u r e 26.  Flow-behavior curves f o r egg albumen a t 10, 20, 30 and H0°C - NV and MVI s p i n d l e s .  - 80 -  TABLE XV.  Spindle  POWER-LAW FLOW-BEHAVIOR  Temperature, °C  NVtt  MVIttt  CURVES FOR EGG ALBUMEN  m  n  r  10  0.351  0.757  0.798**  20  0.457  0.704  0.760**  30  0.433  0.674  0.942**  40  0.424  0.667  0.897**  10  0.256  0.846  0.516**  20  0.260  0.826  0.781**  30  0.298  0.799  0.480**  40  0.250  0.799  0.920**  t  Coefficient  of determination  tt  Shear r a t e s 520 - 3140 s e c  - 1  ttt  Shear r a t e s 220 - 1320 s e c  - 1  **  S i g n i f i c a n t a t P < 0.01 w i t h 22 degrees o f freedom  2  t  - 81 -  It  should be noted t h a t f o r the NV-spindle  f l o w - b e h a v i o r i n d i c e s are s m a l l e r and per 10°C  temperature  Experimental  i d e n t i c a l except t h a t NV-spindle rates.  show g r e a t e r decreases  r i s e than f o r the MVI-spindle  c o n d i t i o n s f o r the two  tests,  tests.  s e t s of t e s t s are  t e s t s are at h i g h e r  shear  Thus, f o r h i g h e r shear r a t e s , egg albumen flow behavior  becomes more p s e u d o p l a s t i c and  furthermore  p s e u d o p l a s t i c i t y per u n i t i n c r e a s e i n The E l l i s  shows g r e a t e r  temperature.  model of flow behavior E l l i s model parameters f o r the flow b e h a v i o r of  albumen at 10, 20,  30 and  40°C are shown i n Table XVI  egg  along  2 w i t h the c o e f f i c i e n t of d e t e r m i n a t i o n ( r ) f o r each c o n d i t i o n . The  average  c o e f f i c i e n t of d e t e r m i n a t i o n (0.625) i n d i c a t e s  t h a t the E l l i s model a c c u r a t e l y (P < 0.01)  d e s c r i b e s albumen  flow p r o p e r t i e s over the range of shear r a t e s s t u d i e d ; however, 2 the power-law p r o v i d e s a b e t t e r f i t of the data (average r 0.762).  Because of lower accuracy o f f i t and  difficulty  i n e v a l u a t i n g flow parameters,  =  relative  the E l l i s  model i s  not j u s t i f i e d as a c o n s t i t u t i v e equation f o r egg albumen over the moderate range of shear r a t e s s t u d i e d . R e s u l t s f o r Temperature E f f e c t s on Flow E f f e c t of temperature  Behavior  on f l o w - b e h a v i o r parameters  The power-law parameters f o r the flow curves of f i g u r e [26] vary w i t h temperature and  [28].  as shown i n f i g u r e s  Consistency c o e f f i c i e n t s  [27]  (m) do not f o l l o w a c l e a r  t r e n d ; however, f l o w - b e h a v i o r i n d i c e s  (n) s t e a d i l y  decrease  - 82 -  TABLE X V I .  Spindle  E L L I S MODEL FLOW-BEHAVIOR  Temperature  NVtt  MVIttt  CURVES FOR EGG  a  ALBUMEN  r t 2  T  l/2  10°C  1.51  - 0.124  78.4  0.76 5*  20  1.72  0.121  56.2  0.72 5*  30  1.76  0.086  51.1  0.9 2 8*  40  1.82  0.081  47. 8  0. 844*  • 10  1.29  0.194  53.6  0.506*  20  1. 35  0.176  44.5  0.755*  30  1.40  0.168  46. 3  0.479*  40  1.42  0 .140  37.2  0.881*  t  Coefficient  of determination  ft  Shear r a t e s 520 - 3140 s e c "  1  ttt  Shear r a t e s 220 - 1320 s e c "  1  **  S i g n i f i c a n t at P < 0.01 w i t h 22 degrees o f freedom  - 83 -  - 84 -  with i n c r e a s i n g temperature. h i g h e r temperatures  Greater p s e u d o p l a s t i c i t y f o r  i n d i c a t e s t h a t egg albumen i s more  s u s c e p t i b l e t o s t r u c t u r a l breakdown at i n c r e a s e d shear r a t e s w i t h i n the 220 t o 3140  sec  range.  As p o i n t e d out i n an  e a r l i e r d i s c u s s i o n , s m a l l e r values of n° and f o r NV-spindle  t e s t s t h a t f o r MVI-spindle  B (Table XVII)  t e s t s , indicate that  egg albumen flow b e h a v i o r i s more p s e u d o p l a s t i c and more s e n s i t i v e t o temperature  changes a t h i g h e r shear  rates.  V a r i a t i o n o f E l l i s model parameters with  temperature  i s shown i n f i g u r e s [29] t o [31] w i t h . v a l u e s of the i n Table XVIII. between 10 and  constants  I n t e r p o l a t i o n t o e v a l u a t e flow parameters 40°C i s p o s s i b l e by means of these  however, these r e s u l t s are based f o u r temperatures  equations;  on t r i p l i c a t e t e s t s a t o n l y  and e x t r a p o l a t i o n beyond t h i s range i s not  recommended. E f f e c t of temperature  on apparent  F i g u r e s [32] and o f apparent  viscosity  [33] show the temperature  dependence  v i s c o s i t y u s i n g shear r a t e as a parameter.  A c t i v a t i o n e n e r g i e s f o r v i s c o u s flow of egg albumen (Table  XIX)  i n c r e a s e w i t h shear r a t e and are c o n s i s t e n t l y lower f o r the MVI-spindle spindle.  data than f o r comparable shear r a t e s w i t h the  NV  A p o s s i b l e "reason f o r t h i s d i f f e r e n c e i s the more  e x t e n s i v e s t r u c t u r a l breakdown o f albumen p r o t e i n s when s u b j e c t e d t o the h i g h e r maximum shear r a t e s i n the NV Since the a c t i v a t i o n energy  apparatus.  f o r v i s c o u s flow i s a r e f l e c t i o n  o f the a t t r a c t i o n s among p a r t i c l e s i n the f l u i d , h i g h e r v a l u e s would be expected w i t h the i n c r e a s e d s u r f a c e area of the disrupted protein  molecules.  - 85 -  TABLE XVII.  TEMPERATURE DEPENDENCE OF POWER-LAW FLOW-BEHAVIOR PARAMETERS  NV S p i n d l e  MVI S p i n d l e  m°t  0.38  0.26  n°  0.75  0.84  A  0.023ns  0.0027ns  -0.030*  B  -0.017*  t  Items i n t h i s column.are d e f i n e d i n equations  ns  Not s i g n i f i c a n t Significant  TABLE XVIII.  TEMPERATURE DEPENDENCE OF ELLIS MODEL FLOWBEHAVIOR PARAMETERS  t  0 T  a t P < 0.10.  a t P < 0.10.  S  T1  [26] and [ 2 7 ] .  NV S p i n d l e  MVI S p i n d l e  0.13  0.19  73.0  1/2  (l/ct)°  0.64  53.0 0.76  C  -0.070*  -0.043*  D  -0.069*  -0.046*  E  -0.035*  -0.022*  t  Items i n t h i s column are d e f i n e d i n equations  *  Significant  at P  < 0.10.  [28] t o [ 3 0 ] .  - 86 -  F i g u r e 30.  Temperature dependence  o f the E l l i s parameter T  F i g u r e 31.  Temperature dependence  o f the E l l i s parameter n .  - 88 -  F i g u r e 32.  Temperature dependence o f apparent v i s c o s i t y u s i n g shear r a t e as a parameter - MV1 s p i n d l e  -0.9  F i g u r e 33.  Temperature dependence o f apparent v i s c o s i t y u s i n g shear r a t e as a parameter - NV s p i n d l e  - 89 -  TABLE XIX.  Spindle NV  MVI  TEMPERATURE DEPENDENCE OF APPARENT VISCOSITY USING SHEAR RATE AS A PARAMETER  *  At  Y 520  AEft  0.0015  22.5  1050  0.00041  28.4  1570  0.00024  30.8  3140  0.00016  32.4  220  0 .0091  14. 3  440  0.0049  16 .8  660  0.0032  19 .0  0.0028  19 .4  1320  t  Constant d e f i n e d by equation [32]  tt  A c t i v a t i o n energy o f v i s c o u s  flow, k c a l  mole  - 1  SUMMARY AND CONCLUSIONS  R h e o l o g i c a l b e h a v i o r o f egg albumen was measured w i t h a narrow-gapped c o n c e n t r i c c y l i n d e r viscometer a t temperatures  o f 10, 20, 30 and 40°C between shear r a t e s o f  220 t o 3140 sec ^.  Apparent  v i s c o s i t y decreases w i t h time  at a constant r a t e o f shear by a combined rheodestructive process.  thixotropic-  Temperature has l i t t l e  e f f e c t on  the r a t e o f s t r u c t u r a l breakdown although apparent v i s c o s i t i e s decrease with i n c r e a s i n g temperature.  Higher  shear r a t e s r e s u l t i n a more r a p i d and e x t e n s i v e breakdown i n egg albumen s t r u c t u r e . Egg albumen d i s p l a y s p s e u d o p l a s t i c flow b e h a v i o r . Storage subsequent  t o t e s t i n g , as w e l l as the use o f  d i f f e r e n t maximum shear r a t e s , r e s u l t s i n d i f f e r e n t flow curves.  The power-law and E l l i s models a c c u r a t e l y d e s c r i b e  the flow d a t a ; however, the power-law i s p r e f e r r e d  because  of a s l i g h t l y b e t t e r f i t t o the data over the range o f shear r a t e s s t u d i e d .  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