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Breadmaking properties of barley proteins Ho, Mary Kwok 1978

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BREADMAKING PROPERTIES OF BARLEY PROTEINS by MARY KWOKJHO B.S., University of Hawaii, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1978 © Mary KwoktHo, 1978 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 requirement fo r advanced degree at the Univers-i t y 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 available f o r reference and study. I further agree that permission f o r exten-sive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copy-ing or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written per-mission. Department of Food Science Faculty of Agriculture The University of B r i t i s h Columbia Vancouver, B.C., Canada V6T 1W5 ABSTRACT S i m i l a r i t i e s can be drawn between protein contents, s o l u b i l i t y p r o f i l e , remix loaf volume, and t o t a l s u l f h y d r y l - d i s u l f i d e contents of barley and wheat f l o u r s . However, the quantitative r e l a t -ship between loaf volume and protein content or protein s o l u b i l i t y d i s t r i b u t i o n used f o r wheat f l o u r did not apply to barley. Barley f l o u r de-monstrated very poor breadmaking a b i l i t y and low loaf volume. El u t i o n patterns from Sephadex G100 showed that the molecular weight of barley gluten was lower than wheat. Sephadex G150 elution p r o f i l e s of barley and wheat g l u t e l i n gave two and three peaks respect-i v e l y . The F 2 f r a c t i o n of wheat glutenin, which d i f f e r e n t i a t e s the breadmaking p o t e n t i a l of d i f f -erent wheat f l o u r s , was missing i n barley. Turbidity studies on protein aggregation behavior revealed that barley proteins had a lower aggregation rate than wheat. Wheat g l i a d i n gave the highest rate of aggregation, while hordein f a i l e d to aggregate. The alcohol soluble protein f r a c t i o n of barley was capable of polymerization v i a d i s u l f i d e bridges at low temperatures, which i r e s u l t e d i n 'gl u t e n ' f o r m a t i o n i n v i t r o . Such a phenomenon was not observed i n wheat. A d d i t i o n of o x i d i z i n g agents d i d not improve the q u a l i t y of b a r l e y dough. These suggested t h a t the t h i o l and d i s u l f i d e groups of b a r l e y are e i t h e r v e r y i n a c c e s s i b l e , and/or d i f f e r e n t from wheat. There-f o r e , i t i s p o s t u l a t e d t h a t the T h e o l o g i c a l l y e f f -e c t i v e s u l f h y d r y l and d i s u l f i d e groups i n b a r l e y are i n f e r i o r i n q u a l i t y and q u a n t i t y than t h a t of wheat. Amino a c i d a n a l y s i s demonstrated t h a t pro-t e i n s o f b a r l e y and wheat were d i f f e r e n t i n s t r u c -t u r e and composition. The amount of glutamic a c i d i n b a r l e y was o n l y about 70fo t h a t of wheat. F l u o -rescence quenching s t u d i e s showed t h a t hydrophobi-c i t y of wheat g l u t e n was h i g h e r than b a r l e y . Bar-l e y i s weak i n the v i t a l chemical f o r c e s which govern breadmaking p o t e n t i a l , and furthermore t h e r e are numerous d i f f e r e n c e s among f u n c t i o n a l p r o t e i n s of these two c e r e a l s . These s t u d i e s would p r o v i d e some i n s i g h t s t o t h e i r d i f f e r e n c e s i n p h y s i c a l p r o p e r t i e s and breadmaking p o t e n t i a l s . LIST OP CONTENTS Page Introduction . . . . . 1 Literature Review ^ 1. Quantitative r e l a t i o n s h i p between lo a f volume and protein content ^ 2 . The composition of proteins 7 3 . Formation of 'gluten'from alcohol soluble barley proteins . . . 11 ^. K i n e t i c studies on aggregation behavior of proteins 12 5 . E f f e c t s of pH and oxidizing agents on dough rheology 1^ 6. Hydrophobicity of protein determined by fluorescence quenching technique . . 15 7 . Importance of amino acid composition, chemical and physical bonds i n dough structure . . . . . . . . . . . 17 Materials and Methods 1. Materials 22 2 . S o l u b i l i t y p r o f i l e of barley and wheat proteins (a) Preparation of chromatographic column . . . . . . . . . . . . . 22 (b) Preparation of f l o u r extract i n AUC . 23 (c) E l u t i o n patterns of proteins . . . 23 3 . S o l u b i l i t y f r a c t i o n a t i o n "of proteins (a) Fractionation of proteins . . . . 23 i i i LIST OF CONTENTS Materials and Methods (Cont.) Page 3 . (b) Determination of protein content 26 4. Determination of s u l f h y d r y l (SH) and d i s u l f i d e (SS) groups i n barley and wheat f l o u r s 26 5 . Preparation of microdoughs (a) E f f e c t s of o x i d i z i n g agents on dough properties . . . . . . . 27 (b) E f f e c t s of oxidizing agents on doughs at pH 12 28 (c) E f f e c t s of pH on barley f l o u r and wheat starch doughs 29 (d) E f f e c t s of d i f f e r e n t proteins on properties of fabricated doughs, . . 29 (e) E f f e c t s of deuterium oxide and urea on wheat dough 30 6 . Formation of 'gluten' from hordein i n v i t r o (a) Extraction of hordein, and formation of 'gluten* i n v i t r o . . 30 (b) Dissolution of p r e c i p i t a t e d protein aggregates 31 (c) Preparation of microdoughs with : dissolved protein aggregates' . . 31 7 . K i n e t i c studies on protein aggregation phenomena (a) Gluten preparation 32 (b) Separation of prolamin and g l u t e l i n by gel f i l t r a t i o n on Sephadex G100 column 32 i v LIST OF CONTENTS Materials and Methods (Cont.) Page 7. (c) Determination of time course curve of t u r b i d i t y 33 (d) Reduction and cyanoethylation of g l u t e l i n f o r Sephadex G150 column . . . . . 3^ (e) Gel f i l t r a t i o n of g l u t e l i n polypeptides on a Sephadex G150 column 35 8. Hydrophobic comparison between barley and wheat proteins using fluorescence quenching technique 35 9. Total amide nitrogen determination . . 37 10. Amino acid analysis 37 Results and Discussion 1. Gel chromatography of barley and wheat proteins 39 2. Quantitative r e l a t i o n s h i p between loa f volume and protein content or protein s o l u b i l i t y d i s t r i b u t i o n (a) S o l u b i l i t y f r a c t i o n a t i o n of endosperm protein 39 (b) Remix lo a f volume (RLV) kZ 3 . Sulfhydryl and d i s u l f i d e groups i n barley and wheat fl o u r s ^6 Preparation of microdoughs (a) E f f e c t s of o x i d i z i n g agents on fabricated doughs ^9 (b) E f f e c t s of oxidizing agents at.pH 12 ; ._'Y" . . . . . ^9 v LIST OF CONTENTS Results and Discussion (Cont.) • Page ^. (c) Ef f e c t s of d i f f e r e n t proteins on properties of fabricated doughs 53 5 . Extraction of hordein and formation of 'gluten' i n v i t r o 56 6 . K i n e t i c studies on aggregation behavior of proteins (a) Physical properties and molecular weight d i s t r i b u t i o n s of glutens 62 (b) Aggregation phenomenon of proteins 63 (c) Gel f i l t r a t i o n of g l u t e l i n polypeptides 65 7 . Amino acid composition and possible chemical bondings i n dough structure (a) Amino acid analysis ?2 (b) E f f e c t of deuterium oxide and urea i n dough-making . . . . . . 72 (c) Hydrophobic comparison using fluorescence quenching method . . 7^ (d) Hydration of proteins 79 Conclusion 83 References 87 v i LIST OF TABLES Table Page 1 T o t a l protein recovered from s o l u b i l i t y f r a c t i o n a t i o n vs. t o t a l protein content 41 2 S o l u b i l i t y f r a c t i o n a t i o n of the endosperm protein - 4 3 3 Prediction of lo a f volume from RLV . . 46 4 T o t a l s u l f h y d r y l - d i s u l f i d e values of barley and wheat fl o u r s 47 5 E f f e c t s of oxidizing agents on fabricated doughs 50 6 E f f e c t s of oxidizing agents on fabricated doughs at pH 12 . . . . . . 51 7 E f f e c t s of pH on. fabricated doughs . . 53 • 8 E f f e c t s of d i f f e r e n t proteins on * properties of fabricated doughs . . . 54 9 Extraction of hordein and formation of 'gluten' i n v i t r o 57 10 Absorbance reading of alcohol soluble proteins . . . . . 59 11 Dissolution of protein aggregates . . 6 l 12 Aggregation behavior of barley and wheat proteins 69 13 Amino acid composition of barley and wheat f l o u r s 73 14 Hydrophobic comparison by fluorescence quenching technique 77 15 Total amide nitrogen content of wheat and barley glutens 81 v i i LIST OF FIGURES Figure Page 1 Flow chart of protein f r a c t i o n a t i o n procedure 25 2 El u t i o n curve of barley and wheat proteins on Sephadex G100 40 3 S o l u b i l i t y d i s t r i b u t i o n of barley, wheat and whole wheat f l o u r s . . . . 44 k E l u t i o n patterns of wheat and barley glutens . 6k 5 Time course of t u r b i d i t y with barley and wheat glutens 66 6 Time course of t u r b i d i t y with barley and wheat g l u t e l i n s 67 7 Time course of t u r b i d i t y with barley and wheat prolamins 68 8 E l u t i o n curve of reduced and cyanoethylated wheat glutenin . . . . 71 9 E l u t i o n curve of reduced and cyanoethylated barley g l u t e l i n . . . 71 10 Fluorescence quenching studies on hydrophobicity of prolamins . . . 76 11 Fluorescence quenching studies on hydrophobicity of g l u t e l i n s . . . 76 v i i i ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to members of the Faculty, e s p e c i a l l y to Dr. S. Nakai f o r his constructive advice and appropriate guidance dur-ing the course of t h i s study. F i n a l l y my deepest appreciation goes to every member of my family, e s p e c i a l l y to my husband, Al a i n , f o r t h e i r patience, consideration and u n f a i l i n g support. ix INTRODUCTION Cult i v a t i o n of barley was f i r s t recorded i n Egypt around 5000 B.C. It i s a grass that belongs taxonomically to the family Gramineae, subfamily Festucoidea t r i b e Hordeae and genus Hordeum. Depend-ing on the arrangement of grains i n the ear, barley can be c l a s s i f i e d into two main types, namely the two and s i x rowed v a r i e t i e s . The former i s predominantly used i n malting while the l a t t e r i s used f o r other purposes. At present the major economic uses of barley are as feed grain, as malt f o r manufacturing beverages or malt-enriched food products. Hulled barley i s usually consumed i n Western countries. Pot or pearl barleys, prepared by removal of the outer husk with abrasion, are used as cereal f o r the preparation of i n v a l i d and infant foods as well as breakfast cereals. The bran or dust r e s u l t i n g from the elimination process i s i n -corporated with other foodstuffs into compounded mix-tures f o r stock-feeding. In the Far-East and i n some developing countries, naked or h u l l - l e s s barleys are usually used i n soups, porridge ^ and f l o u r f o r f l a t type bread. The barley grain consists of the endosperm and the embryo enclosed within the remains of the o r i g i n a l 1 glumes, c a l l e d the husk. On a dry matter basis, bar-ley contains 6 3 to 6 5 $ starch, 1 to 2% sucrose, about 1% other sugars, 1 to 1 . s o l u b l e gums, 8 to 1 0 $ hem-i c e l l u l o s e , 4 to $% c e l l u l o s e , 2 to 3 $ l i p i d s , 8 to 11% protein, 2 to 2 . 5 $ ash and 5 to 6% other components. In regular barley, the l i n e a r starch component comprises 24$ of the t o t a l starch. A high amylose barley i s also available i n which amylose comprises 4 7 $ of the t o t a l starch (Pomeranz 1 9 7 3 ) • The proteins i n barley can be categorized into :' f i v e groups of d i f f e r e n t s o l u b i l i t y properties. The water solublesalbumin f r a c t i o n together with the •salt .soluble globulin f r a c t i o n amount to 20% of the t o t a l soluble protein. The other 80$ are the alcohol soluble f r a c t i o n ( g l i a d i n ) , the acid or alkaline s o l -uble f r a c t i o n (glutenin), and a small amount of i n -soluble protein c a l l e d residue protein (Ewart I 9 6 8 ) . World production of barley i n 1 9 7 0 was 1 1 7 . 0 m i l l i o n tons (compared with 2 8 8 . 1 of wheat, 242 .4 of corn, 1 9 7 . 4 of r i c e , and 2 7 . 0 of rye). Annual per capita consumption of barley as food i n the United states i s only 1 1 . 1 l b (compared with 111 l b of wheat, 7 . 3 lb of r i c e and 1 . 2 lb of rye). (Pomeranz 1 9 7 3 ) Barley has a greater tolerance f o r c l i m a t i c and geographic situations than most other grain crops. 2 It matures f a s t e r than wheat, i t can be grown along h i l l sides and requires l i t t l e attention. In spite of a l l these advantages, the popularity of barley re-mains low compared with wheat. The d i s t i n c t i v e proper-ty of wheat i s i t s excellent breadmaking q u a l i t y . The proteins of barley have a very s i m i l a r s o l u b i l i t y pro-f i l e , nitrogen content and other apparent resemblences of wheat. Nevertheless, i t s breadmaking properties are i n f e r i o r to wheat. Hart et al.(1970) reported that the rheological properties, loaf volume and texture of barley bread could be improved upon addition of gums or g l y c e r y l monostearate. The po t e n t i a l food uses of barley could be broadened i f we can i d e n t i f y the factors a f f e c t i n g loaf volume, and modify them to form doughs and breads rhe o l o g i c a l l y s i m i l a r to those of wheat. These re-sultant products may not be es s e n t i a l i n Western coun-t r i e s , butatheymmayv* veloping countries, or at times when wheat supply i s i n s u f f i c i e n t . 3 LITERATURE REVIEW The protein content of barley l i e s within the range necessary f o r breadmaking requirement. Osborne fr a c t i o n a t i o n of barley f l o u r y i e l d s s o l u b i l i t y p r o f i l e s s i m i l a r to wheat (Whitaker. 1 9 6 3 ) • Barley starch i s a l -most comparable to wheat starch i n breadmaking proper-t i e s . They have s i m i l a r granule sizes and g e l a t i n i z a -t i o n temperatures (Hoseney et.al.1971). However, bar-ley f l o u r demonstrates poor loaf volume and breadmak-ing properties. 1. Quantitative., r e l a t i o n s h i p between loaf volume and protein content. The prime factors a f f e c t i n g the loaf volume of wheat f l o u r have always been controversial. T r a d i t i o n a l b e l i e f favors the use of high protein content f l o u r to improve loaf volume by increasing the water absorption a b i l i t y . Aging of f l o u r , which can be substituted by the addition of small amount of maturing agents such as bromate or ascorbic acid, w i l l also promote greater water absorption a b i l i t y and better breadmaking q u a l i t y (Bloksma- 1 9 7 2 ; Irvine and McMullan I 9 6 0 ; Noguchi et a l . 1 9 7 6 ) • There i s a quantitative r e l a t i o n s h i p between loaf volume and protein s o l u b i l i t y d i s t r i b u t i o n . Loaf volume of a given f l o u r can be predicted by ui-'dn. • using the Remix Loaf Volume (RLV) equation which i s dependent on the protein s o l u b i l i t y d i s t r i b u -t i o n (Orth et a l . 1 9 7 2 ) . Orth and Bushuk ( 1 9 7 2 ) suggested that the loaf volume per unit protein (ULV) would be a bet-te r index of inherent protein q u a l i t y than l o a f volume, since both protein q u a l i t y and quantity would aff e c t l o a f volume. The ULV i s inversely related to the proportion of glutenin protein, and d i r e c t l y related to the proportion of residue protein. Noguchi et a l . ( 1 9 7 6 ) noted that protein con-tent i t s e l f did not correlate with adhesiveness and other rheological properties. Rather, i t i s probable that only certain^ s p e c i f i c proteins are involved (Noguchi et a l . 1 9 7 6 ) . I t i s the t h i o l (SH) groups and d i s u l f i d e (SS-)- groups- i n the network of protein molecules which a f f e c t the dough r i g i d i t y . The d i s u l f i d e cross-links r e s i s t e l a s t i c deforma-t i o n , and t h i o l - d i s u l f i d e interchange reactions provide f o r viscous deformation of the protein network (Bloksma 1 9 7 2 ; Ewart 1 9 7 2 b ) . Glutenin com-ponents form loosely-folded structures through intermolecular d i s u l f i d e bonds, whereas, g l i a d i n molecules form t i g h t l y folded structures through 5 intramolecular d i s u l f i d e bonds. In order to obtain good gluten performance there must be a balance between i n t e r - and intra-chain d i s u l f i d e bonds (Ewart 1 9 6 7 ; 1 9 7 2 b ) . The hydration and configuration of higher mole-cular weight proteins of gluten w i l l a f f e c t the acc-e s s i b i l i t y of some d i s u l f i d e linkages to the attack of SH reagent. Consequently, the exposed SS l i n k -ages are more l a b i l e than those that are buried i n -i side the protein structure (Kuninori 1 9 6 8 )• I t has been postulated that only a very small f r a c t i o n of the a n a l y t i c a l l y determined t h i o l and d i s u l f i d e gr-oups are r h e o l o g i c a l l y e f f e c t i v e . The Theologically e f f e c t i v e d i s u l f i d e bonds are those that r e s i s t the e l a s t i c deformation and the r h e o l o g i c a l l y e f f e c t i v e t h i o l s are those that permit viscous deformation (Bloksma 1 9 7 2 b ; 1 9 7 5 ) - No simple relationships can be drawn between loa f volume, and the t o t a l SS and SH content of f l o u r (Tsen and Bushuk 1 9 6 8 ) . Measurements of the extent of d i s u l f i d e i n t e r -change of wheat, rye, and barley f l o u r s showed that the p o t e n t i a l f o r protein-protein d i s u l f i d e interchange i n wheat i s several times greater than the minimum c a l -culated to be necessary to create a continuous network. Rye just reaches the minimum requirement, whereas proteins i n barley f a l l below the l i m i t i n g extent of interchange required (Redman and Ewart 1 9 & 7 a b ) . 6 The composition of proteins The protein composition of wheat f l o u r i s very-complex and contains many molecular species having d i f f e r e n t s i z e s . These i n d i v i d u a l groups have d i f -ferent properties, and d i f f e r e n t b i o l o g i c a l func-tions. Gluten i s the chief protein that governs the functional properties of bread f l o u r . I t re-presents 60 to 70$ of the t o t a l protein content i n wheat f l o u r , and 50 to 60$ i n barley flour.' .When wheat f l o u r i s mixed and hydrated, the water i n -soluble proteins w i l l form a complex coherent mass, giving bread i t s unique structure, gas retention po t e n t i a l and v i s c o e l a s t i c i t y (Greenwood and Ewart 1975)« Gluten i t s e l f i s a complex of several com-ponents i n t e r a c t i n g together to give i t s s p e c i f i c properties. The predominant proteins i n gluten are high molecular weight glutenin, and compara-t i v e l y low molecular weight g l i a d i n ; some small fractions of albumin or globulin ^proteins may also be present. The glutenin protein f r a c t i o n has a wide mole-cular weight d i s t r i b u t i o n . The average molecular weights reported i n the l i t e r a t u r e range , from 1 5 0 , 0 0 0 to 3 , 0 0 0 , 0 0 0 daltons. G l i a d i n , however, consists of proteins mainly i n the 2 0 , 0 0 0 to 40 , 0 0 0 dalton range (Bushuk 1974; Ewart 1 9 ? 2 a ). Reduced wheat glutenin consists of at least 15 sub-units. Almost a l l of them are unique to that protein, except one major sub-unit which i s s i m i l a r to the 4 4 , 0 0 0 dalton polypeptide i n reduced g l i a d i n . S i g n i f i c a n t differences i n amino acid composition are observed i n the three c h a r a c t e r i s t i c sub-units. The presence of numerous d i f f e r e n t sub-units i n glutenin suggests that only a p a r t i c u l a r combina-t i o n of various sub-units w i l l give the glutenin protein i t s rheological s p e c i f i c i t y (Bietz et a l . 1973)• Reconstitution studies have shown that glutenin governs the mixing requirement and s t a b i -l i t y of f l o u r . During dough formation, the glutenin must in t e r a c t with appropriate l e v e l s of other proteins, e s p e c i a l l y g l i a d i n , to achieve optimum performance and loaf volume during baking (Bietz et a l . 1 9 7 3 ) • Proteins i n the wheat g l i a d i n group are very complex. Fractionation on Sephadex G100, i n a d i s s o c i a t i n g solvent, y i e l d s four major f r a c t i o n s . The majority of these proteins are single polypep-tide chains s t a b i l i z e d by intra-chain d i s u l f i d e bonds, which are c h a r a c t e r i s t i c of the g l i a d i n groups; small f r a c t i o n s of high molecular weight proteins consisting of intermolecular d i s u l f i d e polypeptide chains, s i m i l a r to the glutenin group are also present (Nielson et a l . 19685 Preston and Woodbury 1 9 7 6 ) . The physical conformation^ gliadin 3s a 8 c r i t i c a l f actor f o r mixing s t a b i l i t y . The starch protein residue a f t e r extraction of g l i a d i n , shows extremely long dough development time. Adding back the extracted g l i a d i n restores the o r i g i -n a l mixing properties. Gliadin i s also responsible f o r the differences i n loaf volume of wheat f l o u r s that d i f f e r i n breadmaking , 'potential (Hoseney et a l 1 9 7 1 ) . Therefore, the rheological properties and development time of dough are affected by g l i -adin (Bloksma 1 9 7 2 a ) . L i t t l e research has been done on the compo-s i t i o n of barley gluten, e s p e c i a l l y on g l u t e i i n proteins. The physical properties of barley gluten do not resemble those of wheat gluten. Barley gluten is-loose-non-cohesive and non-elastic. Osborne ( I 8 9 5 ) proposed to name the alcohol soluble proteins of barley as hordein. This hor-dein f r a c t i o n , although s i m i l a r i n many respects to wheat g l i a d i n , appears to be d i f f e r e n t i n ele-mentary composition from alcohol soluble extracts of other cereals. It w i l l polymerize and aggre-gate at low temperature giving a s p e c i f i c coherent mass, s i m i l a r to gluten, with i t s own inherent rheological properties ( Shestakova et.al. 1 9 7 6 ) . Gel f i l t r a t i o n on Sephadex G 1 0 0 y i e l d s three major 9 hordein f r a c t i o n s , and a minor f r a c t i o n of g l u t e l i n may also be present (Whitaker I 9 6 3 ) . Gluten consists of a complex physical mixture of prolamin and g l u t e l i n i n which some interactions between these two proteins have occurred. The max-imum int e r a c t i o n takes place near pH 6 .1 (Hoseney e t - a l l 9 7 1 ; Jones and Carnegie 1 9 7 1 ) . Reconstitu-t i o n studiessshowed that neither g l i a d i n nor glu-tenin alone can give good loaf volume; both of them have to work together to achieve an optimum ef f e c t (Bietz et a l . 1 9 7 3 ; Murthy and Dahle 1 9 6 9 ) . Although gluten i s the functional protein i n breadmaking, bread baked without soluble proteins gives,only 67$ of the volume of those baked with a l l of the protein fractions present. The d i v i s i o n of soluble proteins into albumins and globulins based.on s o l u b i l i t y difference i s not very d i s t i n c t (Enari 1965)• The albumin f r a c t i o n from barley and wheat can be separated by electrophoresis into 7 and 6 f r a c t i o n s , respectively. The amino acid com-positions of the various components are d i f f e r e n t , e s p e c i a l l y t h e i r contents of acid and basic amino acids. The molecular weight of wheat albumin i s about 2 0 , 0 0 0 daltons (Meredith and Wren I 9 6 6 ). In barley, the soluble proteins are very polydisperse, w i 10 with molecular weights ranging between 1 0 , 0 0 0 and 2 0 0 , 0 0 0 daltons (Enari 1 9 6 5 ). I t has not been es-tablished how soluble proteins a f f e c t rheological properties. Neither the albumin nor globulin frac t i o n s are responsible f o r differences i n bak-ing quality; however, they are necessary f o r normal baking c h a r a c t e r i s t i c s and maximum gluten perfor-mance. Therefore, the r o l e of soluble proteins may be a minor but necessary part of the t o t a l f l o u r complex, which gives wheat f l o u r dough i t s c h a r a c t e r i s t i c properties. Formation of "gluten" from alcohol soluble barley proteins. Cooling the alcohol extract of barley f l o u r from which albumin and globulin have been removed during previous extractions, would cause polymer-i z a t i o n of hordein molecules. These protein aggre-gates are firm, rubbery, cohesive and insoluble-. Its physical properties resemble gluten more than hordein proteins, and have rheological properties of a s p e c i f i c type of gluten (Shestakova et a l . 1 9 7 6 ) . Vakar et a l . (1976) term these~protein. agg-regates formed from coaling of ethanolic extracts as 'gluten p r e c i p i t a t e s ' . They d i f f e r considerably from hordein f r a c t i o n s which remain soluble i n the ethanol extract, e s p e c i a l l y with respect to the re-l a t i v e concentrations of a number of amino acids. Electrophoresis studies show that the composition of t o t a l hordein, the hordein remaining ^soluble-at 4°C, and the hordein i s o l a t e d from the 'gluten p r e c i p i t a t e ' are very s i m i l a r i n q u a l i t a t i v e com-position of the protein components. The t o t a l hor-dein and the hordein i s o l a t e d from the 'gluten pre-c i p i t a t e s ' contains the same polypeptide chains. The only difference i s found i n hordein that remains soluble at 4 ° C It does not contain the proteins with the highest molecular weights which have passed from the ethanolic ©xtract into the gluten-like p r e c i p i t a t e s . The g l u t e l i n - l i k e proteins i s o l a t e d from the gluten p r e c i p i t a t e s contain only sub-units which correspond to the polypeptide chain of hordein, whereas the t o t a l g l u t e l i n i n barley f l o u r consists of sub-units which include a l l the polypeptide ch-ains of hordein and several other higher and lower molecular weight sub-units (Shestakova et a l . 1 9 7 6 ) . Kinetic studies on aggregation behavior of proteins. The rheological properties of f l o u r dough are cl o s e l y related to the d i s p e r s i b i l i t y of gluten upon hydration. The dispersion rate of gluten i n mixing doughs varies with f l o u r types. I t i s f a s t e r i n weak f l o u r , and slower i n strong ones. A convenient, i n d i r e c t approach to measure d i s p e r s i b i l i t y of gluten i s to determine the aggregation v e l o c i t y of proteins by recording the time course curve of t u r b i d i t y i n the protein suspension ((Araka-wa et al . 1 9 7 6 ) . Glutens d i f f e r i n g i n protein com-pos i t i o n would exhibit a difference i n aggregation behavior, Aggregation proceeds f a s t e r i n strong f l o u r and slower i n weak ones (Arakawa and Yone-zawa 1975)• Very good correlations were observed when ten types of f l o u r s ranging from weak, medium and strong strength were studied. Aggregation ve-l o c i t y , t u r b i d i t y at the end of the reaction time, farinograph absorption, and development time a l l increase i n agreement with the order of weak, me-dium and strong f l o u r s . No cor r e l a t i o n with f l o u r strength i s found between protein content, and glu-tenin: g l i a d i n r a t i o (Arakawa and Yonezawa 1975)-Therefore, the breadmaking po t e n t i a l of gluten can be predicted by aggregation v e l o c i t y , and t u r b i d i -ty at the end of reaction time. The properties of g l u t e l i n have a profound influence on the rheological properties of f l o u r dough. The difference i n polypeptide composition of g l u t e i i n i s responsible f o r the difference i n g l u t e l i n behavior, which i n turn affects the q u a l i -ty of gluten (Arakawa et a l . 1976)• However, both g l i a d i n and glutenin proteins are responsible f o r determining the mixing requirement of wheat f l o u r . 13 In order toaachieve the best e f f e c t , proper i n t e r -action between these two proteins i s important. (Smith and Mullen 1 9 6 5 ) . 5- E f f e c t s of pH and o x i d i z i n g agents on dough rheology pH w i l l e f f e c t the structure of dough proteins through e l e c t r o s t a t i c a t t r a c t i o n or repulsion as a consequence of changes i n the degree of i o n i z a t i o n of ionizable groups i n the f l o u r proteins. A de-crease i n pHoofstheadoughcwillalefad to a reduction i n e x t e n s i b i l i t y , and an increase i n the relaxation constant. Dough pH would also a f f e c t the r a t e of s u l f h y d r y l - d i s u l f i d e interchange. Extensograms. show that doughs having a pH below k.O give a sharp decrease i n e x t e n s i b i l i t y , while those i n the pH range of 9 . 2 to 1 0 . 2 are short i n extensi-b i l i t y and low i n resistance to extension. Exten-s i b i l i t y of dough increases with increasing pH from 4-.8 to 7 . 3 (Tsen 1966) . Oxidation has a great e f f e c t on rheological properties of wheat dough. Incorporation of small amounts of o x i d i z i n g agents during dough preparat-ion w i l l r e s u l t i n a considerable increase i n loaf volume, and improvement i n rheological properties. It i s generally accepted that oxidation removes t h i o l groups which are e s s e n t i a l f o r the viscous 14-deformation of dough. The concentration of t h i o l groups can be decreased by the addition of o x i d i -zing agents or by atmospheric oxygen (Lauriere et.••a.1.1976) . Rheological properties of dough are very important; they govern the loaf volume and c crumb structure of the baked bread. Hydrophobicity of protein determined by f l u o r e s -cence quenching technique. Fluorescence quenching i s a spectroscopic technique which involves s e l e c t i v e quenching of the fluorescence of trytophan residues by the i n -troduction of external quenchers. The quencher solutions can be of various low molecular weight reagents, and quenching effectiveness may vary "j: greatly depending on the type of quenching reagents used(Ivokova et a l . 1 9 7 1 ; E f t i n k and Ghiron 1 9 7 6 ) . The quenching reaction involves physical contact between the quencher and an excited indole r i n g . It can be k i n e t i c a l l y described i n terms of a c o l -l i s i o n a l and a s t a t i c component. The degree to which a fluorophor i s quenched depends upon i t s "exposure" to the quencher (Eftink and Ghiron 1 9 7 6 ) . The f o l d i n g of polypeptide chains to form glo-bular proteins w i l l r e s u l t i n b u r i a l of certain amino acid residues from the external aqueous en-15 vironment, and c r e a t e a hydrophobic domain of i t s own; while the ot h e r unburied r e s i d u e s w i l l be ex-posed to the s u r f a c e p o l a r s o l v e n t ( E f t i n k and Gh-i r o n 1 9 7 6 ) . T r i c h l o r o e t h a n o l , a l e s s p o l a r n e u t r a l quencher, i s a v e r y s e n s i t i v e hydrophobic probe. I t w i l l i n t e r a c t with the hydrophobic pockets i n the microenvironment of a f l u o r e s c i n g r e s i d u e . T r i c h l o -r o e t h a n o l has an e x a l t e d quenching a b i l i t y ; i t w i l l quench i n d o l e f l u o r e s c e n c e with an e f f i c i e n c y near u n i t y . Furthermore, i t w i l l a l s o d e t e c t the pr e -sence of nearby hydrophobic ( o i l y ) domains t h a t may e x i s t i n p r o t e i n s . The t r i c h l o r e t h a n o l molecules are r e l a t i v e l y f r e e t o d i f f u s e w i t h i n the p r o t e i n . S t a t i c quenching would occur when t r i c h l o r o e t h a n o l molecules are l o c a t e d i n c l o s e p r o x i m i t y t o the i n -do l e r i n g , w h i le the quencher bound a t hydrophobic s i t e s f u r t h e r away would have t o d i f f u s e towards the f l u o r o p h o r i n order t o be d y n a m i c a l l y quenched. T r i c h l o r o e t h a n o l i s t r a n s p a r e n t i n the u l t r a -v i o l e t r e g i o n , i t permits a g r e a t e r s e l e c t i o n of e x c i t a t i o n wavelengths which i n t u r n a l l o w s the study of the e x c i t a t i o n wavelengths dependence of the quenching constant ( E f t i n k e t a l . 1 9 7 7 ) . Since p r o t e i n i t s e l f i s a p o l y e l e c t r o l y t e , e l e c t r o s t a t i c effectsKmay i n f l u e n c e the quenching r e a c t i o n of 16 the charged quenching probes, and induce an over or under-estimation of the exposure of a fl u o r o -phor. In order to overcome t h i s , an uncharged quenching probe such as trichloroethanol i s used. It i s very sensitive to the exposure of tryptophans i n proteins, and able to sense the exposure of re-sidues i n a purely random, k i n e t i c fashion. 7. Importance of amino acid composition, chemical and physical bonds i n dough structure. The complex molecular organization of a protein i s determined by i t s amino acid composition. The sequence of amino acids i s very important i n gover-ning the primary, secondary and t e r t i a r y structure of a protein. This sequence determines the distance and bond types between i n d i v i d u a l amino acids and segments of a protein chain, consequently, giving r i s e to bonds that hold the polypeptide chain to- • gether (Wehrli and Pomeranz 1 9 6 9 ) . The s i z e , shape and s o l u b i l i t y -of" wheat proteins are influenced by several types of chemical and physical forces. They are covalent bonds, i o n i c bonds, hydrogen bonds, Van der Waal forces, and hydrophobic i n t e r -actions. Some of these bonds aris e from the poly-peptide chain, but most of them come from the side chain groups of amino acids ( K r u l l and Wall 1969)• 17 The side chains of amino acids are c l a s s i f i e d into polar and nonpolar groups according to t h e i r p o l a r i t y . The most polar side chains of the amino acid residues arise from a c i d i c and basic amino acids. They have a great influence on the solu-b i l i t y of wheat proteins. The albumin and globu-l i n proteins are r e a d i l y soluble i n aqueous media because of t h e i r high l e v e l s of basic and a c i d i c amino acid content. On the other hand, g l i a d i n and glutenin proteins having a low concentration of ionizable amino acids are insoluble i n water. The non-polar amino acids account f o r 4-0 to 5 0 $ of the amino acid residues i n wheat gluten. Most of these amino acids are f a i r l y insoluble i n water and dissolve more r e a d i l y i n alcoholic or other organic solvents (Wehrli and Pomeranz 1969? K r u l l and Wall I 9 6 9 ) . The breadmaking properties of wheat gluten are greatley affected by the amino acid composition of protein. Cysteine-cystine groups and glutamine-asparagine groups are among the most important ones i n governing the rh e o l o g i c a l properties of bread proteins. In the presence of a i r or oxidizing agents "two adjacent cysteine residues w i l l be oxi£ dized to form cystine, r e s u l t i n g i n d i s u l f i d e bond formation and c r o s s - l i n k i n g of two protein chains 18 or portions of the same protein chain containing cysteine residues. Glutamine and asparagine are the amide protein groups which are present i n f a i r l y large quantities i n wheat gluten. Glu-tamine alone accounts f o r more than 35$ of the amino acids i n wheat gluten. Amide groups are the p r i -mary s i t e s f o r association of wheat protein through hydrogen bonding. The hydrogen bonds are non-cova-lent forces that s t a b i l i z e the folded conformation of protein (Wehrli and Pomeranz 1969? K r u l l and Wall 1 9 6 9 ) • Covalent bonds are high energy, chemical bonds that w i l l not be decomposed at room temperature, except by some chemical reactions. The only im-portant covalent bonds i n dough structure are the d i s u l f i d e bonds between proteins. Their energy level s are about 49 k c a l /mole. Since a network with permanent cross-links cannot give r i s e to v i s -cous flow, this, flow must require opening and re-forming of the r i g i d cross-links(Wehrli and Pom-eranz 1969)• During dough preparation there i s constant interchange of sul f h y d r y l and d i s u l f i d e groups to provide the required cohesiveness and mobility. Hydrogen bonds can be formed from many amino 19 groups on the proteins, such as the terminal amide groups of glutamine and asparagine, hydroxyl groups of serine and threonine, and peptide amide groups on the protein backbone ( K r u l l and Wall I 9 6 9 ) . The amide hydrogen of one amino acid residue and the carbonyl oxygen of another amino acid residue are the primary s i t e s of peptide, hydrogen bond formation. Hydrogen bonds are responsible f o r the insoluble,cohesive nature of wheat gluten. The non-polar amino acids account f o r 40 to 50% of protein residues i n wheat gluten. When d i s -persed i n aqueous media, these non-polar amino acid side chains tend to associate with each other through hydrophobic bonding. The in t e r a c t i o n of non-polar amino acid residues i s merely a small s t a b i l i z i n g force i n wheat gluten. However, when water i s i n -c o r p o r a t e d into f l o u r during dough preparation, the proteins are surrounded by an aqueous environ-? ment and the parameters which dictate protein con-formation are altered. The high d i e l e c t r i c con-stant of water could suppress the e l e c t r o s t a t i c forces, and hydrophobic i n t e r a c t i o n w i l l "begin to play an important r o l e i n protein s t a b i l i t y (Ewart 1972c{Wehrli and Pomeranz I 9 6 9 )• When protein has been stretchbd, and i t s surface area increased, i t 20 has a tendency to resume i t s o r i g i n a l globular con-formation right a f t e r the stress has been released. This e l a s t i c property i s due to the a b i l i t y of hy-drophobic bonds to s t a b i l i z e protein conformations. The energies of hydrophobic bonds are f a i r l y low, thus permitting t h e i r rapid interchange at room temperature, and contribute to dough p l a s t i c i t y . Hydrophobic bonds are also important i n oven-spring formation. During baking a l l chemical bonds are weakened as the temperature increases, however, hydrophobic bond formation i s an endothermic pro-cess favored by increasing temperature up to 6 0 ° C (Tanford 1 9 7 3 ; Wehrli and Pomeranz 1 9 6 9 ) . 21 MATERIALS AND METHODS 1. Materials Barley f l o u r , a product of Canasoy Company, was purchased from a l o c a l health food store. Wheat f l o u r was a commercial product of Five Roses Brand. Sephadex G100, G150 and Blue Dextran-2000 were from Pharmacia Fine Chemicals. U l t r a pure Guanidine hydrochloride (GuHCl) was obtained from Mann Research Laboratories. The 2,2,2 trichloroethanol was obtain-ed from BDH Chemical Ltd. 2. S o l u b i l i t y p r o f i l e of barley and wheat proteins, (a) Preparation of chromatographic column. AUC, an aqueous d i s s o c i a t i n g medium of 0.1M acetic acid, 3M urea and 0.01M cetyltrimethylammonium bromide, was used as the eluent. Sephadex G100 (10 g) was suspended i n 800 ml of AUC f o r ?2 h at 20°C The swollen gel suspension was deaerated and pack-ed into a 2 . 5 x 4-5 cm Pharmacia column. The packed column was equilibrated i n a downward flow d i r e c t -ion with two volumes of AUC During subsequent runs, the column was operated i n a downward flow d i r e c t i o n , and the flow rate was maintained at 20 ml/h. The .void volume was determined by the elution volume of Blue Dextran-2000. Since Blue Dextran exhibited very poor s o l u b i l i t y i n AUC, i t was/first 22 dissolved i n d i s t i l l e d water. After complete d i s -solution, acetic acid, urea and cetyltrimethylammo-niumbromide. were .added to y i e l d concentrations of 0 . 1 M , 3Mi:and O.'OlM respectively. (b) Preparation of f l o u r extract i n AUC Barley or wheat f l o u r (4'g) was s t i r r e d i n 69 ml . of AUC. The AUC flour mixture was blended at top sp-eed with a S o r v a l l omnimixer f o r 2 min. After stand-ing at room temperature f o r 2 h, the mixture was centrifuged at 3 5 , 0 0 0 xG f o r 30 min. The supernat-ant (3 ml) was deaerated, and applied to the column. A fresh extract was prepared f o r each chromatographic run. (c) E l u t i o n patterns of proteins The column was mounted on a direct-volume measuring f r a c t i o n c o l l e c t o r . Fractions of 3 ml were co l l e c t e d . The e l u t i o n pattern of proteins was moinitpre'd'. with a Beckman DB spectrophotometer ; by measuring absorbance. at 280 nm i n a 1 cm c e l l against the solvent, AUC. 3 . S o l u b i l i t y f r a c t i o n a t i o n of proteins, (a) Fractionation of proteins The c l a s s i c a l protein f r a c t i o n a t i o n procedure of Osborne was employed (Chen and Bushuk. 1 9 7 0 ) . In order to minimize;'' the possible side effects of 23 p r o t e o l y t i c enzymes and thermal denaturation, the entire extraction was carried out at 4-°C. Flour was f i r s t defatted with petroleum ether, then 10 g defatted f l o u r was extracted with 4-0 ml of 0.5M NaCl i n a centrifuge tube f o r 2 h. Mild s t i r r i n g with a magnetic s t i r r e r was employed to avoid a r t i f a c t s that might be produced by high shear s t i r -r i n g . Each suspension was centrifuged i n a S o r v a l l Superspeed RC 2B Automatic Centrifuge f o r 30 min at i 8 6 0 xG. The supernatant was decanted. A sec-ond s i m i l a r extraction was carried out on the r e s i -due f o r 1 h. The residue was further extracted with 4-0 ml d i s t i l l e d water f o r 30 min to remove r e s i d u a l s a l t . The three supernatants were combined, and dialyzed against deionized water f o r 48 h i n 4-°C. The precipitated s a l t soluble protein was separated by centrifugation, while the water soluble f r a c t i o n remained i n the supernatant. The residue that re-mained a f t e r extraction with 0.5M NaCl was extract-ed i n a s i m i l a r manner with two portions of 70% ethanol, and the r e s u l t i n g residue was further ex-tracted twice with 4-0 ml of 0.05M acetic acid s o l -ution. Ethanol was removed from the combined sup-ernatants with a f l a s h evaporator. The four soluble protein fra c t i o n s and the f i n a l residue were freeze-dried. A summary of the Osborne method (Tanaka and 24-Defatted Flour extract with 0. centrifuge Supernatant Pre c i p i t a t e r dialyze against deionized water centrifuge extract with 7 0 $ E t o l centrifuge I Supernatant Precipitate Supernatant Precipitate freeze-dry freeze-dry Water Soluble Salt Soluble evaporate off alcohol freeze-dry Alcohol Soluble extract with 0.05M acetic acid centrifuge Supernatant freeze-dry Acid Soluble Precipitate J f g e z e -Residue F i g . 1 Flow Chart of Protein Fractionation Procedure. 25 Bushuk 1972) i s shown i n F i g . 1. (b) Determination of protein content The micro Kjeldahl method was used to determine nitrogen contents of the i n d i v i d u a l protein f r a c t -ions, and the t o t a l protein content of barley and wheat f l o u r s . The d i s t i l l a t i o n process was s u b s t i -tuted by the Technicon method carried out on an auto-analyzer. A conversion factor of 6.25 was used f o r barley, whereas 5*7 was used f o r wheat (Ewart 1968). 4. Determination of s u l f h y d r y l (SH) and d i s u l f i d e (SS) groups i n barley and wheat f l o u r s The method of Beveridge et a l . (1974) was used, with some modifications. Reagent grade chemicals were used to prepare the following: Tris-Glycine buffer (10.4 g T r i s , 6 . 9 g glycine, and 1.2 g EDTA per l i t e r , pH = 8 . 0 , hereafter denoted as T r i s - g l y ) , Urea-GuHCl (8M urea containing 5M GuHCl i n T r i s -gly) , Ellman's reagent (5 - 5'-dithiobis-2-nitroben-zoic acid) i n T r i s - g l y (4mg/ml). A 75 mg sample of f l o u r was dissolved i n 10 ml Urea-GuHCl. For SH determination, 4 ml of Urea-GuHCl was added to 1 ml of the above s l i g h t l y turbid f l o u r solution. Color was developed upon addition of 0 . 0 2 ml of Ellman's reagent. For SS determina-t i o n , 4 ml of Urea-GuHCl and 0 . 0 5 ml of 2-mercapto-26 ethanol were added to 1 ml of the f l o u r dispersion. The mixture was incubated f o r 1 h at 2 5 ° C After an additional 1 h incubation with 10 ml of 12$ TCA (t r i c h l o r o a c e t i c acid), the tubes were centrifuged at 7000 xG f o r 20 min. The pr e c i p i t a t e was t h r i c e suspended i n 5 ml of 12$ TCA, and centrifuged to remove the 2-mercaptoethanol. The p r e c i p i t a t e was dissolved i n 10 ml of 8M urea i n T r i s - g l y . Color was developed with 0.04- ml of Ellman's reagent. Absorbance was measured at 412 nm by a Beckman DB spectrophotometer. SH and t o t a l SH were calculated by the follow-ing equation: SH(uM/g) = 7 3 - 5 3 A^ 1 2D/C where A^ 1 2 = the absorbance at 412 nm, C = the sample concentration i n mg flour/ml, D = the d i -l u t i o n factor, 5-02 f o r SH and 10 f o r t o t a l SH (SH + reduced SS ), and 73«53 was derived from 10 6/(i-3o x 10^), I . 3 6 x 10^ i s the molar absorpti-v i t y units, 10^ i s f o r conversions from the molar basis to the uM/ml basis and from mg so l i d s to g s o l i d s . Preparation of microdoughs. (a) E f f e c t s of oxidizing agents on dough properties 27 Oxidizing agents used were ascorbic acid, potassium persulphate, ammonium persulphate and pot-assium iodate. Quantities of 150 and 250 mg o x i d i -zing agents (ca. 3 and 5$ t o t a l d.b.) were dissolved i n 3 . 5 ml d i s t i l l e d water. Mild heating was emp-loyed to f a c i l i t a t e d i s s o l u t i o n . The solution with dissolved oxidizing agent was added with constant s t i r r i n g into 5 g f l o u r , and the dough was further mixed by hand f o r 3 min. Fermentation took place i n a 38°C water bath f o r 1 .5 h. Doughs were man-u a l l y stressed and compressed to tes t f o r t h e i r cohesiveness and e l a s t i c i t y . The term 'cohesiveness' i n t h i s test i s used to describe the strength of the in t e r n a l bonds making up the body of the dough; 's t r e t c h a b i l i t y * i s used to describe the extent to which a dough can be stretched by manually applied stress before i t reptures; ' e l a s t i c i t y ' i s used to describe the degree to which a dough can be s t r e t -ched, and the extent to which the deformed dough goes back to i t s o r i g i n a l condition afterf the man-u a l l y applied force i s released. (b) E f f e c t s of oxidizing agents on doughs : at ..pH 12 Procedures were s i m i l a r to those outlined\above. The doughs' pH was adjusted vto 12; by replacing 1 ml of d i s t i l l e d water with 1 ml of IN NaOH. Oxidizing 28 agents were incorporated at 1 and 2$ l e v e l s into the dough mixtures. (c) E f f e c t s of*pH, on barley f l o u r and wheat starch doughs. Procedures were s i m i l a r to the above, except that no oxidizing agents were involved. The pH of the mixtures was progressively increased by increa-sing replacement of d i s t i l l e d water with IN NaOH. (d) E f f e c t s of d i f f e r e n t proteins on properties of fabricated doughs. E f f e c t s of d i f f e r e n t proteins on dough proper-t i e s were investigated by recombination studies of selected protein f r a c t i o n s . Basic procedures of dough making were used. Doughs were prepared from recombination and interchange of idifferent Osborne protein f r a c t i o n s . ( i ) Wheat and barley f l o u r doughs were prepared and used as controls ( i i ) 200 mg hordein + 4800 mg barley f l o u r (ca. 10$ protein basis of which 40$ i s the added hordein). ( i i i ) 200 mg hordein + 4 8 0 0 mg wheat f l o u r (iv) 500 mg v i t a l gluten + 4500 mg barley f l o u r (ca. 10$ protein added). (v) 500 mg v i t a l gluten + 4500 mg barley residue (after extraction of proteins). 29 (vi) 500 mg v i t a l gluten + 4-500 mg wheat residue. ( v i i ) 500 mg hordein + 4-500 mg barley residue (e) E f f e c t s of deuterium oxide and urea on wheat dcu'gh Procedures were s i m i l a r to basic dough prepara-t i o n method, except, either deuterium oxide or 3M urea was used i n place of water. Formation of 'gluten' from hordein i n v i t r o . (a) Extraction of hordein, and formation of 'gluten' i n v i t r o . Defatted f l o u r (20 g) was mixed with 100 ml 70% ethanol i n a centrifuge tube, mild s t i r r i n g was applied. Extractions were carried out separately at 80 and 20°C f o r 8 h. The alcohol soluble f r a c t -ion was separated out by centrifuging at 3500 xG f o r 30 min. Supernatants were divided into 10 ml port-ions. Different treatments were applied to these i n d i v i d u a l f r a c t i o n s as follows s (i) leaving overnight at room temperature ( i i ) standing overnight at 4-°C ( i i i ) leaving overnight at 4-°C a f t e r addition ( v i i i ) 500 mg hordein + 4-500 mg wheat residue (iv) of 2-mercaptoethanol (4-$ of t o t a l volume) standing overnight at 4-°C, a f t e r addi-t i o n of 2 drops 30$ hydrogen peroxide (ca. lfo of the t o t a l volume) 30 (v) standing overnight at 4°C, a f t e r add-i t i o n of 5 drops 0.1$ potassium iodate (ca. 0 . 0 0 2 5 $ of the t o t a l volume) Aggregated protein p r e c i p i t a t e s were found at the bottom of fra c t i o n s ( i i ) to (v). Supernatants were separated from the aggregated proteins a f t e r d i a l y z i n g against deionized water at 4°C f o r 48 h. Readings of pH and absorbance at 280 nm were carried out on the supernatants. (b) Dissolution of precipitated protein aggregates. Protein aggregates could not be dissolved or dispersed r e a d i l y i n water. Aliquots of 1 to 3 ml of IN NaOH were used to dissolve the protein aggre^' gates. The pH of the dissolved proteins was meas-ured, and n e u t r a l i z a t i o n was also attempted. (c) Preparation of microdoughs with dissolved protein aggregates. Procedures were s i m i l a r to the dough pre-paration methods. D i s t i l l e d water (1 ml) was r e p l -aced either by 1 ml of dissolved protein aggregates i n IN NaOH, or 1 ml of supernatants from alcohol extraction of d i f f e r e n t treatments. Doughs were manually stressed and compressed to test f o r t h e i r cohesiveness and e l a s t i c i t y . 31 ?. Kinetic studies on protein aggregation phenomena (a) Gluten preparation Flour was defatted with n-butyl alcohol. Star-ch and other non-gluten materials were washed from the f l o u r by hand kneading i n 0.1$ NaCl solution. The gluten b a l l was dispersed at about .5$ protein concentration i n 0.01M acetic acid i n a Waring blen-der, and the r e s u l t i n g dispersion was centrifuged at 20,000 xG. To inactivate p r o t e o l y t i c enzymes, the solution was brought to 9 8 to 100°C by heating on a hot plate, cooled quickly, and then freeze-dried. The r e s u l t -ing white f l u f f y product contained traces of acetic acid which f a c i l i t a t e d i t s s o l u b i l i t y i n water. (b) Separation of prolamin and g l u t e l i n by gel f i l t r a t i o n on Sephadex G100 column. Sixty mg of gluten were dispersed i n 6 ml of 0.1M acetic acid,^with -t a S o r v a l l omnimixer at top speed f o r 5 min. A f t e r the foam had subsided, the gluten solution was applied to a Sephadex Gl00 column (2 . 5 x 4-5 cm) equilibrated with 0.1M acetic acid. Pre-paration of the Sephadex G100 column was i d e n t i c a l to the G100 AUC column used f o r e l u t i o n patterns of proteins, except 0.1M acetic acid was used as'eluent. El u t i o n was made with 0.1M acetic acid, and flow 32 rate was maintained at 20 ml/h. The elution curve was obtained by measuring absorbance at 280 nm, using a Beckman DB spectrophotometer. G l u t e l i n f r a c t i o n s , which were eluted f i r s t , and prolamin f r a c t i o n s , which came off l a t e r were coll e c t e d sep-arately. The i n d i v i d u a l protein f r a c t i o n s were conc-entrated i n d i a l y s i s tubings by pervaporization .and then freeze-dried. The microkjeldahl method was used to determine the nitrogen contents of both prolamin and g l u t e l i n f r a c t i o n s . (c) Determination of time course curve of t u r b i d i t y . An aliquot of 0.2M phosphate buffer, containing 2M NaCl pH 5 • 6 was prepared. Two ml of protein so-l u t i o n i n 0.01M acetic acid (ca. 0.02$ protein) was mixed with an equal volume of phosphate buffer. The time course of"" t u r b i d i t y development was deter-mined by Beckman DB spectrophotometer at 350 nm, and recorded by a Texas Instrument S i l e n t 700 ASR data a c q u i s i t i o n u n i t . Measurement was made at 25°C with quartz c e l l s of 1 cm l i g h t path. Readings were recorded at i n t e r v a l s of 1 .5 sec. The rate constant of aggregation, K, from the early stage of the t u r b i d i t y change was calculated with the aid of L i t t o n Monroe 1880 cal c u l a t o r from 33 the equations below y 3 = 3rt + T; K = r / C 3 •3 o (Arakawa et a l . 1974; Arakawa and Yonezawa 1 9 7 5 ; Arakawa et a l . 1976) where T= t u r b i d i t y , r = constant, t = time i n sec and C = protein concentration i n g/100 ml. A comp-uter l i n e a r regression programme incorporated with p l o t t i n g routine was used to f a c i l i t a t e t u r b i d i t y ( T 5) versus time (t) p l o t , and c a l c u l a t i o n of r values. T and t values of every 7>5 sec were en-tered into L i t t o n Monroe 1880 c a l c u l a t o r . T 5 v e r s u s t was plotted by a L i t t o n Monroe PL-2 p l o t t e r , and the slope of the curve (3r) was calculated simul-taneously. (d) Reduction and cyanoethylation of g l u t e l i n f o r Sephadex G150 column. Phosphate buffer of 0.1M concentration, contain-mg 8M urea and 10 M EDTA at pH 7-4 was prepared. A sample of 25 mg g l u t e l i n was dissolved into 4 ml of the above phosphate buffer. After removal of oxygen by bubbling with nitrogen, 0 . 0 2 5 ml of 2-mereaptoethanol was added to the solution and l e f t overnight at room temperature. The g l u t e l i n solution Mas allowed to stand f o r 4-5 min a f t e r addition of 0 . 0 5 ml of a c r y l o n i t r i d e . Then, the mixture was a c i d i f i e d with 2M acetic acid, and dialyzed against 0.01M acetic acid to remove the reagents. 34 (e) Gel f i l t r a t i o n of g l u t e l i n polypeptides on a Sephadex G150 column. Sephadex G150 (20 g) was suspended i n 1 L 0.1M acetic acid i n 90°C water-bath f o r 5 h. Packing procedures were s i m i l a r to Sephadex G100 columns, except an upward-flow was employed during experimen-t a l operations, and the flow rate was maintained at 15 ml/h. A dialyzed g l u t e l i n sample of 5 ml was applied to the G150 column (25 x 100 cm), and elution was carried out with 0.1M acetic acid. Fractions of 3 ml were collected and the absorbance at 280 nm was measured with a Beckman DB spectro-photometer . 8. Hydrophobic comparison between barley and wheat proteins using fluorescence quenching technique. A mixture of trichloroethanol (TCE) with ethy-lene g l y c o l ( 5 0 : 5 0 , v/v) was prepared and used as a stock solution f o r quenching studies. A; 0--.01M -sodium acetate buffer (pH . 5 . 5 ) . was f r e s h l y prepared each time before the fluorescence studies were per-formed. An excitation wavelength of 295 nm was used during these studies to ensure that the l i g h t was absor-bed almost e n t i r e l y by trytophan groups. A sample of 2 . 5 ml of protein (ca. 10$ concentration) dissolved i n 35 0.01M acetate "buffer, was added i to a fl u o r i m e t r i c cuvette. The fluorescence of a protein, monitored at i t s emission maximum, was quenched by the pro-gressive addition of small aliquots of 5-2M t r i c h l o -roethanol i n ethylene g l y c o l . The solution was then mixed by gentle inversion of the curvette. Blowing of the quencher solution into the aqueous solution was avoided. These precautions were taken t o mini-mize the formation of trichloroethanol droplets i n the solution, which might act to denature the prot-ein. Quenching data were analyzed according to the Stern-Volmer equation F. o - 1 + K_ (Q) ~F~ * ' "sv (Eftink and Ghiron 1976; E f t i n k et a l . 1977) where F Q and F are the fluorescence i n t e n s i t i e s at an appropriate emission wavelength i n the absence and presence of quencher respectively-.while K g v i s the c o l l i s i o n a l quenching constant. By p l o t t i n g F Q/F vs. (Q)» K g v can be obtained. A more complete treatment of the k i n e t i c s of the quenching data can be made by the following mod-i f i e d Stern-Volmer equation F Q/F = (1 + K s v ( Q ) ) e V < Q ) (Eftink and Ghiron 1976} E f t i n k et a l . 1977) 36 The added factor (V) i n t h i s equation describes the quenching of the fluorescence that occurs by a s t a t i c process t. V, ? the s t a t i c quenching constant, i s a parameter that i s related to the p r o b a b i l i t y of f i n d i n g a quencher molecule close enough to a newly formed excited state to quench i t immediately or s t a t i c a l l y . In a randomly di s t r i b u t e d aqueous system there w i l l be a small p r o b a b i l i t y of f i n d i n g the quencher and chromophore molecules very close to each other at the moment that the chromophore becomes excited. I f there i s such an i n t e r a c t i o n , the p r o b a b i l i t y f o r the reaction i s so high that quenching occurs almost instantaneously ( s t a t i c a l l y ) . 9« Total amide nitrogen determination Amide nitrogen was determined according to W i l -cox's procedure (I966). A sample of 100 mg gluten was refluxed i n 50 ml of 2M HCl (ca. 2 mg of protein f o r each ml of acid) f o r 3 h. Amide nitrogen was determined as d i s t i l l a b l e ammonia. D i s t i l l a t i o n and t i t r a t i o n were carried out i n a microkjeldahl appar-atus (Wu et a l . 1976). 10. Amino acid analysis A Phoenix Micro Amino Acid Analyzer M 6800, f i t t e d with a Durrum single column system (Durrum 37 Chem. Corp., Palo Alto, C a l i f . ) was used f o r amino acid analysis. Hydrolysis of protein was done i n the presence of paratoluenesulfuric acid and 3(2-aminoethyl) indole at 110°C f o r Zh h. After hydrolysis the sample was f i l t e r e d with an u l t r a f i n e f i l t e r before applying to the Amino Acid Analyzer (Liu and Chang 1 9 7 1 ) . 38 RESULTS AND DISCUSSION 1. Gel chromatography of barley and wheat proteins. E l u t i o n patterns of wheat and barley proteins were carried out on a Sephadex G100 column. The prot-ein molecules were separated according to t h e i r molecular sizes as they eluted through the cross-linked dextran column. G l u t e l i n , containing mainly large protein molecules, was eluted f i r s t ; followed by prolamin, albumin and globulin. D i s t r i b u t i o n of barley and wheat proteins were s i m i l a r , except proteins of barley were eluted at a s l i g h t l y lower molecular weight region (Fig. 2 ) . 2 . Quantitative r e l a t i o n s h i p between loaf volume and protein content or protein s o l u b i l i t y d i s t r i b u t i o n . (a) S o l u b i l i t y f r a c t i o n a t i o n of endosperm protein Protein content i s believed to be the main factor responsible f o r v a r i a t i o n i n loaf volume within a wheat vari e t y . Although the t o t a l protein content of barley i s lower than wheat (9.6$ com-pared with 14.4$;), i t s t i l l l i e s within the range (8 to 18$ protein, Pomeranz et a l . 1970) of bread-making f l o u r (Table 1). S o l u b i l i t y f r a c t i o n a t i o n of barley and wheat proteins revealed that t h e i r s o l u b i l i t y p r o f i l e s were very s i m i l a r . Not much difference was obser-39 Fife. 2 E l u t i o n Curve of B a r l e y and Wheat P r o t e i n s on Sephadex G100 Table 1. T o t a l P r o t e i n Recovered from S o l u b i l i t y F r a c t i o n a t i o n vs. T o t a l P r o t e i n Content Component T o t a l recovered T o t a l P r o t e i n % P r o t e i n p r o t e i n (•£') content* ($) recovered Barley f l o u r 8 . 8 6 9 . 6 0 + 0 . 1 4 92.29 Wheat f l o u r 13-80 14.42+ 0.93 95-70 Whole wheat 12-30 1 3 - 2 1 + 0 . 5 4 93-11 f l o u r * average of f o u r determinations 41 ved between the protein contents of barley and wheat albumin, globulin and prolamin f r a c t i o n s . The g l u t -e l i n content of barley was lower, whereas i t s r e s i -due protein f r a c t i o n was s l i g h t l y higher than wheat (Table 2 , F i g . 3)» There were some carry-overs i n proteins i s o l a t e d by the s o l u b i l i t y f r a c t i o n a t i o n technique. They were heterogenous, and mutually contaminated to some extent. But, they s t i l l repre-sent f a i r l y d i s t i n c t i n d i v i d u a l groups of protein having t h e i r own s p e c i f i c i t i e s (Chen and Bushuk 1 9 7 0 ) . (b) Remix loaf volume (RLV) Orth et a l . (1972) indicated that there i s a quantitative r e l a t i o n between loaf volume and prot-ein s o l u b i l i t y d i s t r i b u t i o n . The dependence of remix loaf volume on the protein s o l u b i l i t y d i s -t r i b u t i o n and f l o u r protein can be described by the equation: RLV = 65.O x protein + 3 5 - ^ x albumin - 15•7 x globulin + 6 . 7 x g l i a d i n - 11 .3 x glutenin + 13 .1 x residue - 659 Baking studies have shown that the loaf volume of barley i s only about one-third that of wheat bread (Hart et a l . 1 9 7 0 ) . However, the RLV calcu-lati o n s did not show much deviation between them 42 Table 2. S o l u b i l i t y Fractionation of the Endosperm Protein Component Barley Wheat Whole wheat f l o u r * f l o u r * f l o u r * Salt soluble f r a c t i o n Weight, mg 61 ± 2 117 ± 4 115 ± 3 Protein content of the f r a c t i o n , mg 48 74 113 Fraction of t o t a l protein % 5 5 9 Water soluble f r a c t i o n Weight, mg 440 +8 3 2 7 + 4 3 5 0 + 7 Protein content of the f r a c t i o n , mg 132 173 148 Fraction of t o t a l protein fo 15 13 12 Alcohol soluble f r a c t i o n Weight, mg 421+2 504 + 6 570 + 7 Protein content of the f r a c t i o n , mg 263 4l4 453 Fraction of t o t a l protein % 30 30 37 Acid soluble f r a c t i o n Weight, mg 298 + 5 381+7 450 + 4 Protein content of the f r a c t i o n , mg 109 229 202 Fraction of t o t a l protein % 12 17 16 Residue protein Weight, mg 8427 + 4 8319 + 8 8430 + 6 Protein content of the f r a c t i o n , mg 334 490 314 Fraction of t o t a l protein % 38 36 26 Total recovered protein % 8 . 9 13.8 12-3 * average of four determinations 43 i s a l t soluble water soluble alcohol soluble acid soluble residue F i g . 3 S o l u b i l i t y D i s t r i b u t i o n of Barley, Wheat and Whole Wheat Flours (Table 3 ) . Since, the value of RLV i s dependent on the protein s o l u b i l i t y d i s t r i b u t i o n , the solu-b i l i t y d i s t r i b u t i o n of barley and wheat proteins are quite s i m i l a r , i t i s reasonable that t h e i r RLV are c l o s e l y related. 3. Sulfhydryl and d i s u l f i d e groups i n barley and wheat f l o u r s . Sulfhydryl and d i s u l f i d e groups have always been considered as important factors a f f e c t i n g the breadmaking properties of f l o u r protein. They play a v i t a l r ole i n dough structure and vi s c o -e l a s t i c properties. Determination of t o t a l s u l f -hydryl and t o t a l d i s u l f i d e groups did not show a dras t i c difference between barley and wheat f l o u r (Table 4). Wheat f l o u r had s l i g h t l y higher SH and SS values, however, not a l l the a n a l y t i c a l l y deter-mined SH, SS groups are equally l a b i l e to d i s u l f i d e interchange. The a c c e s s i b i l i t y of SH and SS groups i s determined by the s p e c i f i c conformation of that p a r t i c u l a r protein, and i t i s postulated that only small fractions of the a n a l y t i c a l l y determined t h i o l and d i s u l f i d e groups are Theologically e f f e c t i v e (Kuninori I968; Bloksma 1972b). The t o t a l SH and SS contents of barley were 45 Table 3 . Prediction of Loaf Volume from RLV Component RLV Barley f l o u r 9 1 3 . 8 Wheat f l o u r 1 0 7 5 . 4 46 Table 4. Tota l S u l f h y d r y l - d i s u l f i d e Values of Barley and Wheat Flours Flour Protein SH (uM/g dry wt.) SS (uM/g dry wt.) Barley f l o u r 1 . 3 8 ± 0 . 0 8 1 2 . 3 0 ± 0.14 Wheat f l o u r I . 7 2 ± 0.09 13.85 + 0.11 * average of four determinations 47 almost comparable to wheat. However, the T h e o l o g i -c a l l y e f f e c t i v e groups of barley might be i n f e r i o r i n q u a l i t y and quantity, which resulted i n i t s poor breadmaking properties. Studies have shown that more than one-half of the SH content of f l o u r dough i s not involved i n dough mixing properties. They are located i n the albumin and globulin f r a c t i o n s . The least l a b i l e ones are those found i n the glutenin protein. These SH groups are responsible f o r dough mixing properties ( S u l l i v a n et a l . I 9 6 3 ) . Glutenin i s regarded as the functional protein i n breadmaking, and i t i s the only constituent of wheat f l o u r that exhibits s i g n i f i c a n t v i s c o e l a s t i c i t y when i s o l a t e d . Barley f l o u r has a lower g l u t e l i n content (12 . 3 $ on a t o t a l protein basis) than wheat f l o u r (16 . 6 $ ) (Table 1). I f the r h e o l o g i c a l l y e f f e c t i v e t h i o l and d i s u l f i d e groups are located within the g l u t e l -i n protein, t h i s may account f o r the difference i n d i s u l f i d e interchange p o t e n t i a l between barley and wheat proteins since a s l i g h t decrease i n T h e o l o g i -c a l l y e f f e c t i v e groups would have tremendous effect i n breadmaking properties. Furthermore the proteins of barley and wheat have l i t t l e immunochemical s i m i l a r -i t y . ' Thus, t h e i r difference i n t e r t i a r y structure may 48 well a f f e c t the a c c e s s i b i l i t y of d i s u l f i d e bonds and the magnitude of the d i s u l f i d e interchange p o t e n t i a l (Ewart 1968). 4. Preparation of microdoughs. (a) E f f e c t s of oxidizing agents on fabricated doughs. Addition. up.Tto 5$ of oxidizing agents gave some improvements to the cohesiveness and e l a s t i c i t y of barley dough. However, the v i s c o e l a s t i c i t y and ex-t e n s i b i l i t y of barley dough are s t i l l not compara-ble to those of wheat (Table 5)• Oxidation strong-l y a ffects the rheological properties of wheat dough because incorporation of a very minute amount of ox-i d i z i n g agents would r e s u l t i n a considerable i n -crease i n loaf volume (Bloksma 1972). But, the pro-teins of barley do not give a s i m i l a r response. Perhaps the s u l f h y d r y l and d i s u l f i d e groups of bar-ley are very inaccessible, or t h e i r properties are d i f f e r e n t from wheat t h i o l and d i s u l f i d e groups. (b) E f f e c t s of oxidizing agents:; at pH 121. Great improvements i n cohesiveness and e l a s t -i c i t y of barley doughs were observed when oxidizing agents were added, and pH was adjusted to 12. The doughs became brownish i n color, very sof t and s t i c -ky (Table 6). Similar effects were also obtained 49 Table 5« E f f e c t s of Oxidizing Agents on Fabricated Doughs Flour Oxidizing Concen-type agent t r a t i o n (% d.b.) Cohesive- E l a s t - Stretch-ness i c i t y a b i l i t y Barley ascorbic acid Barley potassium iodate Barley Barley Barley Wheat ammonium persulphate potassium persulphate 3 5 3 5 3 5 3 5 o o 2 2 3 3 2 2 2 2 0 10 1 1 0 2 2 2 2 2 0 10 3 2 0 1 1 1 2 1 0 10 Scale i 0 to 10 0 = poor 1 = scarcely, any improvement 2 = very s l i g h t improvement 3 = s l i g h t improvement 10 = best 50 Table 6 . E f f e c t s of Oxidizing Agents on Fabricated Doughs at pH 12 Flour type Oxidizing agent Concen-t r a t i o n ($ d.b.) E l a s t i c i t y Cohesiveness Barley ammonium persulphate 10 Barley potassium persulphate 10 Barley ascorbic acid 3 10 Barley 10 Wheat 10 10 Scale J 1 to 10 1 = poor 10 = best 51 without the presence of oxidizing agents at pH 12 (Table 7 ) • Dough prepared from wheat starch, which was completely devoid of protein, at i t s own o r i g i n a l pH (pH 6 . 3 )» was tough and inextensible. However stickiness and e x t e n s i b i l i t y increased with increa-sing pH. When the pH was at or above 1 0 , excessive stickiness.-was observed (Table 7) • Therefore, i n -creases i n stic k i n e s s and e x t e n s i b i l i t y were not the r e s u l t of protein interactions nor the eff e c t s of oxidizing agents, but rather, they were merely due to starch g e l a t i n i z a t i o n at high pH. (c) E f f e c t s of d i f f e r e n t proteins on properties of fabricated doughs. The proteins of barley and wheat f l o u r are di f f e r e n t i n dough-making properties, although some of t h e i r gross appearances such as s o l u b i l i t y pro-f i l e ; protein content are quite s i m i l a r . Doughs made from barley f l o u r were tough, f l a t and inex-tensible, while wheat f l o u r doughs were s o f t , elas-t i c , cohesive and extensible. Addition of hordein proteins to barley and wheat doughs d i d not improve t h e i r physical properties, and i n f a c t , the hordein proteins interfered with the dough forming proper-t i e s of wheat f l o u r . Incorporation of v i t a l gluten, 52 T a b l e 7 . E f f e c t s o f . pH o n F a b r i c a t e d D o u g h s T y p e o f d o u g h NaOH a d d e d ( m l ) p H C o h e s i v e n e s s B a r l e y f l o u r 0 . 0 5 - 0 0 0 . 5 9 . 6 3 1 .0 1 0 . 5 4 1.5 1 1 . 2 5 2 . 0 11 .5 5 2 . 5 1 1 . 5 5 3 . 0 1 2 . 0 5 B a r l e y r e s i d u e 0 . 0 5 - 8 0 1 .0 9 . 4 3 1.5 1 1 . 0 5 W h e a t s t a r c h 0 . 0 6 . 3 0 1 .0 1 0 . 7 4 1.5 1 1 .6 5 S c a l e J 0 t o 5 0 = n o n c o h e s i v e 5 = v e r y c o h e s i v e 53 which would normally improve the rheology and loaf volume of wheat bread, did not improve barley f l o u r . However, v i t a l gluten greatly improved the dough:-mak-ing properties of wheat and barley residues. V i t a l gluten together with wheat residue gave dough pro-perties s i m i l a r to wheat f l o u r dough. V i t a l gluten and barley residue yielded a s o f t , e l a s t i c dough, which was quite s i m i l a r to the effect of wheat f l o u r , except e x t e n s i b i l i t y was s t i l l quite i n f e r i o r (Table 8). Hordein i t s e l f could not duplicate the role of glu-ten. Its f u n c t i o n a l i t y i n dough-making i s i n f e r i o r to wheat gluten. From the above recombination studies, i t i s obvious that barley protein does not have s u f f i c i e n t dough-making p o t e n t i a l . In f a c t , i t s presence might in t e r f e r e with the other breadmaking properties of the wheat f l o u r dough. It decreases the e l a s t i c i t y and e x t e n s i b i l i t y of wheat flour.dough and does not improve the properties of barley f l o u r . No improve-ment was observed when v i t a l gluten was added to barley f l o u r . However, v i t a l " gluten together with barley residue, which i s free of barley soluble and insoluble proteins, gave dough properties f a i r l y s i m i l a r to wheat f l o u r . •54 Table 8 . E f f e c t s of Different Proteins on Properties of Fabricated. Doughs. .. Type of dough Physical properties o Wheat f l o u r ( 5 . 0 0 0 mg) Barley f l o u r ( 5 . 0 0 0 mg) Hordein and barley f l o u r (200 mg hordein + 4 , 8 0 0 mg barley f l o u r ) Hordein and wheat f l o u r (200 mg hordein + 4 , 8 0 0 mg wheat fl o u r ) V i t a l gluten and barley f l o u r (500 mg v i t a l gluten + 4 , 5 0 0 mg barley f l o u r ) V i t a l gluten and barley residue (-.500 mg v i t a l gluten + 4 , 5 0 0 mg barley residue) V i t a l gluten and wheat residue (500 mg v i t a l gluten + 4 , 5 0 0 mg wheat residue) Hordein and barley residue (500 mg hordein + 4 , 5 0 0 mg barley residue) Hordein and wheat residue ( 5 0 0 mg hordein + 4 , 5 0 0 mg wheat residue) white, cohesive, e l a s t i c and extensible greyish white, tough, loose, inextensible buff, dry, loose, but s l i g h t l y softer than barley dough dry, tough, l o s t the e l a s t i c i t y and extensi-b i l i t y of wheat f l o u r very s l i g h t improvement in cohesiveness and e l a s t i c i t y white, s o f t , e l a s t i c , properties very s i m i l a r to wheat dough, except not so extensible properties were s i m i l a r to wheat f l o u r dough loose, tough, non-e l a s t i c cohesive, tough, non-e l a s t i c and inextensible 55 Extraction of hordein and formation of 'gluten' i n v i t r o . Hot (80°C) alcohol extraction of hordein gave a s l i g h t l y higher y i e l d than extraction c a r r i e d out at 20°G. Storage of these alcohol soluble proteins at k°C f o r several hours would cause agg-regation of hordein molecules at the bottom of the fl a s k . This p a r t i c u l a r phenomenon was not obser-ved when hordein was extracted under s i m i l a r cond-i t i o n s , but l e f t standing at room temperature (Table 9) • Addition of d i s u l f i d e s p l i t t i n g reagents such as 2-mercaptoethanol to the alcohol soluble prot-ein, p r i o r to incubation at 4-°C, greatly reduced the amount of protein aggregates formed. Incorp-oration of a small amount of oxidizing agents, such as hydrogen peroxide and potassium iodate, enhanced the rate of protein polymerization. This indicates that protein d i s u l f i d e groups are involved i n these reactions. Protein d i s u l f i d e groups respond quite d i f f e r e n t l y to various oxidizing agents, hence s l i g h t differences i n the properties of aggregated proteins were observed (Table 9). The minute amount of protein aggregates formed, a f t e r addition of 2-mercaptoethanol, was due to the incomplete s p l i t -56 Table 9 . Extraction of Hordein and Formation of 'Gluten' i n v i t r o Extraction Treatment of Properties of temperature sample aggregated proteins CC) 20 l e f t at room temp. no aggregation was observed 20 l e f t at 4°C white, rubbery, cohesive and f a i r l y e l a s t i c protein aggregates 20 l e f t at 4°C af t e r addition of 2-mercaptoethanol only very minute amount of protein aggregates formed. Properties were quite s i m i l a r to the above 20 l e f t at 4°C af t e r addition of hydrogen peroxide l i g h t yellowish p a r t i c l e s , soft and e l a s t i c 20 l e f t at 4°C af t e r addition of potassium iodate dark brown, loose granules, not very cohesive and e l a s t i c 80 l e f t at room temp. no protein aggre-gation occurred 80 l e f t at 4°C greyish white, loose granules, neither cohesive nor e l a s t i c . Properties are quite d i f f e r e n t from those extracted at 20 C 57 t i n g of the d i s u l f i d e "bridges during storage at 4°C. Reheating of the ethanolic extract containing aggregated proteins to room temperature did not dissolve these protein aggregates. Therefore, the polymerization phenomenon i s not a consequence of an ordinary decrease i n s o l u b i l i t y of protein with a decrease i n temperature. Apparently, i t i s a re s u l t of hordein polymerization through d i s u l f i d e bridges. At low temperature the reversi b l e oxid-ation-reduction reaction leading to the formation of a d i s u l f i d e bridge from two su l f h y d r y l groups s h i f t s i n favor of t h e i r oxidation (Shestakova et a l . 1976). Consequently, polymerization of proteins f a i l s to occur at room temperature. Agg-regation v i a d i s u l f i d e bridges merely takes place at low temperatures (around 4°C), and only a f r a c t -ion of the alcohol soluble protein i s capable of -polymerization. Readings of absorbance at 280nm showed that f a i r l y high protein concentrations s t i l l remained i n the supernatant as soluble hordein (Table 10). Amino acid composition of protein aggregates and hordein that remained soluble at 4°C, were not i d e n t i c a l , yet, electrophoresis patterns revealed 58 Table 10. Absorbance Reading of Alcohol Soluble Proteins* Supernatant from d i f f e r e n t treatments Absorbance 280 Left at room temperature - no polymerization occurred > 2.0 Storage at 4°C a f t e r addition of 2-mercaptoethanol >.2.0 After polymerization at 4°C 1.5 After polymerization at 4°C with addition of hydrogen,peroxide 1.3 After polymerization at 4 C with addition of potassium iodate 0.7 * the supernatant fracti o n s a f t e r protein polymerization 59 that both wereccomposed e n t i r e l y of sub-units which corresponded to the polypeptide chains of hordein (Shestakova et a l . 1976). Evidently, some of the alcohol soluble proteins i n barley are capable of gluten formation i n v i t r o , as a r e s u l t of hordein polymerization through d i s u l f i d e bridges. The aggregated protein p r e c i p i t a t e s extracted from the hordein f r a c t i o n of barley was a complex containing hordein and g l u t e l i n , formed exclusively from polymerization of hordein molecules. E l e c t r o -phoretic analysis performed by Shestakova et a l . (1976) showed that the g l u t e l i n present i n the 'gluten' formed i n v i t r o was not i d e n t i c a l to the natural barley g l u t e l i n . I t appeared to be a polymer com-posed of hordein molecules joined together by d i -s u l f i d e bridges. These g l u t e l i n - l i k e molecules were bound noncovalently to the excess hordein molecules, which resulted i n the formation of gluten-like pro-t e i n aggregates. These white aggregated proteins were s o f t , rubbery, cohesive and quite e l a s t i c . They had s p e c i f i c properties s i m i l a r to gluten rather than hordein. Unlike hordein, i t did not dissolve well i n 70fo ethanol, but, dissolved r e a d i l y under alkaline condition, which i s s i m i l a r to the proper-t i e s of g l u t e l i n . The pH of re-dissolved proteins 60 was very; h i g h ( pH = 11 ) (Table 11). N e u t r a l i z a -t i o n of pH back to 6 caused r e p o l y m e r i z a t i o n and aggregation. Since aggregated p r o t e i n p r e c i p i t a t e s bear many p h y s i c a l resemblances to wheat g l u t e n , r e - d i s s o l v e d p r o t e i n aggregates from d i f f e r e n t treatments were incorporated i n microdough preparations i n order to i n v e s t i g a t e t h e i r p o t e n t i a l i n breadmaking. A d d i t i o n of r e - d i s s o l v e d p r o t e i n s d i d not increase the e l a s t i c i t y of microdoughs when compared wi t h the c o n t r o l ( b a r l e y dough). They became very s t i c k y , cohesive and brownish i n c o l o r . These e f f e c t s were the consequence of s t a r c h g e l a t i n i z a t i o n at high pH, because pH of the r e - d i s s o l v e d p r o t e i n s was very high (pH 11). A d d i t i o n of hordein t h a t remained s o l u b l e at 4°C gave very s l i g h t improvement t o the e l a s t i c i t y of microdough. I t d i d not a f f e c t the c o l o r nor increase the s t i c k i n e s s of the dough. 6. K i n e t i c s t u d i e s on aggregation behavior of p r o t e i n s . (a) P h y s i c a l p r o p e r t i e s and molecular weight d i s t r i b u t i o n s of glu t e n s . Great d i f f e r e n c e s were observed between the p h y s i c a l p r o p e r t i e s of wheat and b a r l e y g l u t e n . Gluten i s o l a t e d from wheat f l o u r was e l a s t i c , 61 Table 11. Dissolution of Protein Aggregates Type of dis s o l v i n g agent Behavior of proteins pH 70$ ethanol sparingly soluble 5«6 IN NaOH a l l proteins from 11.0 d i f f e r e n t treatments dissolved completely i n 1 to 1.5 ml NaOH 62 rubbery, g i v i n g a strong body texture and the a b i -l i t y to form a white s o l i d cohesive mass. Whereas gluten from barley f l o u r was loose, c l a y - l i k e , s o f t , and lacked of cohesiveness and e l a s t i c i t y . There-fore upon hydration, wheat gluten could provide a network holding the starch granules and other prot-ein molecules i n po s i t i o n so as to give bread i t s structure while the barley gluten could not. The elution curves from Sephadex G100 i n d i c a t -ed that barley and wheat glutens had d i f f e r e n t mol-ecular weight d i s t r i b u t i o n s . The peak,area.of barley g l u t e l i n was ' smaller i n si z e than wheat glu-tenin. The hordein f r a c t i o n did not appear at the same pos i t i o n as that of wheat g l i a d i n peak. Its delayed elution volume indicates that the molecular weight of hordein i s lower (Fig. 4). This d i f f e r -ence i n molecular weight d i s t r i b u t i o n may exhibit o a profound influence on breadmaking properties bet-ween barley and wheat proteins. (b) Aggregation phenomenon of proteins. : :„ . The aggregation phenomenon of barley and wheat prolamin, g l u t e l i n and unfractionated gluten were studied by determining the time course curve of t u r -b i d i t y development i n protein suspensions. A consi-derable difference i n the time course curves of 63 30 3o §5 i2o iSo i3b~ ml > F i g . 4 E l u t i o n Patterns of Wheat and Barley Glutens 6k t u r b i d i t y was o b s e r v e d b e t w e e n v a r i o u s p r o t e i n s ( F i g s . 5 » 6 , 7 ) ' The a g g r e g a t i o n b e h a v i o r o f b a r -l e y p r o t e i n s was a l w a y s i n f e r i o r t o w h e a t p r o t e i n s , e s p e c i a l l y i n t h e c a s e o f a l c o h o l s o l u b l e p r o t e i n s . The h o r d e i n o f b a r l e y d i d n o t show a n y s i g n o f a g g r e g a t i o n , w h e r e a s t h e g l i a d i n o f w h e a t g a v e t h e g r e a t e s t a g g r e g a t i o n p o t e n t i a l o f a l l t h e p r o t e i n s i n v e s t i g a t e d ( F i g . 7 ) . When t h e r a t e c o n s t a n t o f a g g r e g a t i o n , K, was c a l c u l a t e d f r o m t h e e a r l y s t a g e o f t h e t u r b i d i t y c h a n g e , t h e v a l u e s o f t h e w h e a t p r o t e i n s w e r e a l w a y s h i g h e r when c o m p a r e d w i t h t h e same p r o t e i n f r a c t i o n s i n b a r l e y ( T a b l e 12) . A s i m i l a r s i t u a t i o n was a l s o o b s e r v e d w i t h t h e t u r -b i d i t y v a l u e s a t t h e l a t e r s t a g e o f a g g r e g a t i o n . The r a t i o s o f T^ 0/C, where i s t h e t u r b i d i t y a t 10 m i n o f r e a c t i o n t i m e , a nd C i s t h e c o n c e n t r a -t i o n o f p r o t e i n , w e r e a l w a y s h i g h e r i n w h e a t p r o t e -i n s ( T a b l e 12 ) . B o t h t h e r a t e o f a g g r e g a t i o n ( K ) , and ^ I Q / C i n c r e a s e d i n t h e o r d e r o f b a r l e y h o r d e i n , b a r l e y g l u t e l i n , w h e at g l u t e n i n , b a r l e y g l u t e n , w h e a t g l u t e n a nd wheat g l i a d i n . ( c ) G e l f i l t r a t i o n o f g l u t e l i n p o l y p e p t i d e s . E l u t i o n c u r v e s o f b a r l e y and w h e a t g l u t e l i n f r o m S e p h a d e x G150, showed t h a t t h e y w e r e d i f f e r e n t i n p o l y p e p t i d e c o m p o s i t i o n s . Wheat g l u t e n i n c o u l d 6 5 F i g . 5 Time C o u r s e o f T u r b i d i t y w i t h B a r l e y and Wheat G l u t e n s F i g . 6 . T i m e C o u r s e o f T u r b i d i t y w i t h B a r l e y and Wheat G l u t e l i r i s Table 12. Aggregation Behavior of Barley and Wheat Proteins 3 * * Source of protein K x 10 'f*/C ** Wheat g l i a d i n Barley g l i a d i n Wheat gluten Barley gluten Wheat glutenin Barley g l u t e l i n 25.02 + 0 .17 19.04 + 0.11 4.85 ± 0.10 2.28 + 0 .07 0.95 ± 0.04 82 .55 + O.36 48 .71 + 0.20 40 .63 + 0.15 34.03 + 0.12 23.13 + 0.11 * no signs of aggregation ** average of four determinations 69 be fractionated into 3 d i s t i n c t peaks (Fig. 8), . whereas only 2 peaks were present i n barley g l u t -e l i n (Fig. 9)« These two peaks were located at sim-i l a r e l u t i o n volumes as the F^ and F^ f r a c t i o n s of wheat glutenin. The F 2 f r a c t i o n of wheat glutenin, which has the highest aggregation p o t e n t i a l of a l l the three f r a c t i o n s , and i s i n f a c t responsible f o r the differences i n breadmaking properties between wheat v a r i e t i e s (Arakawa et a l . 1976), i s missing i n barley. The amount of barley g l u t e l i n protein at the F^ p o s i t i o n i s larger than F^ f r a c t i o n i n wheat, which indicates that barley g l u t e l i n has a comparatively larger amount of proteins i n the lower molecular weight region. The difference i n molecular weight d i s t r i b u t i o n between barley and wheat gluten would contribute to the difference i n breadmaking potentials between these two proteins. The differences i n physical properties and molecular weight d i s t r i b u t i o n s of barley and wheat glutens, the f a i l u r e of hordein to aggregate during t u r b i d i t y studies, and the difference i n polypeptide composition between barley and wheat g l u t e l i n , sug-gested that barley and wheat glutens are d i f f e r e n t i n property and composition. 70 0-24 E c O 00 CN <D <J c o 5°-' < 0 -WHEAT 6 0 9 0 1 2 0 1 5 0 1 8 0 2 1 0 ml F i g . 8 E l u t i o n Curve of Reduced and Cyanoethylated Wheat Glutenin oo ™ 0.1-cu u c o _Q < 0 BARLEY 6 0 ~ i — 9 0 120 —j_ 150 1 8 0 2 1 0 mi F i g . 9 E l u t i o n Curve of Reduced and Cyanoethylated Barley G l u t e l i n 71 ? 4 0 Amino acid composition and possible chemical bondings i n dough structure. (a) Amino acid analysis. Amino acid analysis showed that barley and wheat were d i f f e r e n t i n amino acid composition. Barley has a higher lys i n e content, and a lower content of glutamic acid (Table 1 3 ) . The amount of glutamic acid presented i n barley was only 70% of that i n wheat. Glutamine i s a strong hydrogen bond forming residue, and the insoluble, cohesive nature of wheat gluten i s greatly influenced by hydrogen bond formation. Therefore t h i s would provide some explanations f o r the lack of cohesive-ness of barley gluten. (b) E f f e c t of deuterium oxide and urea i n dough-making. An increase i n e l a s t i c i t y and cohesiveness of barley dough was observed when deuterium oxide was used instead of water. Since deuterium oxide would enhance both hydrogen and hydrophobic bond format-ion (Wehrli-and Pomeranz 1 9 ^ 9 )• we cannot d i s t i n -guish which type of bonding i s r e a l l y responsible f o r t h i s improvement. When water was replaced by urea solution i n wheat, dough'preparation,dough 72 Table 13 • Amino Acid Composition of Barley 1 and Wheat Flour 2 2 Amino Acid Barley * Wheat Aspartic Acid 7.2 4.8 Threonine 3-7 2.9 Serine 4.0 4.6 Glutamic acid 24.6 33.1 Proline 10.2 10.6 Glycine 4.4 4.0 Alanine 4.5 3-8 Valine 5.6 4.0 Methionine 1.4 1.2 Isoleucine 3-9 3-9 Leucine 7-2 6.4 Tyrosine 3-0 2.4 Phenylalanine 5-2 Lysine 4.1 2.4 H i s t i d i n e 2.3 2.3 Ammonia 3-1 3-4 1 g. amino acid/100 g protein 2 average of two analysis 73 structure was completely destroyed. Again, i t i s debatable whether the disruptive e f f e c t was due to the breakage of hydrogen bonds or hydrophobic bonds. According to Levy and Magoulas (1962)» urea forms weaker hydrogen bonds than water, and cannot lower hydrogen bond energy of water. Therefore, i t i s suggestive that urea breaks hydrophobic bonds. But Wu et a l . (1967) believe that since urea i s an amide compound, i t w i l l associate with polar groups on protein and eliminate s i t e s f o r hydrogen bonding. Detergents, having both polar and non-polar portions, l i k e sodium s a l i c y l a t e and sodium dodecyl s u l f a t e , are good solvents f o r gluten. The non-polar port-ion associates with hydrophobic side chains of the protein, while the polar portion i s compatible with the aqueous solvent. A single hydrogen or hydro-phobic bond i s a r e l a t i v e l y weak force, but when multip l i e d by the vast number of hydrogen and hydrophobic bonds i n wheat gluten, they become the ef f e c t i v e force governing cohesiveness and e l a s t -i c i t y . (c) Hydrophobic comparison using fluorescence quenching method. The calculated hydrophobicity values of wheat glutenin and g l i a d i n from data on amino acid comp-74 o s i t i o n revealed that both proteins are capable of forming i n t r a and intermolecular hydrophobic bonds, with the presence of free water (Wehrli and Pomeranz, 1969). In a„ dough system where only about 50$ of the water i s bound, hydrophobic bonds can be r e a d i l y formed i n the remaining 50$ free water ( K r u l l and Wall, 1969). Prolamin proteins have a lower molecular weight than g l u t e l i n proteins. They are comp-osed mainly of single polypeptides joined to-gether by hydrophobic forces and d i s u l f i d e bonds (Nielsen et a l . 1968). Fluorescence quenching studies on prolamin proteins of barley and wheat yielded upward curving Stern-Volmer plots (Fig. 10). This meant that both proteins were f a i r l y hydrophobic and quenched to a reasonable degree (Eftink and Ghiron 1976). The steep upward curving plot of wheat g l i a d i n suggested that i t s indole residues are f a i r l y exposed i n the poly-peptide chain, and they are very e f f e c t i v e l y quenched by the hydrophobic quencher. Its K g v ( c o l l i s i o n a l quenching -constant) value i s app-roximately 2.5 times that of hordein (Table 14). The low K g y value of wheat glutenin indicates that the indole r i n g i n t h i s protein i s extremely 75 5H 0 .002 .004 .006 .008 CTCOM -F i g . 10 F l u o r e s c e n c e Q u e n c h i n g S t u d i e s o n H y d r o p h o b i c i t y o f P r o l a m i n s F i g . 11 F l u o r e s c e n c e Q u e n c h i n g S t u d i e s o n H y d r o p h o b i c i t y o f G l u t e l i n s 76 Table 14. Hydrophobic Comparison by Fluorescence Quenching Technique Protein K * sv V* Wheat g l i a d i n 626 + 7 80 + 4 Barley hordein 301 +1 0 Wheat glutenin 1 8 2 + 1 0 Barley g l u t e l i n 279 ± 2 1 3 + 2 average of four determinations 77 well-insulated from the aqueous environment. Since the degree of quenching by trichloroethanol i s determined predominately by the extent of ex-posure of the fluorescing residue, and the mole-cular weight of wheat glutenin i s very high, i t i s possible that the hydrophobic domains are deep-l y buried inside t h i s high molecular weight poly-peptide; therefore, the a c c e s s i b i l i t y of the quencher to indole residues might be i n t e r f e r e d with i t s high molecular weight polypeptide. Con-sequently, the frequency of c o l l i s i o n with the probe would be reduced, and i t s fluorescence would not be quenched as r e a d i l y . The K g v value f o r t h i s high molecular weight polypeptide i s ex-pected to be lower due to the reduced d i f f u s i o n c o e f f i c i e n t of the indole r i n g when i t i s deeply anchored within the huge, macromolecule. Therefore, the molecular nature of wheat glutenin as d i s -cussed above, the larger amount of g l u t e l i n pro-t e i n present i n wheat than i n barley, and the very high K g y value of wheat g l i a d i n , a l l sugg-ested that the o v e r a l l hydrophobicity of wheat glu-ten i s higher .than that .of. barley, i n spite of the higher observed K value i n barley g l u t e l i n . SV S t a t i c quenching phenomenon was not obser-ved i n barley g l i a d i n and wheat glutenin. The 78 p r o b a b i l i t y of f i n d i n g a quencher molecule i n the v i c i n i t y of a f l u o r e s c i n g group i n both proteins i s almost zero. Since the s t a t i c quenching con-stant, V, i s an estimation of the steady state population of quencher molecules adjacent to the residue, the more an indole r i n g i s shielded by segments of a protein, the less i t w i l l be sub-ject to s t a t i c quenching (Eftink and Ghiron 1976). The absence of s t a t i c quenching i n barley g l i a d i n and wheat glutenin r e f l e c t s the b u r i a l of t h e i r residues j t h e i r indole rings are com-p l e t e l y embraced by the f o l d i n g of the polypep-tide chain. Consequently, steady state contact between the quencher and the residue does not occur. Quencher solution has to penetrate thro-ugh the surrounding protein matrix to the buried residue f o r c o l l i s i o n quenching to take place. Whereas, the very large c o l l i s i o n a l and s t a t i c quenching constants of wheat g l i a d i n (Table 14) suggested that there i s a high l o c a l concentra-t i o n of trichloroethanol i n the immediate v i c i -n i t y of the indole r i n g due to the hydrophobic interactions between the probe and protein ( E f t -ink et a l . 1976). Therefore, both c o l l i s i o n a l and s t a t i c quenching occur quite r e a d i l y between the quencher and wheat g l i a d i n . 79 (d) Hydration of proteins. The f a c t that prolamin proteins are insoluble i n water and dissolve r e a d i l y i n 70$ ethanol sugg-ested that hydrophobic bonds are the predominent force causing association of prolamin proteins i n water. The difference i n hydration properties of barley and wheat revealed t h e i r d i f f e r e n c e s ' i n protein composition . and breadmaking p o t e n t i a l . Hydrated hordein was s o f t , s t i c k y and semi-fluid. Hydrated wheat g l i a d i n yielded a very smooth, el a s t i c , s o l i d s t i c k y mass that had a stronger body than hordein. These differences are due to the strong inherent hydrophobic forces of wheat g l i a -din. Hydrophobic bonds are capable of s t a b i l i z -ing protein conformation, giving wheat dough i t s e l a s t i c i t y and p l a s t i c i t y . Barley g l u t e l i n f a i l -ed to i n t e r a c t with water to form a s o l i d mass. It gave f l a k y deposites showing l i t t l e signs of cohesion; whereas wheat glutenin absorbed water and formed a tough cohesive mass. Glutamine i s a strong hydrogen bond forming residue. The am-ounts of glutamic acid and amide nitrogen (Table 15) present i n barley were less than i n wheat. This suggested that hydrogen bonds which govern the i n s o l u b i l i t y and cohesiveness of gluten had 80 Table 15. Total Amide Nitrogen Content of Wheat and Barley Glutens Type of gluten Amide N ($)* Total N ($)* % of Total Nitrogen Barley gluten 1.80 + 0 .03 10 .75 + 0.18 16.8 Wheat gluten 2.88 + 0.04 13-72 + 0 .23 21.0 * average of three determinations 81 a c o n s i d e r a b l e i n f l u e n c e on the p r o p e r t i e s of b a r l e y and wheat p r o t e i n s . During dough f o r m a t i o n , i t i s v e r y important t h a t the p r o t e i n s w i l l i n t e r a c t w i t h water t o form a c o h e s i v e , v i s c o e l a s t i c network g i v i n g bread i t s s t r u c t u r e . T h i s phenomenon i s an i n t e r a c t i o n of a l l the p o s s i b l e chemical f o r c e s p r e s e n t i n dough. I t s cohesiveness and p l a s t i -c i t y are c o n t r i b u t e d by hydrogen and hydrophobic bonds, w h i l e d i s u l f i d e bonds are the o n l y impor-t a n t c o v a l e n t bonds governing r h e o l o g i c a l propr e r t i e s of dough. The importance of d i s u l f i d e bonds i s not repeated here s i n c e i t has been f u l l y d i s c u s s e d i n p r e v i o u s s e c t i o n s . 82 CONCLUSION t The c l a s s i c a l quantitative r e l a t i o n s h i p between loaf volume and protein content or protein s o l u b i l i t y -d i s t r i b u t i o n exaggerates the differences i n protein content between weak and strong f l o u r within a wheat varie t y . I t gives good prediction of loaf volume be-tween d i f f e r e n t wheat f l o u r s but does not o f f e r rea-sonable comparison between breadmaking potentials of barley and wheat f l o u r s . Barley f l o u r has a f a i r l y high protein content, s i m i l a r s o l u b i l i t y p r o f i l e , and t o t a l s u l f h y d r y l - d i s u l f i d e contents compared with wheat f l o u r . However, baking studies revealed that i t s loaf volume i s only one-third that of wheat. Dur-ing breadmaking not a l l of the f l o u r proteins are equ-a l l y important i n contributing to the f i n a l properties of the product. Rather, i t . i s governed by certa i n s p e c i f i c interactions between functional groups. Many factors can account f o r the differences i n physical properties between barley and wheat gluten. Elution patterns from Sephadex G100 showed that the molecular weight of barley gluten i s lower than wheat gluten, t h i s may have an eff e c t on the breadmaking po t e n t i a l of barley f l o u r . Kinetic studies on aggre-gation behavior of proteins revealed that the rate of 83 aggregation and t u r b i d i t y values of barley proteins are always i n f e r i o r when compared with the same protein f r a -ctions of wheat. This gives good i n d i c a t i o n of the differences i n breadmaking p o t e n t i a l between wheat and barley proteins since aggregation proceeds f a s t e r i n strong f l o u r s than i n weak ones. Wheat g l i a d i n gives the highest K and T 1 0 / c values of a l l the proteins studied, whereas barley hordein f a i l s to show any sign of aggregation. The a b i l i t y of hordein to polymerize at low temperatures suggested that the prolamin proteins of.barley and wheat are unique i n t h e i r own s p e c i f i c properties and composition. A f r a c t i o n of the alcohol soluble proteins i n barley i s capable of 'gluten' form-ation i n v i t r o . This aggregated 'gluten' i s a complex containing hordein and g l u t e l i n , formed exclusively from polymerization of hordein molecules v i a d i s u l f i d e bridges. Rheologically, i t i s more s i m i l a r to the properties of a s p e c i a l type of gluten rather than hor-dein. G l u t e l i n proteins i n barley and wheat are d i f f -erent i n polypeptide composition. E l u t i o n patterns of barley and wheat from Sephadex G150 showed two and three peaks respectively. The Fg f r a c t i o n of wheat, which has the highest aggregation p o t e n t i a l , i s missing i n barley, and most of the barley g l u t e l i n i s d i s t r i b u t e d i n the lower molecular weight region. 84 Amino acid analysis demonstrated that barley and wheat proteins are d i f f e r e n t i n structure and composi-t i o n . Fewer hydrogen and hydrophobic forces are pres-ent i n barley. These would o f f e r some explanation to the lack of cohesiveness and e l a s t i c i t y of barley dough, since hydrogen bonds contribute to the i n s o l u -ble, cohesive nature of gluten, hydrophobic bonds are responsible f o r s t a b i l i z i n g protein conformation, e l a s t i c i t y and p l a s t i c i t y . D i s u l f i d e bonds are the only important covalent bonds a f f e c t i n g dough s t r u c t -ure and v i s c o e l a s t i c i t y . Although the t o t a l s u l f h y d r y l and d i s u l f i d e contents of barley and wheat are s i m i l a r , the p o t e n t i a l of protein-protein d i s u l f i d e interchange i n wheat exceeds several times the calculated minimum necessary to create a continuous network, while the proteins of barley f a l l below the minimum requirement. Addition of oxi d i z i n g agents does not bring much improvement to barley dough. This suggests that the t h i o l and d i s u l f i d e groups of barley are v e r y inaccess-i b l e and/or t h e i r properties are i n f a c t d i f f e r e n t from wheat t h i o l and d i s u l f i d e groups. Furthermore, the alcohol soluble proteins of barley are capable of polymerization v i a d i s u l f i d e bridges, and t h i s pheno-menon i s not observed i n the d i s u l f i d e groups of wheat. It i s believed t h a t t h e T h e o l o g i c a l l y e f f e c t i v e t h i o l 85 and d i s u l f i d e groups are located i n g l u t e l i n protein 0 f r a c t i o n . The g l u t e l i n content of "barley i s lower than wheat. Therefore, together with the above findings, i t i s postulated that the r h e o l o g i c a l l y e f f e c t i v e groups of barley are i n f e r i o r i n q u a l i t y and quantity to wheat. It i s impossible to i d e n t i f y a single f a c t o r or a combination factors that are responsible f o r the bread-making properties of f l o u r dough. It i s a dynamic i n t -eraction between protein functional groups, and a com-plex involvement of a l l the components i n f l o u r , some of which may be of a minor but necessary part of the t o t a l f l o u r complex, which give dough i t s character-i s t i c properties. Moreover, factors that a f f e c t the breadmaking properties of wheat may not be necessary to produce the same eff e c t on barley, since the comp-o s i t i o n , structure, and chemical bondings of both pro-teins are so d i f f e r e n t . 86 REFERENCES; Arakawa, T.f Matsumoto, S. and Yonezawa, D. "Determination of Aggregation, Velocity of Wheat Gluten Particles by Turbidimetry;.'.' Nippon Nogeikagaku Kaishi 48 : 255 1974 Arakawa, T. and Yonezawa, D. "Compositional Diff-erence of Wheat Flour Glutens in Relation to Their Aggregation Behaviors." Agri. Biol. Chem. 39 « 2123 1975 Arakawa, T., Morishita, H. and Yonezawa, D. "Agg-regation Behaviors of Glutens, Glutenins and Gliadins from Various Wheat." Agri. Biol. Chem. 40 : 1217 1976 Beveridge, T., Toma, S.J. and Nakai, S. "Deter-mination of SH and SS Groups in Some Food Proteins Using Ellman's Reagent." J. Food Sci. 39 t 49 1974 Bietz, J.A., Huebner, F.R. and Wall, J.S. "Gluten the Strength Protein of Wheat Flour." Bakers' Digest 47(1) ::26 1973 Bloksma, A.H. "Flour Composition, Dough Rheology, and Baking Quality. " Cereal Sci. Today 17 380 1972'a Bloksma, A.H. "The Relation Between the Thiol and Disulfide Contents of Dough and Its Rheological Properties." Cereal Chem. 49 : 104 1972b Bloksma, A.H. "Thiol and Disulfide Groups in Dough Rheology." Cereal Chem. 52 s 171, 1975 Bushuk, W. "Glutenin: Functions, Properties and Genetics." Bakers' Digest 48(4) : 14 1974 Chen, CH. And Bushuk, W. "Nature of Proteins in Tr i t i c a l e and Its Parental Species. I. Solubility Characteristics and Amino Acid Composition of En-dosperm Proteins." Cereal Chem. 50 : 9 1970 Eftink, M.R. and Ghiron, C.A. "Exposure of Trypto-phanyl Residues in Proteins. Quantitative Deter-mination by Fluorescence Quenching Studies." Biochemistry 15 : 670 1976 8 ? E f t i nk , M.R., Ghiron, C.A. and Za j icek, J . L . "A Hydrophobic Quencher of Prote in Fluorescence: 2,2,2-Trichloroethanol. " Biochim. Biophys. Acta 491 : 471 1977 Ena r i , T.M. "Composition of Albumins and Globul ins of Bar ley . " Cereal S c i . Today 10 : 594 1965 Ewart, J . A .D . "Amino Acid Analys is of Glutenins and G l i a d i n . " J . S c i . Food Agr i c . 18 : 111 1967 Ewart, J . A .D . " F rac t iona l Ext rac t ion of Cereal F lour P ro te ins . " J . S c i . Food Agr i c . 19 t 24l 1968 Ewart, J . A . D . "Further Studies on SS Bonds i n Cereal G l u t e l i n s . " J . S c i . Food Agr i c . 23 : 567 1972a Ewart, J . A . D . "Modif ied Hypothesis f o r the Structure and Rheology of G l u t e l i n s . " J . S c i . Food Agr i c . 23 : 687 1972b Ewart, J . A . D . "Recent Research and Dough Visco-E l a s t i c i t y . " Bakers' Digest 46(4) j 22 1972c Greenwood, C.T. and Ewart, J . A .D . "Hypothesis f o r the Structure of Glutenin and Relat ion to Rheolo-, ,-g ica l Propert ies of Gluten and Dough." Cereal Chem. 52 : 147 1975 Hart , M.R., Graham, R.P. and Gee, M. "Bread from Sorghum and Barley F l o u r s . " J . Food S c i . 35 : 661 1970 Hoseney, R . C , Finney, K.F. , Shogron, M.D. and Pomeranz, Y. "Funct ional (Breadmaking) and B io -chemical Propert ies of Wheat F lour Components. II Role of Water So lub les . " Cereal Chem. 46 : 117 1969 Hoseney, R . C , Finney, K.F. , Pomeranz, Y. and Shogron, M.D. "Funct ional (Breadmaking) and Biochemical Propert ies of Wheat F lour Components. VIII S t a r ch . " Cereal Chem. 48 : 191 1971 I r v ine , G.Nr. and McMullan, M.E. "The 'Remix' Baking T e s t . " Cereal Chem. 37 * 603 i960 Ivokova, M.N., Vedenkina, N.S. and Burs te in , E.A. "Fluorescence of Trytophan Residues i n Serum Albumin." J . Mol . B i o l . 5 : 168, 179 1971 88 Ivokova, M.N., Vedenkina, N.S. and Burstein, E.A. "Fluorescence and the Location of Trytophan Resi-dues i n Protein Molecules." Photochem. Photo-b i o l . 18 : 263 -1973:;; -Jones, I.K. and Carnegie, P.R. "Binding of Oxidized Glutathione to Dough Proteins and a New Explanation, Involving Thiol-Disulphide Exchange, of the Physical Properties of Dough." J . ScURoodL Agric. 22 s 359 Jones, R.W., Taylor, N.W. and Senti, F.R. "Elect-rophoresis and Fractionation of Wheat Gluten." Arc. Biochem. Biophy. 84 : 363 1959 K r u l l , L.H. and Wall, J.S. "Relationship of Amino Acid Composition and Wheat Protein Properties." Bakers' Digest 43(4) : 56 1969 Kuninori, T. "Disulfide-Sulfhydryl Interchange Studies of Wheat Flour. II Reaction of Glut a t h i -one." Cereal Chem. 45 : 486 1968 Lauriere, M., Charbonnier, L. and Mosse, J . "Nature et Fractionnement des Proteines de I'Orge Extraites par I'ethanol, I'isopropanol et l e n-propanol a des T i t r e s D i f f e r e n t s . " Biochim. 58 : 1235 1976 Levy, M. and Magoulas, J.P. "Effects of Urea on Hydrogen Bonding i n Some Dicarboxylic Acids." J . Am. Chem. Soc. 84 : 1345 1962 Liu, T.Y. and Chang Y.H. "Hydrolysis of Proteins with Paratoluenesulfonic Acid." J . B i o l . Chem. 246 t 2842 1971 Meredith, O.B. and Wren, J . J . "Determination of Molecular Weight D i s t r i b u t i o n i n Wheat Flour Proteins by Extraction and Gel F i l t r a t i o n i n a Dissoci a t i n g Medium." Cereal Chem. 48 : 169 1966 Murthy, P.R. and Dahle, L.K. "Studies on a Simpli-f i e d Dough System Composed of G l i a d i n , Glutenin and Starch." Cereal Chem. 46 s 463 1969 Nielsen, H.C, Beckwith, A.C and Wall, J.S. "Effect of Disulfide-Bond Cleaverage on Wheat Glia d i n Fractions Obtained by Gel F i l t r a t i o n . " Cereal Chem. 45 : 37 1968 89 Noguchi, G., Shinya, M., Tanaka, K. and Yoneyama "Correlation of Dough Stickiness with Texturo-meter Reading and with Various Quality Parameters." Cereal Chem. 53 : 72 1976 Orth, R.A., Baker, R.J. and Bushuk, W. " S t a t i s t i -c a l Evaluation of Techniques f o r Predicting Baking Quality of Wheat C u l t i v a r s . " Can. J . Plant S c i . 52 : 139 1972 Orth, R.A. and Bushuk, W. "A Comparative Study of the Proteins of Wheat of Diverse Baking Q u a l i t i e s . " Cereal Chem. 49 : 268 1972 Osborne, T.B. "The Proteids of Barley." J . Am. Chem. S o c 17 t 539 1895 Pomeranz, Y. "Food Uses of Barley." CRC C r i t . Review i n Food Tech. 4(3) t 377 1973 Pomeranz, Y., Finney, K.F. and Hoseney, R.C. "Molecular Approach to Breadmaking." Science 167 : 944 1970 Preston, R. and Woodbury, W. "Properties of Wheat Gliadins Separated by Gel F i l t r a t i o n . " Cereal Chem. 53 : 180 1976 Redman, D.G. and Ewart, J.A.D. "Disulphide Inter-change i n Dough Proteins." J . S c i . Food Agric. 18 : 15 1967a Redman, D.G. and Ewart, J.A.D. "Disulphide Inter-change i n Cereal Proteins." J . S c i . Food A g r i c 18 : 520 1967b Shestakova, N.A., Kolpakova, V.V. and Vakar, A.B. "Formation of Gluten from Hordein i n v i t r o . " Prikladnaya Biokhimiya i Mikrobiologiya 12(5) : 720 1976 Smith, D.E. and Mullen, J.D. "Studies on Short and Long-Mixing Flours. II Relationship of S o l u b i l i t y and Electrophoretic Composition of Flour Proteins to Mixing Properties." Cereal Chem. 42 : 275 1965 S u l l i v a n , B., Dahle, L.K. and Schipke, J.H. "The Oxidation of Wheat Flour. IV Labile and Nonlabile Sulfhydryl Groups." Cereal Chem. 40 : 515 1963 90 Tanaka, K. and Bushuk, W. "Effect of Protein Content and Wheat Variety on S o l u b i l i t y and Electrophoretic Properties of Flour Proteins." Cereal Chem. 49 : 24-7 1972 Tanaka, K. and Bushuk, W. "Changes i n Flour Proteins During Dough-mixing. II Gel F i l t r a t i o n and Electrophoresis Results." Cereal Chem. 50 * 597 1973 Tanford, C The Hydrophobic E f f e c t : Formation of Micelles and B i o l o g i c a l Membrances. Wiley Interscience, U.S.A. 1973 Tsen, C C . "A Note on Eff e c t s of pH on Sulfhydryl Groups and Rheological Properties of Dough and Its Implication with the Sulfhydryl-Disulfide Interchange." Cereal Chem. 4-3 : 4-56 1966 Tsen, C C and Bushuk, W. "Reactive and T o t a l Sulfhydryl and D i s u l f i d e Contents of Flours of Different Mixing Properties." Cereal Chem. 4-5 : 58 1968 Vakar, A.B., Shestakova, N.A. and Kolpakova, V.V. "Formation of Gluten from Hordein i n v i t r o . " General Biochem. 85 1 171 1976 Wehrli, H.P. and Pomeranz, Y. "The Role of Chem-i c a l Bonds i n Dough." Bakers* Digest 4-3(6) : 22 1969 Whitaker, J.R. "Determination of Molecular Weights of Protein by Gel F i l t r a t i o n on Sephadex." A n a l y t i c a l Chem. 35 : 195 1963 Wilcox, P.E. "Determination of Amide Residue by Chemical Methods." Enzymol. 11 : 63 1967 Wu, CH., Nakai, S. and Powrie, W. "Preparation and Properties of Acid-Solubilized Gluten." J . Agric. Food Chem. 24 : 504 1976 Wu, Y.V., Cluskey, J.E. and Sexson, K.R. "Effect of Ionic Strength on the Molecular Weight and Conformation of Wheat Gluten Proteins." Biochim. Biophys. Acta 133 ' 83 1967 91 

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