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Thermal degradation of thiamine in bread 1982

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T H E R M A L D E G R A D A T I O N O F T H I A M I N E I N B R E A D by LOUISE NADEAU B.Sc., U n i v e r s i t e de M o n t r e a l , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f Food S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y 1982 © L o u i s e Nadeau, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library 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 further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Food Science The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date March 1 6 , 1 9 8 2 DE-6 (2/79) ABSTRACT T h i a m i n e i s an i m p o r t a n t n u t r i e n t f o u n d i n s i g n i f i c a n t amounts i n wheat f l o u r s . T h i s v i t a m i n i s h e a t l a b i l e t h u s d e s t r u c t i o n o c c u r s d u r i n g b r e a d b a k i n g . U s i n g a k i n e t i c ap- p r o a c h , t h e e f f e c t o f h e a t and pH on t h i a m i n e d e g r a d a t i o n i n a model s y s t e m were s t u d i e d . In o r d e r t o compare t h e s t a - b i l i t y o f t h i a m i n e from n a t u r a l (whole wheat) and s y n t h e t i c ( e n r i c h e d w h i t e ) s o u r c e s , t h e r m a l d e s t r u c t i o n o f t h i a m i n e i n t h e two b r e a d s was i n v e s t i g a t e d . D e s t r u c t i o n r a t e s o f t h i a m i n e h y d r o c h l o r i d e i n p h o s p h a t e b u f f e r a t pH 6.0 and t e m p e r a t u r e s between 80 and 120°C were m e a s u r e d . The breakdown r e a c t i o n c o u l d be d e s c r i b e d by f i r s t o r d e r k i n e t i c s . An e n e r g y o f a c t i v a t i o n o f 3^-2. k c a l / m o l e was o b t a i n e d . D e s t r u c t i o n r a t e s o f t h i a m i n e hy- d r o c h l o r i d e i n p h o s p h a t e b u f f e r a t 120°C were measured f o r pH v a l u e s between 4.0 and 7.0. The r e a c t i o n r a t e i n c r e a s e d as t h e s y s t e m was made more a l k a l i n e , w i t h g r e a t e r d e s t r u c - t i o n a t pH 6.0 and above. T h i a m i n e l o s s e s i n an e n r i c h e d w h i t e f l o u r s y s t e m baked a t a n o m i n a l t e m p e r a t u r e o f 246°C ( 4 7 5°F) f o r 60, 75 and 90 min were found t o be 2.4, 27.9 and 29.2%, r e s p e c t i v e l y . Two e x p e r i m e n t s were c a r r i e d o u t w i t h 450 g (1 l b ) e n r i c h e d w h i t e l o a v e s baked a t 221°C ( 4 3 0 ° F ) . B a k i n g t i m e s were 30 - i - min for the f i r s t experiment, and 15, 37 and 60 min for the second experiment. No appreciable thiamine destruction were found in either experiment. The main investigation was with a semi-model system of 12g bread loaves made from enriched white and whole wheat fl o u r s . Four d i f f e r e n t nominal oven temperatures of 177, 221, 246 and 288°C (350, 425, 475 and 550°F) were used with four d i f f e r e n t baking times for each run. The pH of the dough and baked bread were determined. Oven, crust and loaf center temperatures were monitored. The mass average temp- erature data of the bread during baking showed a changing rate of temperature r i s e , and because of t h i s , i t was not possible to obtain kinetic data. However, a linear r e l a - tionship was obtained when the logarithm of the percent t h i - amine retention was plotted against time. This experiment showed a lower thiamine s t a b i l i t y with higher oven tempera- ture. Thiamine was less stable in whole wheat bread than in enriched white bread. This might be explained by higher pH and ash content in whole wheat bread. Thiamine losses dur- ing normal baking of whole wheat and enriched white bread were found to be in the range of 28.3 to 47.8%. - i i CONTENTS ABSTRACT i ACKNOWLEDGMENTS i x page I. INTRODUCTION 1 I I . LITERATURE REVIEW 2 Nomenclature and function 2 Thermal destruction of thiamine 5 Thiamine losses in bread 14 III. MATERIALS AND METHODS 18 Model System 18 Vitamin analysis 18 Preparation of buffered thiamine solutions . 19 Treatment of samples 19 Come-up times . . 20 Temperature effect 20 pH e f f e c t 21 Bread System .22 Thiamine analysis . . . 22 pH determination . 23 Moisture determination 23 Apparatus for heating 23 Flour samples 23 Enriched white flour s l u r r y assay 23 One pound loaves (enriched white bread) . . 24 Experiment 1 24 Experiment 2 . . . . 24 12 g loaves (enriched white and whole wheat bread) .24 Treatment of samples 25 Experiment 1 26 Experiment 2 26 IV. RESULTS 31 Model System 31 Temperature effect 31 pH e f f e c t 36 Bread System 42 - i i i - Enriched white flour slurry assay 42 One pound loaves (enriched white bread) . . . 42 Experiment 1 42 Experiment 2 42 12g loaves (enriched white and whole wheat bread) 45 Experiment 1 45 Experiment 2 46 V. DISCUSSION 64 VI. CONCLUSION 72 LITERATURE CITED 73 Appendix page A. THIAMINE LOSSES IN BREAD 77 B. STATISTICAL ANALYSIS - 12G LOAVES (EXPERIMENT 2) . 81 t-tes t 81 F - s t a t i s t i c 82 SNK test 83 Enriched white bread 83 Whole wheat bread 83 Deviation from l i n e a r i t y 83 C. EXAMPLE OF CALCULATION FOR % THIAMINE RETENTION . . 86 Model system . 86 Bread system 86 iv - L I S T OF TABLES Table page 1. Review of kinetic studies for the thermal degradation of thiamine 9 2. Retention of thiamine hydrochloride in phosphate buffer (ph 6.0) at d i f f e r e n t heating times between 80 and 120°C 32 3. k, r and T* 1/2 values for thiamine HCl in phosphate buffer (pH 6.0) in the temperature range 80 to 120°C 35 4. Retention of thiamine hydrochloride in phosphate buffer (120°C) at d i f f e r e n t heating times between pH 4.0 and 7.0 38 5. k, r and f' 1/2 values for thiamine HCl in phosphate buffer (120°C) in the pH range 4.0 to 7.0. . . . 40 6. Thiamine retention for enriched flour slurry for d i f f e r e n t baking times at 246°C (475°F) (nominal) 43 7. Thiamine retention in 450 g (1 lb) loaves of enriched white bread baked at 221°C (430°F) for d i f f e r e n t times 44 8. Thiamine retention of white and whole wheat bread baked at 4 d i f f e r e n t temperatures for several baking times (Exp.1) 47 9. pH values for white and whole wheat dough and bread baked at 252°C (485°F) (nominal) for 17 min (Experiment 2) 48 10. Oven temperatures for enriched white and whole wheat bread baked in d i f f e r e n t loads at 4 nominal oven temperatures 49 11. Thiamine retention of white and whole wheat bread baked at 4 d i f f e r e n t temperatures for several baking times (Exp.2) 54 - v 12. Slope and r values of thiamine destruction curves for white and whole wheat bread baked at 177, 218, 246 and 288°C 60 13. H a l f - l i f e values for thiamine in enriched white and whole wheat bread baked at 177, 218, 246 and 288°C (nominal) 63 - v i L I S T OF FIGURES Figure . page 1. Enriched white and whole wheat loaves baked at 4 di f f e r e n t baking time periods 27 2. Retention curves for thiamine hydrochloride in phosphate buffer (pH 6.0) 33 3. Retention curve for thiamine hydrochloride in phosphate buffer (pH 6.0) 34 4. Arrhenius plot for the thermal degradation of thiamine hydrochloride in phosphate buffer (pH 6.0) 37 5. Retention curves for thiamine hydrochloride in phosphate buffer at 120°C at various pHs 39 6. The ef f e c t of pH on the destruction rate constant for thiamine hydrochloride in phosphate buffer at 120°C 41 7. Heat penetration curves for enriched white bread baked at 4 nominal oven temperatures 50 8. Heat penetration curves for whole wheat bread baked at 4 nominal oven temperatures 51 9. Heat penetration curves for the middle and crust of enriched white bread baked at 218°C (425°F) (nominal) 53 10. Thiamine retention curves for enriched white and whole wheat bread baked at 177°C (350°F) (nominal) . . . 56 11. Thiamine retention curves for enriched white and whole wheat bread baked at 218°C (425°F) (nominal) 57 12. Thiamine retention curves for enriched white and whole wheat bread baked at 246°C (475°F) (nominal) 58 - v i i - 13. Thiamine retention curves for enriched white and whole wheat bread baked at 288°C (550°F) (nominal) 59 14. Thiamine retention curves for enriched white bread baked at 177, 218, 246 and 288°C (nominal). . . . 61 15. Thiamine retention curves for whole wheat bread baked at 177, 218, 246 and 288°C (nominal). . . . 62 16. A review of pH/k curves for thiamine in buffered solutions 66 - v i i i - ACKNOWLEGMENTS F i r s t , I would l i k e t o e x p r e s s my g r a t i t u d e t o Dr. John V a n d e r s t o e p f o r h i s c o n s t a n t s u p p o r t t h r o u g h o u t t h i s s t u d y . I w i s h t o t h a n k H.S. Ramaswamy, E s t e l l a L e e , Lynne R o b i n - son and e s p e c i a l l y Sherman Yee f o r t h e i r h e l p i n t e c h n i c a l m a t t e r s . I must t h a n k my f i a n c e R o b e r t Rempel f o r h i s h e l p i n t h e s t a t i s t i c a l a n a l y s e s and i n t h e p r e p a r a t i o n o f t h e c o m p u t e r - i z e d m a n u s c r i p t and f o r h i s c o n s t a n t e n c o u r a g e m e n t . F i n a l l y , I would a l s o l i k e t o t h a n k my co m m i t t e e members f o r t h e i r s u g g e s t i o n s . T h i s s t u d y was made p o s s i b l e t h r o u g h a s c h o l a r s h i p t o t h e a u t h o r and a g r a n t t o Dr. John V a n d e r s t o e p from t h e N a t u r a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada. - i x - I INTRODUCTION T h i a m i n e i s an i m p o r t a n t n u t r i e n t . The wheat k e r n e l c o n - t a i n s s i g n i f i c a n t amounts o f t h i a m i n e and t h e r e f o r e baked goods made from wheat f l o u r a r e p o t e n t i a l l y a good s o u r c e o f t h i s n u t r i e n t . However, t h i s v i t a m i n i s h e a t l a b i l e and some d e s t r u c t i o n o c c u r s d u r i n g t h e b a k i n g p r o c e s s . I t i s d e s i r a b l e t h e r e f o r e t o d e t e r m i n e t h e s t a b i l i t y o f t h i s n u t - r i e n t i n t h e s e f o o d s d u r i n g t h e p r o d u c t i o n p r o c e d u r e s , p a r - t i c u l a r l y t h e b a k i n g p e r i o d . Knowledge a b o u t t h e mechanisms o f t h i a m i n e d e g r a d a t i o n can be o b t a i n e d by s t u d y i n g t h e b e h a v i o u r o f t h i a m i n e under d i f f e r e n t c o n d i t i o n s o f t e m p e r a t u r e , t i m e and pH i n b r e a d b a k i n g as w e l l as i n model s y s t e m s . Such s t u d i e s can l e a d t o t h e d e f i n i t i o n o f optimum c o n d i t i o n s f o r t h i a m i n e r e t e n - t i o n . I t would a l s o be i n t e r e s t i n g t o know whether d i f f e r - e n t b r e a d s y s t e m s , t h a t i s , b r e a d s made from whole g r a i n f l o u r s o r e n r i c h e d w h i t e f l o u r s , have any e f f e c t on t h e r e - t e n t i o n o f t h i a m i n e . The most r e l i a b l e and s a t i s f a c t o r y ap- p r o a c h t o o b t a i n i n g i n s i g h t i n t o t h i s i s t h r o u g h s i m p l e r e - a c t i o n k i n e t i c s . - 1 - I I L I T E R A T U R E R E V I E W 2 . 1 N O M E N C L A T U R E A N D F U N C T I O N V i t a m i n a c t i v i t y was r e c o g n i z e d i n 1890 by t h e Dutch p h y s i c i a n E i j k m a n ( N e a l and S a u b e r l i c h , 1 9 7 3 ) - P r o f B . C . P . J a n s e n o f Amsterdam w i t h W.F. Donath f i r s t i s o l a t e d t h e s u b s t a n c e from a n a t u r a l s o u r c e i n 1926 ( W i l l i a m s , 1 9 3 8 ; D w i v e d i and A r n o l d , 1 9 7 3 ; N e a l and S a u b e r l i c h , 1 9 7 3 ) . J a n - sen s u g g e s t e d t h e t r i v i a l name A n e u r i n e , w h i c h has come i n t o e x t e n s i v e use i n E u r o p e . However, b e c a u s e o f t h e r a p e u t i c i m p l i c a t i o n s , t h i s name, a n e u r i n e , was e v e n t u a l l y r e p l a c e d by t h i a m i n e ( t h i a m i n ) ( W i l l i a m s , 1 9 3 8 ; N e a l and S a u b e r l i c h . 1 9 7 3 ) . I t was o n l y i n 1936 t h a t R . R . W i l l i a m s and h i s c o l - l e a g u e s i s o l a t e d s u f f i c i a n t q u a n t i t i e s o f t h e v i t a m i n t o f u l l y i d e n t i f y i t s s t r u c t u r e . They e s t a b l i s h e d i t as b e i n g composed o f a p y r i m i d i n e and a t h i a z o l e m o i e t y and as 3 - ( 2 - m e t h y l - 4 ' - a m i n o - 5 ' - p y r i m i d y l m e t h y l ) - 5 - ( 2 - h y d r o x y e - t h y l ) - 4 - m e t h y l t h i a z o l e ( W i l l i a m s , 1 9 3 8 ; H a r p e r , 1 9 7 3 ; N e a l and S a u b e r l i c h , 1 9 7 3 ) . The f o r m u l a b e l o w shows t h e t h i a m i n e h y d r o c h l o r i d e form : - 2 - 3 NH2.HC1 CH3 Pyr i m i d i n e m o i e t y CH3 CH2 C l - M e t h y l e n e b r i d g e -CH2-CH20H T h i a z o l e m o i e t y The r e s t o f t h i s s e c t i o n i s documented from H o l v e y (1972), H a r p e r (1973) and N e a l and S a u b e r l i c h (1973). T h i a - mine i s r e a d i l y a b s o r b e d from t h e s m a l l i n t e s t i n e w i t h no a p p r e c i a b l e s t o r a g e i n t h e t i s s u e s . A l l e x c e s s i n t a k e i s e l i m i n a t e d i n t h e u r i n e . The a c t i v e form o f t h e t h i a m i n e i s TPP ( t h i a m i n e p y r o p h o s p h a t e ) , d i p h o s p h o t h i a m i n e o r c o c a r - b o x y l a s e : 0 0 1 i CH2 - CH2 - 0 - P - O - P - O H i i OH OH P h o s p h o r y l a t i o n , w h i c h i s r e v e r s i b l e , o c c u r s i n t h e i n t e s t i - n a l mucosa, k i d n e y and l i v e r ( H a r p e r , 1973; N e a l and S a u b e r - l i c h , 1973). TPP p a r t i c i p a t e s as a coenzyme i n t h e o x i d a t i v e d e c a r b o x - y l a t i o n o f a l p h a k e t o a c i d s t o a l d e h y d e s . These r e a c t i o n s p l a y a m a j o r r o l e i n e n e r g y p r o d u c t i o n (ATP) i n p l a n t s and a n i m a l s . In y e a s t s , TPP p r o m o t e s a l c o h o l i c f e r m e n t a t i o n . 4 TPP i s a l s o t h e coenzyme o f t r a n s k e t o l a s e t h a t p a r t i c i p a t e s i n t h e d i r e c t o x i d a t i o n o f p e n t o s e s . Some o f t h e f i r s t s i g n s o f d e f i c i e n c y a r e l o s s o f appe- t i t e ( a n o r e x i a ) , w e i g h t l o s s , l o s s o f m u s c u l a r t o n e and de- p r e s s i o n . B e r i - b e r i , t h e c l a s s i c a l syndrome o f t h i a m i n e d e - f i c i e n c y , i s c h a r a c t e r i z e d by p e r i p h e r a l n e u r o l o g i c a l c h a n g e s and c a r d i o v a s c u l a r t r o u b l e s w i t h oedema (wet b e r i - b e r i ) or w i t h o u t ( d r y b e r i - b e r i ) . A c c u m u l a t i o n o f p y r u v i c a c i d and l a c t i c a c i d , as w e l l as p e n t o s e s , i n t h e t i s s u e s i s p r o b a b l y r e s p o n s i b l e f o r t h e s e t r o u b l e s . B e r i - b e r i i s m a i n - l y f o u n d i n t h e c o u n t r i e s where p o l i s h e d r i c e i s t h e p r i n c i - p a l f o o d . In t h e o c c i d e n t a l w o r l d , a d e f i c i e n c y i s o b s e r v e d w i t h a l c o h o l i s m , p a r t i c u l a r l y where t h e f o o d i n t a k e i s v e r y low. T h i s l e a d s t o t h e W e r n i c k e - K o r s a k o f f syndrome ( m e n t a l c o n f u s i o n , p s y c h o s i s ) when t h e d e f i c i e n c y i s s e v e r e and c h r o n i c . T h i a m i n e d e f i c i e n c y can be d e t e c t e d by d e p l e t i o n o f t h i a m i n e i n u r i n e and b l o o d , r e d u c t i o n o f t h e a c t i v i t y o f e r y t h r o c y t e t r a n s k e t o l a s e , a t h i a m i n e p y r o p h o s p h a t e - r e q u i r - i n g enzyme, and a b normal e l e v a t i o n o f p y r u v i c a c i d and a l p h a k e t o g l u t a r a t e i n b l o o d and u r i n e . The r e q u i r e m e n t s f o r t h i a m i n e i n human n u t r i t i o n a r e u s u - a l l y b a s e d on c a l o r i c i n t a k e . They a r e 0.23 t o 0.50 mg per 1000 k i l o c a l o r i e s . F o r added s a f e t y , t h e Food and N u t r i t i o n Board (1980) recommends 0.5 mg o f t h i a m i n e f o r e a c h 1000 k i - l o c a l o r i e s i n t h e d i e t . T h e r e f o r e t h e recommended a v e r a g e d a i l y i n t a k e f o r men i s 1.2 t o 1.4 mg and 1.0 mg f o r women. 5 Lipids seem to have a saving ef f e c t on thiamine requirements while carbohydrates have an opposite e f f e c t . In the d i f f e r e n t regions of the world, thiamine i s pro- vided by d i f f e r e n t foods. In North America, cereals provide 35% of the thiamine intake; meat (especially pork), 30%; f r u i t s and vegetables, 15% and dairy products,10%. 2.2 THERMAL DESTRUCTION OF THIAMINE Many researchers have worked on the thermal degradation of thiamine for several years. At f i r s t , the r e s u l t s were not very accurate because of the poor methods of thiamine determination, for example, rat growth and diazotation meth- ods. The thiochrome procedure i s now the most widely used method. Only recently has the k i n e t i c approach been used to determine the degradation of thiamine in several food sys- tems. (Farrer, 1955). Some of the degradation reaction products in food or in model systems have just been i d e n t i f i e d (Dwivedi and Arnold, 1973). The main mechanism involves hydrolytic cleavage of the C-N bond of the methyl bridge between thiazole and p y r i - midine moieties of thiamine, leading to a pyrimidine deriva- t i v e , probably 2- methyl- 4- amino- 5- hydroxymethyl- p y r i - midine, and 4- methyl- 5- (B hydroxyethyl) thiazole (Williams, 1938; Dwivedi and Arnold, 1972, 1973). A second reaction involves the breakdown of the thiazole ring with hydrogen s u l f i d e as the major degradation product and other v o l a t i l e products (Dwivedi and Arnold, 1972, 1973). Many d i f f e r e n t factors a f f e c t the thermal degradation of thiamine, and among them, temperature, time of heating and pH are the most important ones. According to a number of d i f f e r e n t authors, as reviewed by Farrer (1955), 1 F e l i c i o t t i and Esselen (1957) and Mulley et a l . ( 1975), the degradation reaction for thiamine can be described as following f i r s t order k i n e t i c s and adhering to the Arrhenius equation. The rate of a f i r s t order reaction is d i r e c t l y proportional to the concentration of the reac- tant. The rate expression which describes a f i r s t - o r d e r re- action i s : - dc = k c dt where c = concentration of reactant t = time k = reaction rate constant or ve l o c i t y c o e f f i c i e n t (expressed in units of time ~\) Upon rearranging: - dc — = k dt c and integrating, t h i s becomes: - In c = k t + constant This author as well as Coppock et a l . (1956) have written excellent reviews and much of the information obtained prior to 1955 i s in these reviews. 7 I n t e g r a t i n g t h i s e q u a t i o n between l i m i t s o f c o n c e n t r a t i o n c 1 a t t 1 and c 2 a t a l a t e r t i m e t 2 , g i v e s : C2 - Cdc c 1 - I n c 2 - (- In c.j) = k ( t 2 - t ̂ ) 2.303 l o g c 1 T h i s c an be m o d i f i e d t o : t 2 - t l c. 2.303 l o g c Q ( 1 ) where C q = c o n c e n t r a t i o n a t t h e b e g i n n i n g (when t i s 0) c = c o n c e n t r a t i o n a f t e r t i m e t T h i s e q u a t i o n w r i t t e n i n e x p o n e n t i a l form i s : - k t c = c Q e The p e r i o d o f h a l f - l i f e , f 1/2, w h i c h i s t h e t i m e n e c e s s a r y f o r h a l f a g i v e n q u a n t i t y o f m a t e r i a l t o decompose, can be c a l c u l a t e d by t h e s u b s t i t u t i o n o f t h e a p p r o p r i a t e n u m e r i c a l v a l u e s i n t o e q u a t i o n ( 1 ) : 2 .303 l o g 1 k = Y 1/2 1/2 0.693 << 1/2 k 4 1/2 i s u s u a l l y i n d e p e n d e n t o f t h e c o n c e n t r a t i o n o f t h e i n i t i a l s u b s t a n c e . 8 The rate constant generally depends on the absolute temperature following the law f i r s t proposed by Arrhenius in 1889: - Ea/RT k = A e where k = reaction rate constant A = frequency factor Ea = energy of activation (kcal/mole) R = gas constant (1.987 kcal/mole K°) T = absolute temperature (K°) (Daniel and Alberty, 1958; Boudart, 1968; Capellos and B i e l - s k i , 1972; Adamson, 1979) A review of the kin e t i c s of thiamine in buffer systems and in foodstuffs i s given in Table 1. Many researchers reported the well known fact that t h i a - mine i s rapidly destroyed in neutral and alkaline medium. Farrer (1941) plotted log k against the corresponding pH and showed an abrupt increase in the slope of the curve, forming two d i s t i n c t straight l i n e s . He concluded that the reaction rate constant was inversely proportional to the hydrogen ion concentration. Lincoln et. _al. (1944) studied the loss of thiamine during cooking (at 100°C) of flour enriched with thiamine hydrochloride and cocarboxyla se at f i v e d i f f e r e n t pH values ranging from 5.8 to 7-5. Cooking losses increased with pH. They also looked at the same two forms of thiamine in phosphate buffer solutions at pH values of 3-5 to 7.0 at Table 1 Review of kinetic studies for the thermal degradatation of thiamine Reference Median pH Temperature range Ea range Kcal/tole (min-1) Watanabe (1939)1 aqueous solution 248°F Greenwood et al. (1944) pork (lunch meat) 210-250°F Rice and Beuk (1945) pork (lean) 49-121°C Farrer and Morrison (1949) phosphate buffer 120-230°F Bendix et aJL_ (1951) whole peas natural PH 220-270°F 21.2 0.0058-0.0351 Farrer (1953) potatoes carrots peas cabbage 6.0 5.7 6.5 5.5 212°F 212 F 212°F 212 F 0.0026 0.0022 0.0021 0.0027 Garrett (1956)2 Bi-HCl liquid multivitamin prep. 3.2 39-158°F 26 0.00118a Feliciotti and Esselen (1957) aqueous solution phosphate buffer carrot puree green bean purge pea puree spinach puree beef heart puree beef liver puree lamb puree pork puree 3.5 4.5-7.0 6.1 5.8 6.8 6.7 6.1 6.1 6.2 6.2 228-300°F 228-300°F 228-300°F 228-300°F 228-300°F 228-300°F 228-300 F 228-300°F 228-300 F 228-300°F 0.0026-0.0944 28.8 0.0024-2.092 27.0 27.0 27.0 27.0 0.0049-0.2326 27.0 27.0 27.0 27.0 V O Table 1 (continued) Reference Medium pH Temperature Ea range range Kcal/frtole (min x) Gillepsy (1962)2 20.0 Mulley et al. (1975a) phosphate buffer 6.0 250-280°F 29.4 0.015-0.067a pea puree natural 250-280 F 27.5 0.0093-0.038a beef puree pH 250-280°F 27.4 0.0091-0.037a peas-in-brine " 250-280°F 27.0 0.010-0.039a puree aCalculated from the D values (k=2.303/TJ). "Steferenced in Farrer (1955). 2Referenced in Lund (1975). 11 120°C. Losses increased with pH, with greater losses be- tween pH 6.25 and 7 . 0 . Pace and Whitacre (1952) studied the pH e f f e c t in corn bread. At some pH value between 6 .2 and 6.6 for the batter (corresponding to pH 7 . 2 to 8 . 9 for the resultant breads) there appeared to be a c r i t i c a l point at which thiamine was rapidly destroyed during baking. With increasing pH from 4 .5 to 7 - 0 in a phosphate buffer at 228, 246, 264 and 282°F ( 1 0 9 , 119, 129 and 139°C), F e l i c i o t t i and Esselen ( 1957 ) observed an increase in the rate of thiamine destruction. The most pronounced change was between pH 6 .0 and 6 . 5 , as indicated by a change in the slope of the log k versus pH curves (similar to Farrer, 1941). The most recent study on the pH eff e c t in a phosphate buffer solution i s by Mulley e_t al. ( 1 9 7 5 b ) . The rates of destruction curves at 265°F (129°C) were determined for thiamine hydrochloride, cocarboxylase and mixtures of the two at pH 4 . 5 , 5 . 0 , 6 .0 and 6 .5 - Log D values (time for 90% destruction) were plot- ted against pH. the D values for pH 4 .0 and 5 - 0 were simi- lar for each system i n d i v i d u a l l y . However, when the pH ex- ceeded 6 . 0 , the D values decreased sharply showing a decreased s t a b i l i t y of the thiamine molecule. In the case of cocarboxylase, the change in the slope of the curve oc- curred at a lower pH than i t did for the thiamine hydrochlo- ri d e . Dwivedi and Arnold ( 1972 ) suggested that thiamine would be more stable at pH 3 . 5 than pH 5 . 0 or 6 .0 because the protonated form of thiamine, which predominates at pH 3 . 5 , i s less prone to thermal destruction. 12 It i s well recognized that thiamine can occur in three forms: free thiamine, pyrophosphate ester (cocarboxylase) and protein-bound thiamine (Booth, 1943; Greenwood e_t a l . , 1943; Lincoln et a l . , 1944; Farrer, 1955). Greenwood et a l . (1943) showed that cocarboxylase was s l i g h t l y more resistant than thiamine, however other workers (Booth, 1943; Lincoln et a l . , 1944; Farrer, 1945; Mulley et a l . , 1975b) found the opposite to be true, i . e . cocarboxylase was much more sus- ceptible to degradation whether in buffer solutions or in foodstuffs. Mulley e_t a_l. ( 1975b) also mentioned that con- centrations of less than 35% of the cocarboxylase form (which i s the amount normally present in foods) did not af- fect the k i n e t i c s of the thermal destruction of t h i s v i t a - min. The protein-bound thiamine has a greater thermal sta- b i l i t y , according to Farrer (1955). However, F e l i c i o t t i and Esselen (1957) mentioned that the combined form (probably the protein-bound form) was found to be less stable, at a given pH, than the free form. These authors suggested that thiamine degradation in foods may be dependent on the i n t e r - relationship of pH and the proportion of the free and the combined form of the vitamin. The work of these d i f f e r e n t researchers has demonstrated that thiamine in food products i s notably more resist a n t to heat than pure thiamine in aqueous or buffer solutions. This would indicate that many factors other than the ones mentioned previously could affect the thermal degradation of 13 thiamine. These are s t i l l not very well established, how- ever they cannot be ignored, and they w i l l be discussed b r i e f l y . Farrer (1955) and several others since then reported that acids, g e l a t i n , albumin, proteins, a and g amino acids, c a l - cium hydrogen phosphate, soluble starch, gums, dextrins, fructose, invertase and i n o s i t o l have a protective or s t a b i - l i z i n g e ffect upon thiamine. Borate, t h i o s u l f a t e , acetate, carbonate, monohydrogen phosphate, potassium dihydrogen phosphate, oxidizing agents ( l i k e potassium bromate, a dough improver), y , 6 , c - a l i p h a t i c amino acids, p-aminobenzoic acid, copper, gamma radi a t i o n , u l t r a v i o l e t l i g h t and u l t r a - sonic waves would accelerate thiamine destruction. Fe, Zn, Al, Sn and Ni could modify the destruction reaction or be without e f f e c t , depending on the conditions obtained in the solutions. No e f f e c t on thiamine s t a b i l i t y has been ob- served with lithium, sodium, potassium, chloride, potassium n i t r i t e , s u l f a t e , iodide, magnesium ions, glucose, ethyl a l - cohol, glycine, xanthine and r i b o f l a v i n . Thiaminases, en- zymes found mainly in fresh water f i s h , s h e l l f i s h and mol- luscs, are capable of destroying thiamine. The percent of moisture in the food, together with other factors, also i n - fluences thiamine destruction. The rate of destruction i s more l i k e l y to increase as the product becomes more concen- trated. F i n a l l y , Farrer(1955) commented that under normal conditions found in foods, most of these factors do not play a very s i g n i f i c a n t role in thiamine degradation. 14 2.3 T H I A M I N E L O S S E S I N B R E A D Even though no work has been done on thiamine kinetics in bread, many studies on thiamine losses in bread and baked products were reported. The data of these studies are pre- sented in Appendix A. A review of the data in the l i t e r a t u r e (as presented in Appendix A) allows one to conclude that the thiamine losses of baked bread are of the order of 20%, whatever the condi- tions. The only exceptions are the 43 and 47% losses found by Tabekhia and D'Appolonia (1979). Among a l l these au- thors, two d i f f e r e n t conclusions were reached. Some au- thors, Schultz et a l . (1942) and Coppock et a l . (1956). agreed that bread made from flours of d i f f e r e n t extractions show similar retention rates, whereas Farrer (1949) and Daw- son and Martin (1942) found higher losses from high-extrac- tion f l o u r s . Goldberg and Thorpe ( 1946) cited in Coppock e_t a l . (1956) also agreed with the l a t t e r , but attributed t h i s r e s u l t to a longer baking time for the wholemeal (100% ex- traction) bread. There are several theories explaining the differences in thiamine retention between low and high extractions f l o u r s . Dawson and Martin (1942) suggested two hypotheses. The f i r s t was that these differences were due to changes in pH, although Farrer (1949) cited in Coppock et al. (1956) com- mented that in Dawson and Martin's study l i t t l e difference in the pH values were detected in the bread. The second hy- 15 pothesis was that the free thiamine was absorbed by the yeast during fermentation and then was protected during sub- sequent baking. They suggested that t h i s would explain higher losses at high extraction rates, e s p e c i a l l y i f the vitamin in the yeast was less susceptible to heat and i f thiamine was bound to bran, and therefore less available to the yeast. Here again Farrer (1949) c r i t i c i z e d them by say- ing that thiamine in yeast was in the cocarboxylase form, which was more thermolabile than the free form. An interesting explanation for higher losses at higher extraction rates i s related to the ash or mineral content of the f l o u r s . Dawson and Martin (1942) and Farrer (1955) showed a linear r e l a t i o n s h i p when ash content, which i s higher as extraction rates increased, was plotted against thiamine destruction. Farrer (1955) also observed that the pH increased with the percent of extraction (from pH 5.68 with 75% extraction to pH 5-98 with 100%). He concluded that because he had shown in previous work (Farrer, 1945) only a s l i g h t increase in the k values between pH 5.0 and 6.0 in phosphate buffer solutions (which i s the predominant anion in cereals) then the increase in the destruction rates may be due not only to the pH but also to the increase in inorganic constituents. Coppock et a l . (1956) commented that they disagreed with the ash theory since they showed similar thiamine losses for a l l the d i f f e r e n t extractions. 16 Only one study by Van der M i j l Dekker (1951) cited in Coppock et al. (1956) showed smaller losses at higher ex- traction rates. This was attributed by other authors (Far- rer, 1955; Coppock et. a l . , 1956) to the use of the diazota- tion procedure, a very poor method for thiamine analysis. The source of thiamine would be a factor that affects i t s s t a b i l i t y . As previously mentioned, the cocarboxylase form (which i s the form of thiamine in yeast) i s more vulnerable to heat. Farrer (1955) suggested that more cocarboxylase could be present in the higher extraction f l o u r s , which would explain the higher thiamine losses. However, some au- thors referenced by Farrer (1955), agreed that wheat con- tains l i t t l e cocarboxylase and l i t t l e or no protein bound thiamine. The cocarboxylase content of 74.9, 85, 98 and 100% extraction flours were reported to be 8.2, 12.9, 11.3 and 10% respectively. A study described by Coppock e_t a l . ( 1956) using i n t e r - nal temperatures of various 450 g (1 lb) bread loaves, cakes and b i s c u i t s found a linear relationship between the product of minutes heated above 80°C and f i n a l temperature minus 80°C versus average percent l o s s . This experiment took into consideration the bulk of the product. And f i n a l l y , Coppock e_t _al. ( 1956) concluded from t h i s l a t t e r study, as well as other studies and their own work, that 'thiamine destruction in baked products i s mainly ther- mal and only to a r e l a t i v e l y small extent affected by other factors'. 17 Although a number of studies have been carried out on thiamine losses in bread, these have been conducted over a wide range of conditions, making i t d i f f i c u l t to compare them. In addition, there have been only a few studies at- tempting to investigate the mechanism of thiamine degrada- tion in food using the kinetic approach, and none have been done on bread. As stated e a r l i e r , the kinetic approach a l - lows one to obtain standardized data to make comparative studies. The objectives of t h i s thesis were to study the destruc- tion of thiamine (using the k i n e t i c approach) i n : 1. a phosphate buffer solution at pH 6.0 over the temp- erature range 80 to 120°C. 2. a phosphate buffer solution at 120°C over the pH range 1.0 to 7.0. 3- in a semi-model enriched white and whole wheat bread system at natural pH over the nominal oven tempera- ture range 177 to 288°C (350 to 550°F). Furthermore, thermal destruction in bread was to be stud- ied to determine whether any difference in the destruction of the natural and synthetic sources of thiamine occurred by comparing their rate of destruction. I l l MATERIALS AND METHODS A l l thiamine analyses were done by the thiochrome method described by the Association of Vitamin Chemists ( 19 6 6). The fluorescence was measured with an Aminco-Bowman Spectro- fluorometer (American Instruments Co. Inc, Silver Spring, MD) and was recorded on a photomultiplier- microphotometer. The wave lengths used were 365 nm for exci t a t i o n , and 435 nm for the output. Blanks were used in each run. An Accumet Model 230 pH/ion meter (Fisher Co., Pittsburg, PA) was used to check pH values. A l l chemicals used were reagent grade and/or prepared as specified in the procedure. 3. 1 MODEL SYSTEM 3.1.1 Vitamin analysis Because the analyses were made on pure solutions, the ex- traction and p u r i f i c a t i o n steps were omitted from the th i o - chrome method. For each run, two standards and two re f e r - ence controls (unheated samples representing 100% thiamine) were measured. - 18 - 19 3.1.2 Preparation o f b u f f e r e d thiamine solutions Intermediate buffered thiamine solutions were made by adding 5.0 ml of stock thiamine solution (see Thiochrome method, p.130 in Assoc. of V i t . Chem., 1966) to a buffer so- lution and d i l u t i n g to 100 ml. Two d i f f e r e n t working buffer solutions were used. One, for the test tubes, was made by adding 5.0 ml of intermediate thiamine solution to a buffer solution and d i l u t i n g to 100 ml. The concentration of thi s working buffer was 0.25 yg thiamine/ml. The other working buffered thiamine solution, for the TDT (thermal death time) tubes, was made with 30.0 ml of intermediate buffered t h i a - mine diluted to 100 ml with a buffer solution, giving a con- centration of 1.5 yg thiamine/ml. 3-1.3 Treatment of samples The samples heated at temperatures lower than 100°C were heated in a water bath using test tubes closed with a metal cap. Those heated at temperatures higher than 100°C were heated in a o i l bath using TDT tubes sealed in an oxy/ace- tylene flame. Both baths were equipped with temperature controls. After heating, the tubes were cooled immediately in an ice bath. HCl (0.01N) solution was added to the sam- ples to adjust the pH to between 3-5 and 4.5. The f i n a l volume of a l l samples was 6.8 ml. The f i n a l concentration of a l l unheated samples was 0.2193 JJ g thiamine/ml. The sam- ples were then stored at 4°C in an ice bath u n t i l needed for analysis. 20 P r e l i m i n a r y work showed t h a t t h e i c e h o l d i n g t i m e and s t o r a g e a t 4°C up t o 30 h r d i d n o t have any s i g n i f i c a n t e f - f e c t on t h i a m i n e c o n c e n t r a t i o n . Fo r e a c h h e a t i n g t i m e , a t o t a l o f 2 t o 6 r e p l i c a t e s were done o v e r s e v e r a l d a y s . 3.1.4 Come-up t i m e s Come-up t i m e s f o r 80, 90 and 100°C were e s t i m a t e d from h e a t p e n e t r a t i o n d a t a o b t a i n e d from two t e s t t u b e s m o n i t o r e d w i t h t h e r m o c o u p l e s . The come-up t i m e s were found t o be 3-6 min f o r 80°C, 4.0 min f o r 90°C, and 2.3 min f o r 100°C. In th e a s s a y s u s i n g t h e TDT t u b e s , t h e come-up t i m e s were e s t i - mated t o be 0.6 min a c c o r d i n g t o t h e p r o c e d u r e d e s c r i b e d by S o g n e f e s t and B e n j a m i n ( 1 9 4 4 ) . In t h e p r e s e n t s t u d y t h e come-up t i m e c o r r e c t i o n s were n o t a p p l i e d t o t h e d a t a , s i n c e t h e e s t i m a t e d come-up t i m e s were v e r y s m a l l and t h e c o o l i n g l a g f a c t o r w i l l compensate f o r t h e h e a t i n g l a g f a c t o r . 3.1.5 T e m p e r a t u r e e f f e c t The b u f f e r s y s t e m used was a 0.2 M p h o s p h a t e b u f f e r ( S o r - ensen) ( G o m o r i , 1955) a t pH 6.0. D e s t r u c t i o n r a t e s were de- t e r m i n e d a t t e m p e r a t u r e s o f 80, 90, 100, 110, 115 and 120°C. The k i n e t i c r a t e d a t a were o b t a i n e d g r a p h i c a l l y by p l o t - t i n g t h e l o g a r i t h m o f p e r c e n t t h i a m i n e r e t e n t i o n a g a i n s t t i m e f o r d i f f e r e n t t e m p e r a t u r e s . The l i n e s were a l l f o r c e d t h r o u g h 100% t h i a m i n e r e t e n t i o n . S l o p e s and 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 (r2s w e r e c a l c u l a t e d by l i n e a r r e g r e s s i o n 21 equation using appropriate formula for slopes forced through the o r i g i n (Zar, 1974, p.214). The destruction rates, k, expressed in reciprocal minutes, were calculated by the for- mula: k = - 2.303 slope H a l f - l i f e values ( I * 1/2) were calculated by the formula: 0.693 Y 1/2 = k Log k values were plotted against the reciprocal of the absolute temperature (°K) x 1000. Slopes were determined by the method of least squares. The energy of a c t i v a t i o n , Ea, expressed in k i l o c a l o r i e s per mole, was calculated by the formula: - Ea/RT k = A e Ea = - 2.303 R (slope) where R (gas constant) = 1.987 kcal/mole K° 3.1.6 pH effect The destruction rates were determined at 120°C and pH of 4.0, 4.5, 5.0, 6.0 and 7.0. This temperature was taken as being representative of average bread temperature while bak- ing. The pH 6.0 data were previously determined in section 2.1.5. Phosphate buffer (0.2M) (Sorensen) (Gomori, 1955) was used for pH 6.0 and 7.0 and 0.2M phosphate-0. 1M c i t r i c acid buffer (Mcllvaine, 1921) for the pH below 6.0. The k i - netic rate data and ^ 1/2 values for the pH e f f e c t were ob- 22 t a i n e d i n t h e same way as f o r t h e d e t e r m i n a t i o n o f t h e temp- e r a t u r e e f f e c t . To e v a l u a t e t h e e f f e c t o f t h e pH on t h e s t a b i l i t y o f t h i - amine, l o g k v a l u e s were p l o t e d a g a i n s t pH. 3.2 BREAD SYSTEM 3.2.1 T h i a m i n e a n a l y s i s The enzyme used f o r t h e e x t r a c t i o n s t e p was a c i d p h o s p h a - t a s e (0.4 u n i t s / m g ) a t a c o n c e n t r a t i o n o f 0.25 g/100 ml. T h i s s t e p was n o t o m i t t e d s i n c e p r e l i m i n a r y work showed h i g h e r t h i a m i n e r e c o v e r y f o r c o m m e r c i a l e n r i c h e d w h i t e and whole wheat b r e a d s when u s i n g t h i s enzyme. The p u r i f i c a t i o n p r o c e d u r e was c a r r i e d o u t as w e l l , b e c a u s e p r e l i m i n a r y de- t e r m i n a t i o n showed t h a t w i t h o u t t h i s s t e p , t h e s o l u t i o n s were n o t c l e a r and b l a n k v a l u e s were v e r y h i g h ( s e e t h i o - chrome p r o c e d u r e 2 ( b ) ( 6 ) , i n A s s o c . o f V i t a m i n C h e m i s t s , 1966). Ionac C-102 ( m o d i f i e d a l u m i n o s i l i c a t e r e s i n 30-80 mesh-85%, by MCB, Los A n g e l e s , CA) and a c t i v a t e d D e c a l s o ( P e r m u t i t - T by t h e F i s h e r Co., P i t t s b u r g , PA) were used as column m a t e r i a l . Two s t a n d a r d s were used f o r e a c h r u n . Two samples o f dough a f t e r p r o o f i n g ( b e f o r e b a k i n g ) were a n a l y s e d a t e a c h r u n as t h e r e f e r e n c e r e p r e s e n t i n g 100% t h i a m i n e r e t e n t i o n . Each sample was f i n e l y g r o u n d i n a C u i s i n a r t Food P r o c e s s o r i n p r e p a r a t i o n f o r t h e e x t r a c t i o n p r o c e d u r e . 23 3.2.2 pH determination In the 12 g bread assays, the pH of the bread was meas- ured in a constantly s t i r r e d s l u r r y made of bread crumbs, or dough, and d i s t i l l e d water. 3.2.3 Moisture determination The moisture in the samples (dough and baked product) were determined by the standard method (AOAC, 1980) using the ground up material l e f t over from the extraction step. Results could therefore be calculated on dry weight basis. 3.2.4 Apparatus for heating A Despatch laboratory e l e c t r i c oven (Minneapolis, MN) with Robertshaw thermostat control was used to heat a l l the samples. 3.2.5 Flour samples The commercial enriched a l l purpose white flour used in a l l assays was labelled as containing 0.45 mg of thiamine per 100 g of flour . 3.2.6 Enriched white flour s l u r r y assay Mixtures of 100 g of enriched flour and 150 g of tap wa- ter were heated at a nominal oven temperature of 246°C (475°F) for 60, 75 and 90 min. Duplicate determinations for each baking time were used. Moisture analysis was done on 24 each baking time mixture for each pair of duplicates. Anal- ysi s was performed right after cooling the samples in an ice bath. Percent thiamine retentions were calculated. 3.2.7 One pound loaves (enriched white bread) The bread formula and procedure were those used by Tabek- hia and D'Appolonia (1979). Bread loaves were also baked at an oven temperature of 2 2 1°C (430°F). Two s l i c e s from each loaf were stored at - 1 0°C prior to analysis. 3.2.7.1 Experiment 1 Two loaves of bread were baked for 3 0 minutes. An aver- age moisture content, using duplicates, was determined on s l i c e s from both loaves. Percent thiamine retentions were recorded for each bread. 3.2.7-2 Experiment 2 Three loaves of bread were baked for 15, 37 and 60 min. No moisture analyses were done. Percent thiamine retentions were recorded for each duplicate for the d i f f e r e n t baking times . 3.2.8 12 g loaves (enriched white and whole wheat bread) The following formula, similar to straight-dough formula from AACC (1969), was used: Flour 120.0 g Water 70.0 g 25 Sugar 6.5 g Shortening 4.0 g Yeast 2.7 g Salt 1.5 g A straight-dough method with 3 hr fermentation and 55 min proofing done in a fermentation cabinet at 36-38°C was used. After the fermentation period, the dough was allowed to rest for 10 minutes, then i t was divided into approximately 20 g loaves (12 g flour each), and f i n a l l y molded by hand and placed into bread pans (dimensions: top: 5.8 cm x 3.1 cm; bottom: 5.0 cm x 2.3 cm; height: 2.3 cm). 3.2.8.1 Treatment of samples Bread loaves were baked for 4 di f f e r e n t time periods at 4 dif f e r e n t nominal oven temperatures. Enriched white and whole wheat bread loaves were baked in d i f f e r e n t batches. The bread was considered to be underbaked for the 2 f i r s t time i n t e r v a l s . For the 3rd time, i t was normal baked, and the l a s t one was overbaked. Normal bake refers to the stage during baking where the colour of the crust and degree of doneness are optimum for consumption in terms of i t s func- t i o n a l and organoleptic properties. Underbaked means when the bread has not been baked long enough to reach the normal bake stage, and overbaked when i t has been baked beyond the normal bake stage. An i l l u s t r a t i o n , on the following page (Figure 1), of the enriched white and whole wheat bread 26 shows the loaves baked at the 4 di f f e r e n t baking time p e r i - ods . After each baking time, the loaves were removed from the oven, allowed to cool at room temperature, and then were frozen (-10°C). Just before the extraction procedure, the bread was thawed at room temperature, or at 37°C The en- t i r e bread lo a f was used for the extraction step in the thiochrome procedure. Each run included two re p l i c a t e s of each baking time at a given temperature for each type of bread . 3-2.8.2 Experiment 1 The nominal oven temperatures used were 163, 191, 218 and 246°C (325, 375, 425 and 475°F) . Two r e p l i c a t e s , baked in the same oven load, for each time and temperature treatment were used. No moisture analyses were done. Only the oven temperature was monitored, using thermocouples. pH of bread after baking was recorded. The percent thiamine retentions were calculated from these experimental r e s u l t s . 3.2.8-3 Experiment 2 The nominal oven temperatures used were 177, 218, 246 and 288°C (350, 425, 475 and 550°F). Four r e p l i c a t e s , baked in the same oven load, were used for each time and temperature treatment. An average moisture content, using duplicates, was determined on each pair of r e p l i c a t e s analysed in the 27 F i g u r e 1: E n r i c h e d w h i t e and whole wheat l o a v e s baked a t 4 d i f f e r e n t b a k i n g t i m e p e r i o d s . 28 the same run. pH of dough before proofing, dough after proofing and bread after baking were recorded. Heat penetration data. The oven, crust and bread crumb temperatures were moni- tored with a 0.12 mm diam. (crust) and 0.60 mm diam. (middle and oven) copper-constantan thermocouple on a Digitec Datal- ogger (United Systems Corporation, Dayton, OH), which re- corded the temperature at one minute i n t e r v a l s . In each oven load, two bread pans were equipped with thermocouples. One thermocouple was placed in the middle of the l o a f , and another was inserted just beneath the top surface of the dough. One thermocouple monitored the oven temperature in the middle of the oven, just above the loaves. The mass av- erage temperatures (MAT) at minute in t e r v a l s for each bread treatment were calculated by using the Stumbo (1965) formula that applies to a cylinder shaped mass: MAT (°C) = °Tcrust - 0.27(°Tcrust - °Tmiddle) This Stumbo formula i s only an approximation of the re a l av- erage bread temperature for a given time. Heat penetration curves (mass average temperature versus time) were plotted for each oven load, taking the average of the two bread MAT data. In addition, the heat penetration data for the crust and the middle were plotted for enriched white bread baked at 218°C (425°F) (nominal temperature). The average oven temperature for each load was also calculated. 29 T r e a t m e n t o f d a t a . D e g r a d a t i o n r e a c t i o n r a t e s were o b t a i n e d g r a p h i c a l l y by p l o t t i n g t h e l o g a r i t h m o f p e r c e n t t h i a m i n e r e t e n t i o n a g a i n s t t i m e f o r t h e v a r i o u s b a k i n g t e m p e r a t u r e s . A l l t h e l i n e s were f o r c e d t h r o u g h 100% t h i a m i n e r e t e n t i o n . A l l s l o p e s and r were c a l c u l a t e d by t h e method o f l e a s t s q u a r e s ( Z a r , 1974, p. 2 1 4 ) . H a l f - l i f e v a l u e s ( Y1/2) were c a l c u l a t e d by t h e f o r m u l a : 0.301 < 1/2 = s l o p e I n t e r p r e t a t i o n o f d a t a . The s l o p e s were used t o compare t h e s t a b i l i t y o f t h i a m i n e i n 1) t h e e n r i c h e d w h i t e and whole wheat b r e a d s f o r e a c h d i f f e r e n t t e m p e r a t u r e ; 2) t h e same t y p e o f b r e a d a t d i f f e r - ent oven t e m p e r a t u r e s . S t a t i s t i c a l a n a l y s i s S l o p e s f o r t h e r e s u l t s o f d a t a p e r t a i n i n g t o b r e a d t y p e s (1 a bove) were compared u s i n g t h e S t u d e n t s ' t - t e s t . S l o p e s f o r t h e r e s u l t s o f d a t a f o r d i f f e r e n t oven t e m p e r a t u r e s (2 above) were compared u s i n g F - t e s t and SNK (Student-Newman- K e u l ) . D e v i a t i o n from l i n e a r i t y , v i z . whether Y i s a l i n e a r f u n c t i o n o f X, was t e s t e d f o r e a c h s l o p e . The t e s t i s a n a l - ogous t o a one-way ANOVA. E s s e n t i a l l y , t h e t o t a l v a r i a n c e i s d i v i d e d i n t o between g r o u p s SS and w i t h i n g r o u p s SS, and t h e between g r o u p s SS was f u r t h e r d i v i d e d so t h a t t h e d e v i a - 30 tion from l i n e a r i t y SS i s equal to the between groups SS minus the regression SS. If the F value i s high, v i z . the deviation from l i n e a r i t y MS i s greater than the within groups MS, then the n u l l hypothesis that the regression i s line a r i s rejected. Results were considered to be s i g n i f i - cant at the maximum l i m i t of the 5% probability l e v e l . (Zar, 1974, chap.16 and 17, and Appendix B) IV RESULTS 4.1 MODEL SYSTEM A sample calculation of percent thiamine retention is given in Appendix C. 4.1.1 Temperature effect Table 2 shows the average percent retention - the stan- dard deviation for thiamine hydrochloride in phosphate buff- er (pH 6.0) for different times at temperatures ranging from 80 to 120°C. Coefficients of variation for the percent t h i - amine retention data varied from 0.0 to 29.8%. Thermal de- struction curves for these data are given in Figure 2 and 3- The extremely good f i t of the experimental points to a straight line is strong evidence that the thermal destruc- tion of thiamine in phosphate buffer is f i r s t order in na- ture. Table 3 gives the k values and the coefficients of deter- mination (r ) for each of these destruction curves. The very high r values also demonstrate the excellent f i t of the curves. These results reveal that thiamine st a b i l i t y decreases as temperature increases. - 31 - Table 2 Retention of thiamine hydrochloride in phosphate buffer (pH 6.0) at different heating times between 80 and 120 C. Thiamine retention (%) Time 80°C 90°C 100°C 110°C 115°C 120°C 10 min 20 min 30 min 40 min 50 min 60 min 87.0+1.9(6) 77.6±3.8(4) 80 min 90 min 100 min 120 min 80.6±4.2(4) 66.9±2.6(4) 3 hrs 76.Oil.5(4) 55.5±3.4(4) 4 hrs 64.9±1.8(3) 47.1±4.6(4) 5 hrs 38.3±5.1(4) 6 hrs 58.9±4.0(4) 32.4±2.1(4) 7 hrs 49.2±3.5(4) 8 hrs 80.0±4.4(4) 12 hrs 32.8±3.8(2) 24 hrs 45.5±1.8(4) 46 hrs 22.4±3.5(4) 72 hrs 9.8±1.8(4) 72.6±3.8(4) 49.9±2.2(3) 37.3±5.7(4) 25.9±1.8(4) 13.6±1.2(4) 69.4±2.5(3) 49.4±8.6(4) 30.6±6.0(4) 29.1±0.3(2) 17.1±1.7(3) 9.610.7(2) 72.0+0.0(2) 51.814.5(4) 37.011.8(4) 27.218.1(4) 24.113.9(3) 15.612.2(3) Values are presented as means 1 standard deviations. Numbers in parentheses refer to number of replicates. 33 Figure 2. Retention curves for rMamine hydrochloride in phosphate buffer (pH 6.0). 34 TIME (HR) LEGEND i TEMP°C • • • 60 Figure 3. Retention curve for thiamine hydrochloride in phosphate buffer (pH 6.0). 35 TABLE 3 k, r 2 and < 1/2 values for thiamine HCl in phosphate buffer (pH 6.0) in the temperature range 80 to 120°C. (°C) (min - 1) (min) 80 0.0005477 0.9952 1265 90 0.001602 0.9862 433 100 0.003199 0.9894 217 110 0.01116 0.9952 62 115 0.01827 0.9871 38 120 0.03132 0.9845 22 Table 3 gives ttllae h a l f — l i f e values f o r the d i f f e r emit heating temperatures. In Figure 4, ttoe l i n e a r i t y of the ^srhenius plot i n d i - cates that the data conform to the Arrheinius equation. Ttoe energy of ac t i v a t i o n , Ea, ©Ibtained from t h i s curve for t'ftie phosphate buffer-system i s .3-4.2 kcal/mole, with a r valuae of 0.8375, which shows a good f i t of t'he data to the curve. 4.1.2 pH e f f e c t The average percent thiamine retention i the standard de- viation for thiamine hydrochloride in phosphate buffer at piH 4.0 to 7-0 heated at 120°C for different, lengths of time are given-in Table 4. Coefficients of variation- for the percent thiamine retention values among the d i f f e r e n t r e p l i c a t e s vary from 0.0 to 25.5%. Figure 5 stows the destruction curves plotted from these data. Like ita the temperature as- say, the experimental points f i t very well to the l i n e . Ttae o k values of these destruction curves wifcih the r values are '2 given in Table 5- High r values confirm the good f i t ©f the l i n e s . H a l f - l i v e s for the d i f f e r e n t pH are given in Table 5. When the k valuaes are plotted against pH (Figure 6), tthe graph shows that tihiamine s t a b i l i t y decreases with pH, with greater losses when the ;piH reaches 6-©. One can observe that the experimental points f i t on a irregular curve shaped l i n e . 37 i i i ' 2.54 2.58 2.62 2.66 2.70 2.74 2.78 2.82 1/T X 1000 (°K~1) Figure 4. Arrhenius plot for the thermal degradation of thiamine hydrochloride in phosphate buffer (pH 6.0). Table 4 at different heating times between pH 4.0 and 7.0. Thiamine retention (%) 4.0 4.5 5.0 5.5 6.0 7.0 (Min) 3 48.4±3.9(3) 7 11.2±1.2(3) 1 0 72.0±0.0(2) 5.6±1.1(2) 1 2 3.7±0.2(2) 15 83.2±4.9(4) 20 51.8±4.5(4) 30 63.0±4.6(3) 63.1±2.8(4) 37.0±1.8(4) 32 61.5 (1) 38 58.3±0.5(2) 40 62.8±2.5(4) 27.2±8.1(4) 45 55.7±0.7(2) 50 24.1±3.9(3) 60 39.2+7.4(6) 42.3±4.1(6) 15.6±2.2(3) 78 36.8+1.5(2) 80 43.9±0.8(3) 90 25.5±3.1(6) 36.1±3.9(3) 110 27.1±6.9(2) 120 32.312.3(2) 21.5il.8(2) 19.6+3.4(5) 160 21.812.5(3) Values are presented as means 1 standard deviations. Numbers in parenthesis refer to number of replicates. Figure 5. Retention curves for thiamine hydrochloride in phosphate buffer at 120°C at various pHs. 40 TABLE 5 p k, r and Y 1/2 (120 v a l u e s f o r °C) i n t h e t h i a m i n e pH r a n g e HCl i n p h o s p h a t e 4.0 t o 7.0. b u f f e r PH k 1 (min ] ) r 2 < 1/2 (min) 4.0 0.009733 0.9942 71 4.5 0.01261 0.9915 55 5.0 0.01459 0.9838 47 5.5 0.01299 0.9769 53 6.0 0.03132 0.9845 22 7-0 0.2870 0.9951 3 41 Figure 6. The effect of pH on the destruction rate constant for thiamine hydrochloride in phosphate buffer at 120°C. 42 4.2 BREAD SYSTEM A s a m p l e c a l c u l a t i o n o f p e r c e n t t h i a m i n e r e t e n t i o n i s g i v e n i n A p p e n d i x C. i 4.2.1 E n r i c h e d w h i t e f l o u r s l u r r y a s s a y R e s u l t s f o r t h e a v e r a g e t h i a m i n e r e t e n t i o n a r e shown i n T a b l e 6. A f t e r 60 m i n u t e s o f b a k i n g a t 246°C (475°F), no a p p r e c i a b l e t h i a m i n e d e s t r u c t i o n was f o u n d , w h e r e a s f o r b o t h 75 and 90 m i n u t e s o f b a k i n g , a b o u t 30% o f t h i a m i n e was d e - s t r o y e d . 4.2.2 One pound l o a v e s ( e n r i c h e d w h i t e b r e a d ) 4.2.2.1 E x p e r i m e n t 1 A f t e r b a k i n g 450 g (1 l b ) l o a v e s o f b r e a d f o r 30 m i n u t e s a t 221°C (430°F) ( o v e n t e m p e r a t u r e ) , no d e s t r u c t i o n o f t h i a - mine was f o u n d . The p e r c e n t t h i a m i n e r e s u l t s were a c t u a l l y s l i g h t l y h i g h e r t h a n 100%: 103-3%, f o r l o a f 1, and 105.2%, f o r l o a f 2. 4.2.2.2 E x p e r i m e n t 2 T a b l e 7 shows t h e mean p e r c e n t t h i a m i n e r e t e n t i o n o f 450 g (1 l b ) l o a v e s made f r o m e n r i c h e d w h i t e b r e a d and b a k e d f o r d i f f e r e n t t i m e s . The r e s u l t s a r e i n c o n c l u s i v e b e c a u s e t h e y a r e on a wet w e i g h t b a s i s . B e c a u s e b r e a d becomes d r y e r as b a k i n g t i m e i n c r e a s e s , and b e c a u s e p e r c e n t t h i a m i n e r e s u l t s 43 TABLE 6 Thiamine retention for enriched flour s l u r r y for d i f f e r e n t baking times at 246°C (475 F) (nominal). Baking time Thiamine (min) retention* 60 97-6 ± 6.0 75 72.1 + 16.7 90 70.8 ± 6.9 *Mean of duplicates ± standard deviations. 44 TABLE 7 Thiamine retention in 450 g (1 lb) loaves of enriched white bread baked at 221°C (430°F) for d i f f e r e n t times. Baking time Thiamine (min) retention* (%) 15 126.3 ± 4.8 37 112.3 ± 21.3 60 97.9 ± 1.3 *Means of duplicates ± standard deviations. 45 are based on the bread dough, which has a higher moisture content than the baked bread, each percent thiamine r e s u l t i s therefore calculated on a d i f f e r e n t weight basis and thus cannot be compared to each other. However, i f the dry weight data of the dough and bread at 30 min baking from Ex- periment 1 are considered in the calculation of the % t h i a - mine retention for the 37 min baking, the resu l t i s 97.6% retention. This res u l t agrees very well with the one from Experiment 1. 4.2.3 12g loaves (enriched white and whole wheat bread) 4.2.3.1 Experiment 1 The mean pH value of two repl i c a t e s (one baked at 191°C and the other one at 246°C - nominal temperatures) was 5.08 for enriched white bread and 5.63 for whole wheat bread. pH of whole wheat bread was slig h t y higher than enriched bread. Table 8 shows the mean percent thiamine retention for en- riched white and whole wheat bread at four d i f f e r e n t oven temperatures, 163, 191, 218 and 246°C (325, 375, 425 and 475°F) , for di f f e r e n t baking times. Because the results were calculated on a wet weight basis instead of on a dry weight basis, they are inconclusive (see explanation in pre- vious section). This i s shown by the very irregular pat- terns of thiamine destruction with time and di f f e r e n t oven temperatures. The greatest thiamine destruction obtained was 26.1% for enriched white bread at 163°C (325°F) for 45 minutes. 46 4.2.3.2 Experiment 2 The pH values of dough and baked bread for enriched and whole wheat bread are given in Table 9. Whole wheat bread as well as the dough have a s l i g h t l y higher pH than enriched white bread and dough. The dough after proofing and the baked bread, for each type of bread, have e s s e n t i a l l y the same pH values. The higher degree of a c i d i t y demonstrated by the dough after proofing, compared to the dough before proofing, i s a res u l t of yeast a c t i v i t y during the fermenta- tion process. Heat penetration data. The recorded average oven temperatures during the baking of the enriched white and whole wheat bread are shown in Ta- ble 10. The oven temperatures for both types of bread at each nominal temperature are in good agreement. Consider- ing that the oven doors were opened four times during each baking period and that the oven has i t s own temperature cy- c l e , the standard deviations are quite small. The c o e f f i - cients of variation for 177, 218 and 246°C (350, 425 and 475°F) are less than 5% and less than 6% for 288°C (550°F). The heat penetration curves, where the mass average temp- erature (MAT) i s plotted against time, are given for a l l the d i f f e r e n t oven temperatures for enriched white bread in Fi g - ure 7, and for whole wheat bread in Figure 8. 47 TABLE 8 Thiamine retention of white and whole wheat bread baked at 4 dif f e r e n t temperatures for several baking times (Exp.1). Nominal Enriched white Whole wheat oven temp (°C) Baking Thiamine Baking Thiamine time retention* time retention* (min) (%) (min) (%) 10 90.7 10 115.1 (325°F) 20 81.7 20 122.5 32 101.1 31 98.3 45 73.7 43 102. 3 191 7 97.2 7 131.8 (375°F) 13 98.8 13 108.5 20 95.4 20 120.8 27 97.8 27 79.7 218 4 94.5 4 93.6 (425°F) 8 80.5 8 98.5 13 82.3 13 104. 1 19 94.2 19 94.9 246 3 119.9 3 71.9 (475°F) 7 102.3 7 95.8 9 88.4 10 109.3 12 100. 6 13 104.7 *Values are means of 2 r e p l i c a t e s . 48 TABLE 9 pH v a l u e s f o r w h i t e and whole wheat dough and b r e a d baked at 252°C (485°F) ( n o m i n a l ) ' f o r 17 min ( E x p e r i m e n t 2 ) . pH* E n r i c h e d w h i t e Whole wheat Dough b e f o r e p r o o f i n g 5 . 3 5 6 . 0 3 Dough a f t e r p r o o f i n g 4 . 9 3 5. 40 Baked b r e a d 5 . 0 3 5 . 3 8 * V a l u e s a r e means o f 2 r e p l i c a t e s . 49 TABLE 10 Oven temperatures for enriched white and whole wheat bread baked in d i f f e r e n t loads at 4 nominal oven temperatures. Nominal Oven temperature ( C) oven temp (°C) Enriched white* Whole wheat* 177 0 152.2 + 7.3 154.9 + 6.8 (350°F) 218 172.6 5.6 177.6 ± 6.9 (425°F) 246 189. 1 ± 8.3 189.5 + 6.3 (475°F) 288 215.8 + 12. 1 217-7 + 11.9 (550°F) *Values are means + standard deviations. 50 Figure 7. Heat penetration curves f o r enriched white bread baked at 4 nominal oven temperatures. 51 160 H 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 60 65 90 95 TIME (MIN) LEGENOi OVEN°TeF 350 e-B-e 425 475 550 Figure 8. Heat penetration curves f o r whole wheat bread baked at 4 nominal oven temperatures. 52 Figure 9 shows the heat penetration curves for the middle and crust of enriched white bread at 218°C (425°F) (nominal temperature). 4 Thiamine retention r e s u l t s . Table 11 summarizes the results of thiamine retention in enriched white and whole wheat bread at d i f f e r e n t baking times at 177, 218, 246 and 288°C (350, 425, 475 and 550°F (nominal oven temperatures). V a r i a b i l i t y in % thiamine re- tention values among the 4 rep l i c a t e s as expressed by the c o e f f i c i e n t s of variation were 2.8 to 35.7% for enriched white.bread, and 3.7 to 23.4% for whole wheat bread. V a r i - a b i l i t y of thiamine retention for whole wheat bread appears to be smaller than for enriched white bread. Figures 10, 11, 12 and 13 show the thiamine destruction curves for each d i f f e r e n t oven temperature comparing en- riched white and whole wheat bread on the same graph. From these experimental data, i t i s evident that thiamine reten- tion for both types of bread decreases with baking time for a l l baking temperatures. Since most of the data points seemed to form a straight l i n e , regression equations were calculated from the data and the l i n e s were drawn through the points and the slopes were calculated. The slopes with r values are given in Table 12. When the s t a t i s t i c a l hy- pothesis Ho: the population regression i s linear i s tested (Zar,1974, p. 215), only the c u r v e for the enriched white 53 Figure 9. Heat penetration curves for the middle and crust of enriched white bread baked at 218 C (425 F) (nominal). 54 TABLE 11 Thiamine retention of white and whole wheat bread baked at 4 dif f e r e n t temperatures for several baking times (Exp.2). Nominal Enriched white Whole wheat oven _______ temp (°C) Baking Thiamine Baking Thiamine time retention** time retention** (min) (%) (min) (%) 175^ 15 72.3 + 1.7 16 78.0 ± 4.4 (350°F) 35 63-6 ± 6.6 40 66. 1 + 2.5* 55 53-3 ± 1.5 65 52.2 ± 1.9 92 51.6 ± 7.1 90 43.5 + 9.4 220 10 88.3 ± 31.5 10 92.9 ± 11.2 (425°F) 20 89.0 ± 18.3 25 71.0 ± 4.2 35 57.2 + 7.9 40 59.0 + 10.1 55 42. 3 + 3. 1 71 20.7 + 4.3 245 6 76.2 + 21.0 7 83-0 + 7.3 (475°F) 15 72.4 ± 16.2 20 70.2 + 7.2 25 71.7 ± 10.8 35 52.2 + 8.2* 40 56. 1 + 8.1 60 26.3 + 8.2 280 5 91.5 ± 12.4 6 83-0 ± 9.4 (550°F) 12 86.8 ± 10.3 15 67.0 + 5.7 22 65.3 + 7.0 22 60.4 + 14. 1 31 45.0 ± 7.8 31 35.3 ± 7.7 **Values are means + standard deviations for 4 r e p l i c a t e s . *0nly 3 rep l i c a t e s were used. 55 bread at 177°C (350°F) deviates s i g n i f i c a n t l y from l i n e a r i t y o at the 5% probability l e v e l . Values of r are quite s i g n i f - 2 icant, e s p e c i a l l y for the whole wheat bread. The lowest r value obtained was for enriched white bread at 246°C (475°F) (r 2=0.7618). For a l l oven temperatures, the slopes for whole wheat bread are higher than the ones for enriched white bread. Only the curves for 177°C (350°F) did not show any s i g n i f i - cant difference (see Appendix B). These results demonstrate that thiamine in whole wheat bread (a natural source) i s less stable than thiamine in enriched white bread (a syn- theti c form) . Thiamine destruction l i n e s for a l l d i f f e r e n t oven temper- atures for each type of bread were also drawn (see Figures 14 and 15). When comparing the slopes from each graph, the slope values increase as oven temperature increases, which means that thiamine become less stable as oven temperature r i s e s . When each l i n e i s compared to each other (SNK test) at 5% l e v e l , 218 and 246°C (425 and 475°F) li n e s for en- riched white and whole wheat bread do not show a s i g n i f i c a n t difference (see Appendix B). H a l f - l i f e values are given in Table 13. 56 Figure 10. Thiamine retention curves for enriched white and whole wheat bread baked at 177 C (350 F) (nominal). 57 Figure 11. Thiamine retention curves for enriched white and whole wheat bread baked at 218 C (425 F) (ncminal). TIME WIN) LEGEND i TYPE • o • ENR.MHIT * * • M. HHEBT Figure 12. Thiamine retention curves for enriched white and whole wheat bread baked at 246°C (475°F) (rcminal). 59 Figure 13. Thiamine retention curves for enriched white and whole wheat bread baked at 288 C (550 C) (nominal). 6© TABLE 12 Slope and r 2 values of thiamine destruction curves for white and whole wheat bread baked at 177, 218, 246 and 288°C. Nominal oven Enriched white Whole wheat temp (°C) Slope (min - 1) r 2 Slope (min~ n) r 2 177„ (350°F) 0.003931 0.8956 0.004264 0 .9619 218 (425°F) 0.006545* 0.8576 0.008491* 0 .9333 246 (475°F) 0.006800** 0.7618 0.009475** 0 .9487 288 (550°F) 0.01039* 0.9327 0.01317* 0 .9187 *Difference between slopes in the same row i s s i g n i f i c a n t at 0.02<p<0.05. **Difference between slopes in the same row i s s i g n i f i c a n t at 0.01<p<0.02. 61 Figure 14. Thiamine retention curves for enriched white bread baked at 177, 218, 246 and 288 C (roriinal). 62 •i 11111111111 i i " " i i 0 5 10 15 20 25 30 35 40 45 SO 55 60 65 70 75 80 85 90 TIME (MIN) LEGEND i TEMP"F • • • 350 * * * 425 A A A 475 O D D 550 Figure 15. Thiamine retention curves for whole wheat bread baked at 177, 218, 246 and 288°C (nominal). 63 TABLE 13 H a l f - l i f e values for thiamine in enriched white and whole wheat bread baked at 177, 218, 246 and 288°C (nominal). i 1/2 Nominal (min) oven temp (°C) Enriched White Whole wheat 177„ (350°F) 77 71 218 (425°F) 46 36 246 (475°F) 44 32 288 (550°F) 29 23 V DISCUSSION This study on kinetics of thiamine degradation in a phos- phate buffer confirms again the work done by the many au- thors mentioned e a r l i e r where thermal degradation of t h i a - mine follows a f i r s t order reaction and adheres to the Arrhenius equation. The Ea value of 34.2 kcal/mole found in th i s experiment i s s l i g h t l y higher than values recorded by F e l i c i o t t i and Esselen (1957) and Mulley et a l . (1975) in the same buffer system (see Table 1). A l i k e l y explanation for the higher value i s that a l l of the destruction curves were forced through 100% retention, whereas these other workers did not correct their curves accordingly. When the present data were not forced through 100% an Ea value of 27.8 kcal/mole was obtained, which i s closer to the values found by these authors. The pH experiment agrees with the findings of the re- searchers cited e a r l i e r , where thiamine destruction i s greater at higher pH. Furthermore, the results show a high- er loss between 6.0 and 7.0, which i s also found by Lincoln et a l . (1944), Pace and Whitacre (1953) and F e l i c i o t t i and Esselen (1957). The pattern of the curve of log k versus pH in Figure 16 agrees f a i r l y well with the ones of Farrer - 64 - 65 (1945), F e l i c i o t t i and Esselen ( 1957) and Mulley e_t a l . (1975b). The only obvious difference i s that in t h i s study there appears to be a small drop in the k value at pH 5.5 when compared to the trend of the 3 preceeding points. A l l the k and f 1 / 2 values found in the model system are in the same range as those found in other studies (Table 1). When the re s u l t s of d i f f e r e n t authors, using similar sys- tems, are compared, the values obtained are not i d e n t i c a l . These differences among k and 4 1/2 values of d i f f e r e n t au- thors and t h i s work may be due to experimental variation in the buffer system used, in the thiamine analysis procedure and/or in the considerations for the treatment of data (e.g. forcing slopes through 100% thiamine retention). The pH values of baked white bread in experiment 1 and 2 agree with the values given by Pomeranz and Shellenberger ( 1973 ) where pH of white bread crumb varies from pH 5.1 to 5.4. It i s surprising to see that there i s no loss of thiamine during normal baking of 450 g (1 lb) loaves of enriched white bread. This i s es p e c i a l l y interesting because 30 to 50 percent thiamine destruction was found in the 12g bread formula at normal baking and 30% in the s l u r r y assay at 75 and 90 minutes (although 0% was found at 60 minutes). Cer- t a i n l y t h i s 0% destruction in 1 lb loaves does not corre- spond well with the 20% destruction results of other work- ers, and i s in sharp contrast to the 50% destruction r e s u l t 66 0.31623 - | Figure 16. A review of pH/k curves for thiamine in buffered solutions. (1) from this work. (2) Mulley'et'al. 11979b) - phosphate buffer at 129 C. (3) Feliciotti and Esselen (1957) - phosphate buffer at 119°C. (4) Farrer (1945) - phosphate buffer at 100°C. (5) Farrer (1945) - phosphate-citric acid buffer at 100°C. 67 of Tabekhia and D'Appolonia (1979), where the same condi- tions were used . One explanation for the 0% destruction i s that yeast might have been active s t i l l during the i n i t i a l period of baking. Because the mass of the bread i s large, i t takes a while for the middle of the bread to reach a temperature where the yeast i s k i l l e d . Some thiamine would then be pro- duced during t h i s i n i t i a l period, which could account for an error in the percent retention c a l c u l a t i o n . The reason why other workers obtained up to 20% destruction could be be- cause the production of thiamine might vary with the variety of yeast, and the type they used produced less thiamine. More 450 g (1 lb) loaf experiments should be done at the normal bake and over bake stage to confirm these r e s u l t s . The 12 g bread formula was used instead of the 450 g (1 lb) loaves mainly because i t was easier to handle. However, these breads are very small and the proportion of surface crust to volume i s much higher compared to 450 g ( 1 lb) loaves. Because most of the thiamine destruction occurs in the crust, one would expect more destruction in these smaller loaves than in the 450 g (1 lb) loaves. This i s well demonstrated by the 30 to 50 percent thiamine destruc- tion found in the 12 g loaves at the normal bake time com- pared to the 0% found in the 1 lb loaves here and the 20% average found by the other workers cited in Appendix A. 68 Because of the unsteady temperatures in the bread during baking, as shown by the heat penetration curves, i t was not possible to calculate the kinetic rate constant (k) and the energy of activation (Ea) for thiamine destruction in bread by means of the steady state approach. Bread being a con- duction heating food with changing temperatures, i t i s prob- ably better analyzed by the a more complex non-steady state approach. This was not done for this work. However, exper- imental results showed that log percent thiamine retention decreased at a constant rate with time. Thus these slopes made i t possible to compare mathematically the difference between whole wheat and enriched white bread. When comparing the slopes for enriched white and whole wheat bread baked at d i f f e r e n t oven temperatures, the 218 and 246°C (425 and 475°F) slopes were found to be not s i g - n i f i c a n t l y d i f f e r e n t . This can be explained for the whole wheat by the fact that their MAT curves are similar for the two temperatures, therefore the same percent destruction would be expected. However, t h i s explanation cannot be true for the enriched white bread since their MAT curves for 218 and 246°C (425 and 475°F) are quite d i f f e r e n t . However, the difference in oven temperature between these two nominal oven temperatures i s only 16.5°C for enriched white and 11.9°C for whole wheat, whereas the difference between the other temperatures are much higher (see Table 1 0 ) . This l a t - er observation i s a more l i k e l y explanation for the non-sig- 69 n i f i c a n t difference between the slopes for 218 and 246°C (425 and 475°F). Slopes show a higher thiamine i n s t a b i l i t y as oven temper- ature increases. But because bread i s baked in less time as oven temperature increases, during normal baking of bread there i s not more destruction of thiamine at the higher temperature. For a l l curves, i t appears that the MAT increases accord- ing to a pattern approximated by two straight l i n e s . F i r s t , the MAT increases very rapidly for 6 to 15 minutes, with slopes increasing as oven nominal temperature increases. Then, forming a second l i n e , the MAT continues to increase at a constant rate, but slower than the f i r s t l i n e . This rate also i s greater with increasingly higher oven tempera- tures. The 288°C (550°F) curve i s the only curve where the experimental points would not f i t well on t h i s hypothetical 2nd straight l i n e pattern. Also, when curves for enriched white and whole wheat bread are compared for the same oven temperature, they do not agree very well with each other, esp e c i a l l y the curves at 246°C (475°F). MAT curves for en- riched white bread are a l l steeper than the whole wheat bread curves. This i s l i k e l y due to the d i f f e r e n t composi- tion and moisture retention of the two types of bread, hav- ing d i f f e r e n t heat penetration properties. Whole wheat bread would have a greater moisture retention, because of the high fiber content. Like for Figure 7 and 8, the two 70 s t r a i g h t l i n e s p a t t e r n can be o b s e r v e d . Both c u r v e s show a r a p i d i n c r e a s e i n t e m p e r a t u r e ( b e i n g s l i g h t y h i g h e r f o r t h e c r u s t ) f o r t h e 12 f i r s t m i n u t e s - A f t e r w a r d s , t h e c u r v e f o r m i d d l e o f t h e b r e a d i s a l m o s t h o r i z o n t a l ( s l i g h t y p o s i t i v e s l o p e ) , whereas f o r t h e c r u s t , t h e t e m p e r a t u r e i n c r e a s e s a t a c o n s t a n t r a t e . T h i s a l m o s t c o n s t a n t t e m p e r a t u r e f o r t h e m i d d l e o f t h e b r e a d i s about 100°C on t h e g r a p h . The temp- e r a t u r e d a t a f o r a l l t h e o t h e r b r e a d t r e a t m e n t s a l s o r e v e a l t h a t t h e m i d d l e t e m p e r a t u r e d a t a f o l l o w a v e r y s i m i l a r p a t - t e r n w i t h t h e t e m p e r a t u r e n e v e r e x c e e d i n g 100 t o 108°C. From the MAT f o r m u l a and g r a p h s , i t i s o b v i o u s t h a t t h e c r u s t t e m p e r a t u r e i s t h e one t h a t g o v e r n s t h e MAT o f t h e b r e a d l o a f . B e c a u s e MAT c u r v e s were a l l l o w e r f o r whole wheat b r e a d compared t o e n r i c h e d w h i t e , l e s s t h i a m i n e d e s t r u c t i o n s h o u l d be e x p e c t e d . But i n f a c t t h e d e s t r u c t i o n c u r v e s d e m o n s t r a t - ed more t h i a m i n e d e s t r u c t i o n f o r whole wheat b r e a d . T h i s o b s e r v a t i o n on MAT c u r v e s r e i n f o r c e s t h e t h i a m i n e d e s t r u c - t i o n d a t a which showed t h a t t h i a m i n e i n whole wheat b r e a d i s more v u l n e r a b l e t o t e m p e r a t u r e t h a n s y n t h e t i c t h i a m i n e i n e n r i c h e d w h i t e b r e a d . Two d i f f e r e n t t h e o r i e s c o u l d be p r o p o s e d t o e x p l a i n t h e l o w e r s t a b i l i t y o f t h i a m i n e t o h e a t f o r whole wheat b r e a d . The f i r s t i s t h e pH e f f e c t . In t h i s s t u d y , whole wheat b r e a d had a s l i g h t l y h i g h e r pH t h a n e n r i c h e d w h i t e b r e a d , and i t has been d e m o n s t r a t e d t h a t t h i a m i n e s t a b i l i t y d e - 71 creases with an increase in pH. As a second theory there is the ash or inorganic constituents explanation proposed by Dawson and Martin (1942) and Farrer (1955), where higher thiamine losses are found with higher extractions of f l o u r . Since k values are very similar between pH 5.0 and 5.5, higher thiamine losses in whole wheat are most probably caused by both higher pH and an increase in the inorganic constituents. This theory was also proposed by Farrer ( 1955). To take into consideration the pH e f f e c t only, other ex- periments should be done using the same type of bread but varying the pH, for example, by adding an acid l i k e vinegar to the bread, or using a sour-dough type bread. To v e r i f y the ash theory, d i f f e r e n t percent extraction flours with controlled pH could be used. VI CONCLUSION T h i a m i n e d e s t r u c t i o n i n p h o s p h a t e b u f f e r f o l l o w s a f i r s t o r d e r r e a c t i o n and t h e A r r h e n i u s e q u a t i o n , where Ea was 34.2 k c a l / m o l e . I t i n c r e a s e d as pH i n c r e a s e d , w i t h g r e a t e r de- s t r u c t i o n a t pH 6.0 and above. K i n e t i c d a t a c o u l d n o t be o b t a i n e d w i t h b r e a d s y s t e m be- ca u s e o f u n s t e a d y t e m p e r a t u r e change o f t h e b r e a d d u r i n g b a k i n g . However, a l i n e a r r e l a t i o n s h i p was o b t a i n e d when l o g a r i t h m o f p e r c e n t t h i a m i n e r e t e n t i o n was p l o t t e d a g a i n s t t i m e , showing h i g h e r d e s t r u c t i o n w i t h t i m e . When s l o p e s were compared, t h e s t a b i l i t y o f t h i a m i n e d e c r e a s e d w i t h h i g h e r oven t e m p e r a t u r e s , and t h i a m i n e i n whole wheat b r e a d ( a n a t u r a l s o u r c e ) was l e s s s t a b l e t h a n i n e n r i c h e d w h i t e b r e a d ( a s y n t h e t i c s o u r c e ) . T h i s d i f f e r e n c e m i g h t be ex- p l a i n e d by h i g h e r pH and h i g h e r ash c o n t e n t i n t h e whole wheat b r e a d . B a k i n g l o s s e s were f o u n d t o be 30 t o 50% f o r no r m a l bak- i n g o f 12g l o a v e s . To i n t e r p r e t t h e s e r e s u l t s i n terms o f a no r m a l l o a f (450 g o r 1 l b ) , one would e x p e c t t o g e t l e s s d e s t r u c t i o n i n l a r g e r l o a v e s o f b o t h t y p e s o f b r e a d , a l - t h o u g h n o t n e c e s s a r i l y 0%. - 72 _ LITERATURE CITED AACC. 1969. Methods of Analysis of the American Association of Cereal Chemists. St-Paul, MN. Adamson, A.W. 1979- A Textbook of Physical Chemistry, 2nd ed. Academic Press, New York, NY. p.543- AOAC. 1980. O f f i c i a l Methods of Analysis, 13th ed. Association of O f f i c i a l Analytical Chemists, Washington, DC. Association of Vitamin Chemists. 1966. Methods of Vitamin Assay, 3rd ed. Interscience Publishers, New York, NY. p.123- Aughey, E. and Daniel, E.P. 1940. Effect of cooking upon the thiamine content of foods. J. Nutrition 19: 285. Bendix, G.H., Heberlein, D.G., Ptak, L.R. and C l i f c o r n , L.E. 1951. Factors influencing the s t a b i l i t y of thiamine during heat s t e r i l i z a t i o n . Food Res. 16: 494. Booth, R.G. 1943. The thermal decomposition of aneurin and cocarboxylase at varying hydrogen ion concentrations. Biochem. J. 37: 518. Boudart, M. 1968. Kinetics of Chemical Processes. Prentice- Hall Inc., Englewood C l i f f s , NJ. Capellos, C. and B i e l s k i , B.H.J. 1972. Kinetic systems: Mathematical Description of Chemical Kinetics in Solution. John Wiley & Sons Inc., New York, NY. p.5. Coppock, J.B.M., Carpenter, B.R. and Knight, R.A. 1956. Cereal product f o r t i f i c a t i o n : The B vitamins with special reference to thiamine losses in baked products. J. S c i . Food Agric. 7: 457. Daniels, F. and Alberty, R.A. 1958. Physical Chemistry. Wiley-Interscience, New York, NY. p.318. Dawson, E.R. and Martin, G.W. 1941. Vitamin B.. Estimation in yeast and bread and s t a b i l i t y during breadmaking. J. Soc. Chem. Ind. (Transactions). 60: 241. - 73 - 74 Dawson, E.R. and Martin, G.W. 1942. Vitamin B.. Estimation in wheatmeal and brown bread and s t a b i l i t y of d i f f e r e n t forms of vitamin B. during bread-baking. J. Soc. Chem. Ind. (Transactions;. 61: 13. Dwivedi, B.K. and Arnold, R.G. 1972. Chemistry of thiamine degradation. Mechanisms of thiamine degradation in a model system. J. Food S c i . 37: 886. Dwivedi, B.K. and Arnold, R.G. 1973- Chemistry of thiamine degradation in food products and model systems: A review. J. Agric. Food Chem. 21(1): 54. Farrer, K.T.H. 1941. The influence of pH on the destruction of aneurin at 100°C. Austr. Chem. Inst. J. Proc. 8: 113. Farrer, K.T.H. 1945- The thermal destruction of vitamin B^. 1. The influence of buffer s a l t s on the rate of destruction of aneurin at 100°C. Biochem. J. 39: 128-132. Farrer, K.T.H. 1953- The thermal destruction of vitamin B 1 in vegetables. Austr. J. S c i . 16: 62. Farrer, K.T.H. 1955. The thermal destruction of vitamin B̂ in foods. Adv. in Food Res. 6: 257. Farrer, K.T.H. and Morrison, P.G. 1949- The thermal destruction of vitamin B.. 6. The ef f e c t of temperature and oxygen on the rate of destruction of aneurin. Austr. J. Exptl. B i o l . Med. S c i . 27: 517. F e l i c i o t t i , E. and Esselen, W.B. 1957. Thermal destruction rates of thiamine in pureed meats and vegetables. Food Technol. 11 (Feb.): 77. Food and Nutrition Board. 1980. Recommended Dietary Allowances, 9th ed. National Research Council, National Academy of Sciences, Washington, DC. Gomori, G. 1955. Preparation of buffers for use in enzyme studies. In: Methods in Enzymology, Volume I. S.P. Colowick and N.O. Kaplan (Eds). Academic Press Inc., New York, NY. p. 143- Greenwood, D.A., Beadle, B.W. and K r a y b i l l , H.R. 1943. S t a b i l i t y of thiamine to heat. II. Effect of meat-curing ingredients in aqueous solutions and in meat. J. B i o l . Chem. 149: 349. 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A p p e n d i x A THIAMINE LOSSES IN BREAD Type of bread White Whole wheat Rye Regular_white High B- white Whole wheat Regular white High B. white Whole wheat Regular white High B 1 white Whole wheat Regular white High B- white Whole wheat Melba toast Reg. white High B 1 Whole wheat White White High B 1 white Enriched white 85% extraction 95% extraction 100% extraction (stone ground) Hovis(wheat germ) Baking Baking % loss Reference time temperature (min) l i g h t toasting medium toasting heavy toasting almost 0 8 5 9 0 4, 12 0 0 10, 17 3 0 24,21 40 9 26 45 20 30 190-218 C 15 425°F 475°F 45-48 510-520UF 6 - 8 6 - 8 11 3-14 22 27 33 35 19 Morgan and Frederick (1935)*^ Hoffman et al (1940) Aughey and Daniel (1940) Ha r r e l 1 e t a l . (1941) 1 Dawson and Martin (1941) Dawson and Martin (1942) - 77 ~ 78 (continued) Type of bread Baking Baking % loss Reference time temperature (min) Enriched white (1 lb) Whole wheat (1 lb) High B 1 white (1 lb) ' Commercial scale Enriched white High B. white Whole wheat 10 410°F 3 20 13 30 21 40 33 10 2 20 17 30 23 40 33 10 6 20 14 30 23 40 32 'commercial conditions' 21 (corresponding 22 to 30 min 26 labor atory bakes) Schultz et al (1942) White (1.25 lb) White (10 lb) White (4 lb) 35 44 80 75 80 440°F 360-440°F 360-475°F 17 14 21 19 24 Meckel and Anderson (1945) 1 B r i t i s h 85% extr B r i t i s h 80% extr South Af r i c a 95% extr. Canned bread Good baking cond i t i o n s 16 15 29 15 Goldberg and Thorpe (1946) 1 Brenner.et a l . (1948a) 1 White Bread r o l l s (various) 30 40 50 15 20 25 400°F 17 20 26 7- 12 8- 16 12-22 Zaehringer and Personius (1949) 75% extraction (0.35% ashes) 85% extraction (0.49% ashes) 18.0 21.7 Farrer ( 1949)' 79 ( c o n t i n u e d ) Type o f b r e a d B a k i n g B a k i n g % l o s s R e f e r e n c e t i m e t e m p e r a t u r e (min) 98% e x t r a c t i o n ( 0 . 9 7 % a s h e s ) 100% e x t r a c t i o n ( 1 . 0 6 % a s h e s ) Wheat Rye Melba t o a s t L o a v e s M u f f i n s S t i c k s Wheat Rye White p a t e n t f l o u r 80% e x t r . ( u n b l e a - ched , u n t r e a t e d ) 80% e x t r . ( w i t h dough i m p r o v e r ) 80% e x t r . ( w i t h c h l o r i n e d i o x i d e ) 100% e x t r . (no a d d i t i v e s ) A m e r i c a n army t y p e canned b r e a d N a t i o n a l b r e a d 5mm t h i c k n e s s 9mm 12mm ( a l l l o a v e s were 1 E n r i c h e d w h i t e (1 l b ) C o n v e n t i o n a l method 30 45 2 2 5 - 2 4 0 ° F 2 4 0 - 2 6 0 ° F 30 475°F T o a s t i n g l b l o a v e s ) 8 13 18 23 30 430°F 30.0 31.0 11- 20 15-25 20 15 21 34 12- 14 30 21 .0 17-3 20.7 19- 1 23-3 19.6 3 1 . 0 14.7 13.4 4 10 10 21 47 P u l k k i e t a l . ( 1950) 1 Menger ( 1 9 5 2 ) 2 Pace and W h i t a c r e ( 1953)' B u k i n e t a l . ( 1 9 5 4 ) ^ Coppock e t a l . (1956) T a b e k h i a and D ' A p p o l o n i a ( 1 9 7 9 ) * * 80 (continued) Type of bread Baking Baking % loss Reference time temperature (min) Continuous 8 430°F 8 Tabekhia and method 13 12 D'Appolonia 18 15 (1979)** 23 27 30 43 •Feeding t e s t s . **Approximate values from their Fig. 1. ^Referenced in Farrer (1955). Referenced in Coppock et a l . (1956). A p p e n d i x B STATISTICAL ANALYSIS - 12G LOAVES (EXPERIMENT 2) B. 1 T-TEST b1 - b2 S b 1 - b 2 350°F 475°F Enr. white W. wheat Enr. white W. wheat s x ; 51756 55124 9944 19871 ZY^ 0.8931 1 .0419 0.6035 1.8804 ZXY 203.4679 235.0443 67-6186 188.2795 n 16 15 16 15 b 0.003931 . 0 .004264 0.006800 0 .009475 Residual SS 0.09323 0 .03973 0.1437 0 .09640 Residual DF (S^y.x)p 15 14 15 14 0. 004585 0. 008281 S b ^ b p t exp 0. 0004144 0. 001118 -0 .8035 -2 .3931 V 29 29 t.05(2),29 +2 .045 +2 .045 Conclusion Bew=Bww (p=0. 2140) Beŵ Bww (p=0. 0204) 425°F 550°F Enr. white W. wheat Enr. white W. wheat EY EXY n b Residual SS Residual DF (S y.x)p 19000 0. 9491 124.3641 16 0.006545 0.1351 15 29464 2.2763 250.1912 16 0.008491 0.1519 15 6120 0.7081 63-5770 16 0.01039 0.04765 15 6824 1.2881 89.8601 16 0.01317 0. 1048 15 0.009566 0.0050807 - 81 - 82 ( c o n t i n u e d ) M25°F 5 5 0 ° F E n r . w h i t e W. wheat E n r . w h i t e W. wheat Sb . . -bp 't i x p t . 0 5 ( 2 ) , 3 0 C o n c l u s i o n 0 . 0 0 0 9 1 0 0 2 . 1 3 8 4 30 ± 2 . 0 4 2 Bew^Bww ( p s 0 . 0 1 1 7 ) 0 . 0 0 1 2 5 5 2 . 2 1 5 4 30 ±2 .0 .42 Bew^Bww (p = 0 . 0 1 7 2 0 ) B . 2 F - S T A T I S T I C SSc - SSp k - 1 SSp DFp E n r . w h i t e W. wheat Res. SS Res. DF Res. SS Res. DF R e g r e s s i o n 350 R e g r e s s i o n 425 R e g r e s s i o n 475 R e g r e s s i o n 550 P o o l e d r e g r e s s i o n Common r e g r e s s i o n F e x p e r i m e n t a l F . 0 5 ( 1 ) , 3 , D F p C o n c l u s i o n 0 . 0 9 3 2 3 15 0 . 0 3 9 7 3 14 0 . 1351 . 15 0 . 1 5 1 9 15 0 . 1437 15 0 . 0 9 6 4 14 0 . 0 4 7 6 5 15 0 . 1 0 4 8 15 0 . 4197 60 0 . 3 9 2 8 58 0 . 7 2 7 0 59 1 . 2 5 0 2 57 1 4 . 6 3 9 6 4 2 . 2 0 3 4 2 . 7 6 b 3 5 0 * b 4 2 5 * b 4 7 5 * b 5 5 0 (p< < 0 . 0 0 0 1 ) 2 . 7 7 b 3 5 0 * b 4 2 5 * b 4 7 5 * b 5 5 0 (p< < 0 . 0 0 0 1 ) 83 B.3 SNK TEST q S.E. B.3•1 Enriched white bread See following page. B.3.2 Whole wheat bread See following page. B.4 DEVIATION FROM LINEARITY H Q: The population regression i s l i n e a r . H.,: The population regression i s not l i n e a r . MS deviation from l i n e a r i t y F MS within groups Example of anova table for whole wheat at 550°F: Source of variation SS DF MS Total Between groups Linear regression Deviation from l i n e a r i t y Within groups 1.288089 1.211828 1.183316 0.0285119 0.0762604 2 13 0.014256 0.0058662 F = 2.4302 F.05(1),2,13 = 3-89 conclusion: accept H Q (p=0.1269) Enriched white bread Comparison (1 vs 2) Difference 0>L - b 2) S.E. q P q.05,60,p P r o b a b i l i t y P Conclusion 0.0007995 8.0792 4 3.737 p<0.001 b550f*b350 0.0008693 4.4266 3 3.399 0.01<p<0.005 b550^j425 0.0009609 3.7361 2 2.829 0.025<p<0.01 b550#3475 0.0006476 4.4304 3 3.399 0.01<p<0.005 b475?^350 0.0007320 0.3483 2 2.829 p>0.50 b475=b425 0.0005017 5.2105 2 2.829 p<0.001 b425^350 550 vs 350 550 vs 425 550 vs 475 475 vs 350 475 vs 425 425 vs 350 0.006459 0.003848 0.003590 0.002869 0.0002550 0.002614 Whole wheat bread 350 425 475 550 Comparison (1 vs 2) Difference ( b l " b2> S.E. p q.05,58,p P r o b a b i l i t y P Conclusion 550 vs 350 0.008906 0.0007467 11.9267 4 3.737 p<0.001 550 vs 425 0.004679 0.0007817 5.9855 3 3.399 p<0.001 550 vs 475 0.003695 0.0008164 4.5257 2 2.829 0.001<p<.005 475 vs 350 0.005211 0.0004815 10.8230 3 3.399 p<0.001 475 vs 425 0.0009840 0.0005341 1.8428 2 2.829 O.l0<p<0;20 425 vs 350 0.004227 0.0004199 10.0650 2 2.829 p<0.001 b550#3350 b550#>425 b550#>475 b475#>350 b475-b425 b425#>350 350 425 475 550 85 Summary: F exp F.0 5 ( 1 ) , 2 , D F w i C o n c l u s i o n p E n r . w h i t e 350 26. 050 3. 89 r e j e c t E n r . w h i t e 425 1. 395 3. 89 a c c e p t E n r . w h i t e 475 2. 908 3. 89 a c c e p t E n r . w h i t e 550 2. 107 3. 89 a c c e p t W.wheat 350 1. 432 3. 81 a c c e p t W.wheat 425 1. 027 3. 89 a c c e p t W.wheat 475 1 . 252 3. 81 a c c e p t p<<0.0001 0.2826 0.0904 0.1612 0.2769 0.3853 0.3207 Appendix C EXAMPLE OF CALCULATION FOR % THIAMINE RETENTION C.1 MODEL SYSTEM Example: Sample at 100°C after 4 hrs of heating. Photomultiplier reading '0' (no heating) 50.5 •0' blank 0.3 Standard 50.0 Standard blank 0.3 Sample 25.8 Sample blank 0.3 Thiamine yg/ml Sample reading - Sample blank reading 1 x Standard reading - Stand.blank reading 5 50.5 - 0.3 1 yg thiamine/ml '0' = x — = 0.2020 yg/ml ( 1) 50.0 - 0.3 5 25.8 - 0.3 1 yg thiamine/ml sample r x — = 0.1026 yg/ml (2) 50.0 - 0.3 5 (2) % thiamine retention = x 100 = 50.8% (1) C.2 BREAD SYSTEM Example: Whole wheat bread baked at 350°F for 65 min (2nd run). Photomultiplier reading Dough 93.0 Dough blank 1.8 Standard 85.85 Sample 63.5 - 86 - 87 Sample blank 5.6 Sample weight: 9-0 g dry weight: 0.8506 g/g Dough weight: 10.0 g dry weight: 0.5906 g/g S.reading - S.blank read. 1 100 Thiam.yg/g sample = x — x Stan .read .-St.blank read. 5 dry sa- mple wt (g) (63.5 - 5.6) 1 100 yg thiam./g sample = x x (4) (85.85-0.65) 5 (9 x 0.8506) = 1.7745 yg/g (3) (93.0 - 1.8) 1 100 yg thiam./g dough x x (85.58-0.65) 5 (10 x 0.5906) = 3-5950 yg/g (3) % thiamine retention = x 100 = 49.4% (4) Dry weight c a l c u l a t i o n : (dry sample wt + container wt) - container wt wet sample wt dry wt g/g 4.7082 - 1.2991 Duplicate 1 : = 0.8523 g/g (1) 4.0000 4.7004 - 1.3047 Duplicate 2 : = 0.8589 g/g (2) 4.0000 (1) + (2) Average dry wt g/g = = 0.8506 g/g

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