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Preparation, characterization and functionality of acid-modified gluten Wu, Chiu Hui 1975

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PREPARATION, CHARACTERIZATION AND FUNCTIONALITY OP ACID-MODIFIED GLUTEN BY CHIU HUI WU B.S.A. National Taiwan University, Taiwan, China M.Sc. University of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Food Science We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i ABSTRACT The i n s o l u b i l i t y of gluten i n aqueous solutions i s one of the major l i m i t a t i o n s of i t s more extensive use i n food industry. This property i s due to the presence of the high concentration of nonpolar amino acid residues such as p r o l i n e , leucine and glutamine; and the lack of many ionizable side chains such as l y s i n e , arginine, glutamic acid and aspartic acid. The hydrogen bond interactions between glutamine and asparagine side chains play an important role i n promoting association between g l i a d i n and glutenin molecules. The purpose of t h i s study i s to search f o r the best condition f o r s o l u b l l i z i n g gluten by mild acid treatment; t h i s treatment i s expected to decrease the amount of side chain amides, thereby weakening the hydrogen bond interactions between the molecules. Gluten solutions containing various concentrations of HCl (0.02 to 0.5 N) and acetic acid (1.75 to 8.75 N) were heated at d i f f e r e n t temperatures (from 100 to 121 C) f o r d i f f e r e n t lengths of time. The following r e s u l t s were observed from t h i s study. (1) Soluble gluten was recovered with y i e l d s of 85 to 92% of the o r i g i n a l gluten protein by d i a l y s i s and y i e l d s of 70 to 78% by i s o e l e c t r i c p r e c i p i t a t i o n . (2) The amount of amide nitrogen i n the treated samples was dependent mainly on the concentration of the acid used. Gluten can be s o i u b i l i z e d when the amide content was reduced by approximately 10$. (3) Gel f i l t r a t i o n through Sepharose 6 B columns showed better i i i separation of higher molecular weight components than through Sephadex G-100. The r e s u l t s suggested that with the increasing HC1 concentrations the amount of higher molecular weight components i n s o l u b i l i z e d gluten decreased; acetic acid was considerably milder f o r gluten modification, (lj.) The c h l o r a n i l test proved that a small amount of low molecular weight peptides and free amino acids had been formed during the acid treatment. (5) The molecular weight d i s t r i b u t i o n of the products was further evaluated by Trautman p l o t f o r the approach-to-equili-brium method of u l t r a c e n t r i f u g a t i o n . The r e s u l t s showed that the HC1-treated gluten was polydisperse and that when the concentration of the acid used to treat gluten was low, weight average molecular weight was much greater. (6) The 2,1).,6 t r i -nitrobenzenesulphonic acid method and the c h l o r a n i l test were used to determine the amino groups exposed by peptide hydroly-s i s . As the concentration of acid was increased f o r acid solu-b i l i z a t i o n of gluten, more amino groups were exposed i n s o l u b i -l i z e d gluten. (7) No s i g n i f i c a n t changes were detected i n the content of SS-groups of gluten during acid modification. (8) Amino acid analysis revealed no s i g n i f i c a n t changes i n the pattern with the acid treatments. (9) The acid modification of gluten remarkably improved the emulsifying capacity and the emulsion s t a b i l i t y ; the emulsifying capacity of the gluten modified by d i l u t e HC1 and acetic acid was equal to or i n some cases better than that of soybean protein i s o l a t e s . (J>Q) The pH S»k soluble f r a c t i o n prepared from the i s o e l e c t r i c a l l y i v p r e c i p i t a t e d gluten modified with 0,02 and 0 .05N HC1 exhibited excellent ioamability and s t a b i l i t y . (11) A warm activated carbon column was e f f e c t i v e i n elimination of wheaty flavour of the acid-modified gluten solutions. The best conditions f o r the acid modification of gluten were found to be the heat treatment of 5% gluten suspen-sion i n 0.02H HC1 at 121 C f o r 30 min or i n 0 .05N HC1 f o r 15 min. These conditions were adequate f o r achieving s o l u b i l i t y of gluten while minimizing the hydrolysis of p r o t e i n molecules. TABLE OF CONTENTS INTRODUCTION Kfe LITERATURE REVIEW 3 MATERIALS AND METHODS 10 Source of Gluten 10 Preparation of Acid-modified Gluten Samples 10 Deodorization of Modified Gluten by Activated Carbon Treatment 10 1. Treatment 10 2. Preliminary Evaluation of the E f f e c t of the Activated Carbon Treatment by Taste Panel 11 Methods of Chemical Analysis '.,.,/ 1. Methods of Proximate Analysis 11 2. Amide Nitrogen Determination 12 3. C h l o r a n i l Test 12 l±. Trinitrobenzene Sulphonic Acid Test 12 5. Determination of SS- and SH- Groups 13 Gel Electrophoresis lij. Gel F i l t r a t i o n 15 Methods of U l t r a c e n t r i f u g a l Analysis \.;^'~t 1. Determination of Sedimentation C o e f f i c i e n t 16 2. The Trautman p l o t by Sedimentation Equilibrium Technique 16 Amino Acid Analysis 19 Determination of Functional Properties 20 page 1. Emulsifying Capacity 20 2. Emulsion S t a b i l i t y Rating 21 3. Poamability and Foam S t a b i l i t y 21 RESULTS AND DISCUSSION Preparation 1. Recovery of S o l u b i l i z e d Gluten 23 2. Carbon Column Treatment of Acid-modified Gluten 29 Characterization 1 . Amide Nitrogen Content 31 2. Disc Gel Electrophoresis 33 3» Determination of Molecular Weight D i s t r i b u t i o n A. Gel F i l t r a t i o n 35 B. U l t r a c e n t r i f u g a l Sedimentation Study 6ii It,. Determination of the Amino Groups Exposed by Acid-modification A. Tririitroberizene Sulphonic Acid Method 89 B. C h l o r a n i l Test 91 5. SS- and SH- Group Determination 92 6. Amino Acid Composition 96 Functional Properties 96 1. Emulsifying Capacity and the S t a b i l i t y Rating 98 2. Foamability and Foam S t a b i l i t y 99 GENERAL DISCUSSION 105 CONCLUSION 109 LITERATURE CITED 111 LIST OP FIGURES Figure 1. Typical UV scanner chart f o r u l t r a c e n t r i f u -gation 2. Procedure f o r the preparation of solub&lized gluten by p r e c i p i t a t i o n method 3. Disc electrophoresis pattern of (1) untreated gluten (2) 0.02N HCl-, (3) 0.05N HG1-, (L\ 0.1N HC1-, (5) 0.5N HCl-treated gluten I4.. Disc electrophoresis pattern of (1) untreated gluten (2) 0.02N HCl pH 5 soluble f r a c t i o n (3) 0.05N HCl pH 5 soluble f r a c t i o n (li) 0.02N HCl-treated gluten 5 . Gel f i l t r a t i o n pattern on Sephadex G-100 f o r untreated gluten 6. Gel f i l t r a t i o n pattern on Sepharose 6B f o r r • untreated gluten 7. Gel f i l t r a t i o n pattern on Sephadex G-100 f o r 0.02N HCl-modified gluten 8. Gel f i l t r a t i o n pattern on Sephadex G-100 f o r O.O^N HCl-modified gluten 9. Gel f i l t r a t i o n patterns on Sephadex G-100 f o r 0.02N HCl-modified gluten and pH 5 soluble f r a c t i o n of 0.02N HCl-treated gluten 10. Gel f i l t r a t i o n patterns on Sephadex G-100 f o r 0.05N HCl-modified gluten and pH 5 soluble f r a c t i o n of 0.05N HCl-treated gluten 11. Gel f i l t r a t i o n pattern on Sepharose 6B f o r 0.02N HCl-modified gluten 12. Gel f i l t r a t i o n pattern on Sepharose 6B f o r 0.05N HCl-modified gluten 13. Gel f i l t r a t i o n pattern on Sepharose 6B f o r 0.1N HCl-modified gluten 11L. Gel f i l t r a t i o n pattern on Sepharose 6B f o r 0.5N HCl-modified gluten 15» Gel f i l t r a t i o n pattern on Sepharose 6B f o r 1.75N acetic acid-modified gluten 16. Gel f i l t r a t i o n pattern on Sepharose 6B f o r 3.5N acetic acid-modified gluten 17. Gel f i l t r a t i o n patterns on Sepharose 6B f o r 6.IN HCl-modified gluten: (1) refluxed at 100 G f o r 6 hr (2) heated at 121 C f o r 15 min. 18. Gel f i l t r a t i o n patterns on Sepharose 6B f o r 0.07N HCl-modified gluten: (1) heated at 121 C fo r 30 min (2) heated.at 121 C f o r 5 min. 19. Gel f i l t r a t i o n patterns on Sepharose 6B f o r 0.5N HCl-modified gluten: (1) heated at 121 C f o r 15 min (2) heated at 121 C f o r 30 min. 20. C h l o r a n i l t e s t of 0.02N HCl-modified gluten 21. C h l o r a n i l test of 0.05N HCl-modified gluten 22. C h l o r a n i l test of 0.1N HCl-modified gluten 23. C h l o r a n i l test of 0.5N HCl-modified gluten 2k* T y p i c a l schlieren pattern of 1% acid-modified gluten solution a f t e r reaching 59,000 rpm fo r 120 min. 25. Typical schlieren pictures of.1% acid-modified gluten sol u t i o n f o r Trautman p l o t 26. UV scanning patterns of 0.02N HCl-modified gluten (I) 27. UV scanning patterns of 0.02N HCl-modified gluten (II) 28. UV scanning patterns of 0.5N HCl-modified gluten (I) 29. UV scanning patterns of 0.5N HCl-modified gluten (II) 30. Trautman p l o t of 0.02N HCl-modified gluten 31. Trautman plot.Sf 0.05N HCl-modified gluten 32. Trautman p l o t of 0.1N HCl-modified gluten i33-i Trautman p l o t of 0.5N HCl-modified gluten 3k» Trautman plots of HCl-modified gluten 79 35. Typical Trautman plots f o r various polymeric system with no concentration dependence 81 36. Weight concentration r a t i o analysis of the Trautman p l o t of 0.02N HCl-modified gluten 86 37. Weight concentration r a t i o analysis of the Trautman p l o t of 0.5N HCl-modified gluten 87 X LIST OP TABLES Table page 1. Protein content, pH f o r maximum p r e c i p i t a t i o n arid colour of the acid-modified gluten 25 2. Y i e l d of s o l u b i l i z e d gluten (dry weight basis) after autoclaving at 121 C 27 3. Taste panel score of the wheaty flavour of the acid-modified gluten af t e r activated carbon treatment 30 ij.. Amide nitrogen i n acid-modified g l u t e i n protein 32 5. The el u t i o n volume of the peaks from gel f i l t r a t i o n of d i f f e r e n t acid-modified gluten samples 5k 6. Data from UV scanning pattern and calculated X ,Y f o r 0.05N HCl-treated gluten 7k 7. Amino groups exposed by peptide hydrolysis i n acid-modified gluten samples as determined by the TNBS method 90 8. The absorbance at 350 nm of 0.1$ untreated and acid-modified gluten solutions (the c h l o r a n i l test) 93 9. Sulfhydryl and d i s u l f i d e values of acid-modified gluten samples 95 10. Amino acid composition of acid-modified gluten 97 1. Emulsifying capacity of acid-modified gluten 10 12. S t a b i l i t y r a t i n g of oil-in-water emulsions containing 1% of various acid-modified gluten 101 13. Poamability and foam s t a b i l i t y of acid-modified gluten 103 I4. Trends i n molecular weight, released small peptides and free amino acids, and released amino groups of the HCl-modified gluten 107 x i ACKNOWLEDGEMENTS The author would l i k e to express her sincere gratitude to Dr. W. D. Powrie and Dr. S. Nakai of the Depart-ment of Pood Science f o r t h e i r u n f a i l i n g i n t e r e s t , constructive c r i t i c i s m s , and appropriate guidance throughout the course of t h i s study. She also wishes to acknowledge her appreciation to Dr. J. P. Richards, Dr. M. A. Tung of the Department of Pood Science, and Dr. M. Lee of the School of Home Economics f o r t h e i r valuable suggestions and assistance. Thanks are also extended to her husband, whose under-standing and encouragements have made th i s study possible. INTRODUCTION Around the world, wheat i s the p r i n c i p a l food cereal and i t provides more nutrients than any other single food source. The c u l t i v a t i o n of wheat and the use of wheat f l o u r f o r making bread and pastry have had a hi s t o r y as long as the hi s t o r y of mankind. Canada has a long and enviable record of producing 300-700 m i l l i o n bushels of wheat grain of high bread-making qu a l i t y on 15-25 m i l l i o n acres per year and has demonstrated the c a p a b i l i t y to produce large quantities of competitively priced grain f o r the world market. Wheat proteins are generally credited with attaining the f i n a l objectives of baker, t h i s i s due to the a b i l i t y of wheat proteins to form a strong v i s c o - e l a s t i c network. This protein network, i n combination with other materials, i s capable of gas retention during the baking of bread. In addition, wheat proteins l a r g e l y govern the f l o u r ' s water absorption, oxidation requirement, and mixing and fermentation tolerance. Man's search f o r protein continues and expands into new areas as the demands of the world population continue to grow. To increase the cereal and legume production with improved n u t r i t i v e value i n order to prevent starvation and malnutrition i s a mandatory objective i n the developing countries. In the nations experiencing an upward cycle of of development, where the basic needs to avoid famine are s a t i s f i e d , thoughts move to the p o s s i b i l i t y of increasing the supply of foods of animal o r i g i n , processed foods and convenience i n foods. The f e a s i b i l i t y of commercial processing of plant proteins f o r use i n the manufacture of food products has been established i n t e r n a t i o n a l l y f o r some years. Low cost, function-a l proteins from soybeans and wheat are marketed as basic food ingredients and are used i n a variety of food products. It i s predicted that the demand for proteins from plant sources w i l l continue to increase. , Gluten, a major wheat protein f r a c t i o n , i s produced commercially and i s by f a r the major vegetable protein current-l y manufactured i n Canada, more research on gluten i s required to enlarge the knowledge of i t s basic structure, properties and the p o t e n t i a l f o r more extensive i n d u s t r i a l use. The i n s o l u b i l i t y of gluten i n aqueous solutions i s one of the major l i m i t a t i o n s of i t s use i n the food industry. The objective of t h i s study i s to determine optimum conditions f o r the s o l u b i l i z a t i o n of gluten by mild acid treatment without an appreciable change i n the molecular size of gluten proteins. The p o t e n t i a l of acid-modified gluten f o r more use by the food industry has been explored. 3 LITERATURE REVIEW When wheat flour is mixed with water, proteins inter-act to form an elastic gluten dough. Gluten can be purified by washing the dough with water or a dilute salt solution during which time starch granules and water-soluble constituents are removed. Commercial gluten obtained by one of several washing processes contains on a dry weight basis 75-80% proteins (Nx5.7), 5 to 15% carbohydrates and 5 to 15% of l i p i d s which are combined with protein 3 2'^"'k 2. These l i p i d s , ?iiiiiJai|y are phospholipids, are not extractable with either ethyl ether or petroleum ether32'**"2. Wheat gluten is made up of two major protein frac-tions: gliadin, an alcohol-soluble fraction and glutenin, an alkali-soluble fraction. Only a small amount of water-soluble protein present as a contaminant. The amino acid compositions of gliadin and glutenin dif f e r very slightly. In gliadin and glutenin, about one of every three amino acid residues is glutamine, and about one of every seven residues is proline^*'^ 7. The concentration of ionizable amino acid residues such as glutamic acid and aspartic acid, lysine, histidine and arginine is low in wheat glu t e n 1 3 ' ^ 7 . Purified wheat gluten is not soluble in water but does dissolve in aqueous urea or acidified aqueous solvents with low ionic strengths*^'''*. The nature and number of the side chain groups i n wheat gluten proteins determine the s o l u b i l i t y c h a r a c t e r i s t i c s and molecular association i n the various solutions. The glutamine molecule has a terminal polar amino group which i s capable of i n t e r a c t i n g with other amide groups and with hydrophilic groups of proteins through hydrogen bonding'^'* ^ # Water molecules i n hydrated gluten perform an important function by s a t i s f y i n g some of the hydrogen-bonding s i t e s and f a c i l i t a t i n g interchange of hydrogen bonds among proteins. The presence of a large number of nonpolar amino acids may explain the s o l u b i l i t y of one f r a c t i o n of gluten i n 70% ethanol. In aqueous solutions, these nonpolar groups tend to associate by hydrophobic bonding. Cystine residues i n gluten proteins are important to gluten structure since cystine residues can l i n k polypeptide chains together through S-S bonds. Chemical reagents such as mercaptans can breakdown dough r a p i d l y ^ . It has been suggested that reducing agents break intermolecular S-S bonds and that o x i d i z i n g improvers encourage the formation of intermolecular S-S bonds. Reduction of d i s u l f i d e bonds of glutenin r e s u l t s i n a substantial decrease i n molecular weight 2^'^ 1. The major fr a c t i o n s of reduced glutenin have molecular weights of 1+0,000 and 100,000 whereas the molecular weight of glutenin varies from 1.2 to 2 m i l l i o n 1 * * ' ^ 1 . Evidently glutenin consists of r e l a t i v e l y low molecular weight protein components cross-linked intermolecularly by d i s u l f i d e bonds**-1. Woychiic- et al-*-* have suggested that glutenin may consist of polymerized g l i a d i n 5 components joined by d i s u l f i d e c r o s s - l i n k s . However more recent work provides evidence f o r differences between the 9 10 component proteins of glutenin and g l i a d i n ' . Though g l i a d i n and the components of reduced glutenin have s i m i l a r molecular weights and frequently s i m i l a r amino .acid compositions, and may even have p a r t i a l l y i d e n t i c a l sequences of amino acids i n t h e i r polypeptide chains 1-^, there are some s i g n i f i c a n t d i f f e -rences between them. For example, glutenin components form loosely-folded structures through intermolecular S-S bonds, whereas g l i a d i n molecules form t i g h t l y - f o l d e d structures through intramolecular S-S bonds^'^. Grosskreutz proposed a hypothesis i n 1961 to explain the c h a r a c t e r i s t i c rheological properties of gluten. He suggested that the e l a s t i c i t y of gluten can be attributed to the c o i l e d and folded protein molecules, while the p l a s t i c flow can be explained as imperfect r i g i d i t y due to s l i p p i n g i n the protein molecular arrangement. He proposed that gluten forms a p o l y p l a t e l e t sheet structure during the hydration period and that these p l a t e l e t s consist of c o i l e d polypeptide chains with t h e i r hydrophilic side chains oriented outwards and t h e i r hydrophobic side chains oriented inwards. In t h i s model, a small proportion of l i p o p r o t e i n complex which consists of two or more protein chains bound by phospholipid material i s present i n the form of bimolecular l e a f l e t s . The linkage between the l i p i d and the protein i s considered to be a s a l t -l i k e bond between the aci d i c group of the phospholipid and a basic amino acid group. These l i p o p r o t e i n l e a f l e t s occur as random f i l l i n g among the protein p l a t e l e t s . Assuming that the bonding between the layers of the bimolecular phospholipid l e a f l e t s i s weaker than the bonding between the protein p l a t e -l e t s , there i s a random weakness throughout the protein struc-ture wherever the phospholipid f i l l i n g occurs. Therefore an applied stress to the gluten structure w i l l r e s u l t i n s l i p along the interfaces of phospholipid layers before the rupture strength of the i n t e r p r o t e i n bond i s reached. This gives the p l a s t i c flow properties i n the gluten complex. Hydrophobic bonding probably contributes to the cohesiveness of the gluten proteins. Amino acids with nonpolar groups are probably involved i n l i p i d binding as well as protein-ic protein interactions . The nonpolar part of phospholipid molecules may interact with nonpolar s i t e s on the protein molecules, leaving the polar group free on the surface. Such polar groups can change the surface p o t e n t i a l of the proteins by adding charges or by forming i o n i c bonds with oppositely charged side c h a i n s 3 2 . Obviously, binding of either polar or nonpolar l i p i d s can a f f e c t protein aggregation i n a number of ways. Experiments have been c a r r i e d out to modify the proper-t i e s of wheat gluten proteins. In 1959, Holme and Briggs-' found that when selective mild acid hydrolysis (0.008 to O.OI4. W H C 1 ) of the amide groups of the glutamine residues i n g l i a d i n occurred, an increase i n the protein s o l u b i l i t y and a decrease protein i n t e r a c t i o n was apparent. This study supports the > theory that hydrogen bonding between glutamine amide groups i s important i n protein association within the gluten system. In o 1963, Beckwith et a l found that by treating the gluten protein with 0.6N HCl i n methanol, glutamine residues were converted to methyl glutamate esters. The methoxyl groups introduced i n the protein during the reaction p a r a l l e l e d the amide group l o s s , and the hydrated gluten protein was no longer cohesive. This chemical change i n the proteins increased the s o l u b i l i t y of \ gluten i n aqueous methanol but reduced i t s s o l u b i l i t y i n urea solutions. Beckwith et a l stated that methanol may be involved i n breaking hydrophobic bonds, whereas urea, which contains amide groups, appears to be responsible f o r breaking hydrogen bond8 between polar groups of d i f f e r e n t protein molecules. In the studies of K r u l l and W a l l ^ with glutamine-glutamic acid * copolymers and glutamine-glutamyl ester copolymers, they conclu-ded that the amide groups of gluten proteins play an important r o l e i n the aggregation of the gluten proteins through strong hydrogen bonding. L a s z t i t y ^ studied the Theological proper-t i e s of gluten modified by deamidation, methylation, e s t e r i f i -cation and acylation and by blocking of t h i o l groups with N-ethylinaleimide. He reported that blocking of t h i o l groups and e s t e r i f i c a t i o n of free carboxyl groups had no s i g n i f i c a n t e f f e c t on the rheological properties of gluten. Interference with the amide groups, and acylation of primary amino groups caused r a d i c a l changes i n the rheological behaviour of gluten. 8 OA Lasztity-^ concluded that: the free t h i o l groups have no s i g n i f i c a n t e f f e c t on the Theological properties of glutenj the t h i o l - d i s u l p h i d e interchange i s not important i n dough formation; free carboxyls do not interact s i g n i f i c a n t l y to form secondary bonds; primary amino groups play a substantial r o l e i n the formation of intermolecular non-covalent bonds; removal of amide groups reduces the number of hydrogen bonds and as a consequence a looser structure i s formed, probably caused by a substantial increase of free carboxyl groups which create repulsive forces within the molecule;; G r a n t 2 7 modified wheat f l o u r with succinic anhydride suspended i n aqueous dioxane arid found that 95% of the protein f r a c t i o n became water-soluble. Succinic anhydride reacts with free amino groups, t y r o s y l hydroxyl groups, and sulfhydryl groups i n proteins, forming amide, esters and thioesters linkages, respectively. The t y r o s y l esters and thioesters are hydrolyzed spontaneously i n aqueous media within few hours. As a r e s u l t of the reaction, each amino group with i t s p o t e n t i a l p o s i t i v e charge i s masked, while at the same time an additional carboxyl group i s i n t r o -duced, and therefore an increase i n s o l u b i l i t y occurs. The p o s s i b i l i t y of chemical modification of gluten f o r wider use i n food products has arisen i n recent years. McDonald and Pence 3 <^ have prepared deamidized g l i a d i n by mild hydrochloric acid ( 0 . 0 7 N ) hydrolysis and tested i t s foamabili-ty. They found that f o r meringue dessert s h e l l s , butter cream cake i c i n g , f l u f f y cake i c i n g and d i v i n i t y candy, deamidized g l i a d i n performed as well as egg white. Gagen and Holme*10 have obtained deamidized gluten by using hydrochloric acid at various concentrations (0 .15 to IH) under r e f l u x . The s o l u b i -l i t y of the treated gluten was greatly increased as deamidation proceeded to completion. The foamability, whippability and baking performance of deamidized gluten were determined and a l l the performances were poorer compared to v i t a l gluten (powdered gluten, obtained by f i r s t d i s s o l v i n g i n BH^OH and then spray dried) except the whippability of the insoluble residue obtained aft e r hydrolysis. 10 MATERIALS AND METHODS Source of Gluten V i t a l gluten f o r t h i s study was obtained from John Labatt Ltd Research Laboratory through the courtesy of Dr. J. Holme. Preparation of Acid-modified Gluten Samples Gluten (5% suspension) was modified by acid hydroly-s i s with either hydrochloric or acetic acid. The concentration of hydrochloric acid varied from 0.02N to 0.5N and the c o n c e n t t r a t i o n of acetic acid varied from 1.75N to 8.75N. Hydrochloric acid ( a n a l y t i c a l grade) was supplied by MallinckrOdt Chemical Works and g l a c i a l acetic acid ( a n a l y t i c a l grade) was supplied by A l l i e d Chemical Canada Ltd. Acid hydrolysis of gluten was carr i e d out .using 100 ml samples i n 2j?0 ml Erlenmeyer fl a s k s heated at d i f f e r e n t temperature, from 100 C (at normal atmospheric pressure) to 121 C (at 15 psig steam pressure). A hot plate was used f o r the ref l u x i n g process at b o i l i n g temperature, and a Barnstead autoclave was used f o r heating at temperature higher than b o i l i n g points. It took about 25 min f o r the temperature of the suspension to r i s e to 121 C from room temperature and about an hour f o r the heated suspensions to cool down to room temperature. Deodorization of Modified Gluten by Activated Carbon Treatment 1. Treatment Granular activated carbon from Witco Chemical Company was f i r s t washed to eliminate the f l o a t i n g f i n e p a r t i c l e s and then packed into a column (1.2x8 cm). Deodorization was c a r r i e d out by passing 5% modified gluten solution (pH 7.5) through t h i s column at 25 C or 100 C. 2 . Preliminary Evaluation of the E f f e c t of the Activated  Carbon Treatment by Taste Panel A six-member taste panel was formed fo r the p r e l i m i -nary evaluation of the e f f e c t of the deodorization of modified gluten by activated carbon treatment. The panelists were select* ed i n t h i s laboratory from students, s t a f f and f a c u l t y members at random; two female and four male. Samples were served to the panelists at the same time. They were asked to rank the samples on a 3-point scale of which 1 was weakest wheaty flavour and 3 was strongest whwaty flavour, and to repeat the examination f o r three times. Methods of Chemical Analysis 1. Methods of Proximate Analysis Moisture and ash were determined according to the standard A.O.A.C. procedure^". Ether extractable f a t was detervl' mined according to the standard A.O.A.C. procedure . The amount of nitrogen contained i n the untreated gluten and modified gluten samples was determined by microkjel-dahl method^, and the amount of protein was calculated by multiplying the amount of nitrogen by a fac t o r of 5»7« 2. Amide Hitrogen Determination Total amide nitrogen was determined by re f l u x i n g 100 mg of gluten or modified gluten i n 50 ml of 2H HC1 f o r three 52 hours • The cooled hydrolyzed material was transferred to a mc r o k j e l d a h l d i s t i l l a t i o n apparatus and the amount of ammonia was determined according to the method of A.O.A.C. standard procedure . This amount of ammonia formed during three hour of hydrolysis was taken as a measure of t o t a l amide nitrogen i n the protein sample, 3. Chjoranj l y Q a t p The c h l o r a n i l method was employed to determine the micro-gram amounts of amino acids and small peptides present i n the efflu e n t s obtained during gel f i l t r a t i o n . C h l o r a n i l i s in s e n s i t i v e to urea, which i s present i n the e l u t i n g solution. To 1 ml of each f r a c t i o n , 5 ml of water, 2 ml each of c h l o r a n i l and borate buffer (pH 9) solutions were added. The tubes were placed i n a water bath at 65*1 C a f t e r mixing thoroughly and kept i n t h i s condition f o r 1 hr to complete the color develop-ment. The absorbance of the solution was measured at 350 nm i n a 1-cm c e l l against a blank containing no amino acid. k.. Trinitrobenzene Sulphonic Acid Test F i e l d s f method p using 2,14.,6-trinitrobenzene sulphonic 13' acid (TABS) f o r determining the amount of unblocked amino groups was used f o r the gluten and modified gluten samples. Ten milligrams of the 0.02N-, 0.05N-, 0.1N-, and 0.5H-HC1 and 1.75N-aeetic acid treated gluten samples were dissolved i n 15 ml of water separately. To 0.5 ml of sample, 0.5 ml of 0.1 M N a 2 \ ° 7 i n G , 1 N N a 0 H a n d ° » 0 2 m l 1.8M TNBS solution were added, mixed thoroughly and r a p i d l y , and the tubes l e f t at room tempe-rature f o r f i v e minutes. After the incubation, 2 ml of 0.1 M NaHgPQ^ containing 1.5 mM sodium sulphite were added to neutra-l i z e the solution. The absorbance at 1±20 nm of the solution was measured i n a 1-cm c e l l against water. 5. Determination of SS- and SM- groups The SS- and SH- groups i n gluten and modified gluten 12 samples were determined by the method of Beveridge et a l . A 75 mg sample was suspended i n 1 ml of t r i s - g l y buffer, i±..7 g of guanidine hydrochloric (GuHCl) was added, and the volume made up to 10 ml. For the determination of SH- groups, ii ml of urea-GuHCl (bM urea and 5M GuHCl i n t r i s - g l y buffer) was added to 1 ml of t h i s s l i g h t l y t u r b i d solution and then 0.05 ml of E l l m a ^ s reagent was added. For the determination of SS-groups, 0.05 ml of 2-mercaptoethanol and Ij. ml of urea-GuHGl were added to 1 ml of the sample solution. The mixture was v£**?3 incubated f o r 1 hr at 25 C. After an additional 1 hr incubation with 10 ml of added 12% t r i c h l o r o a c e t i c acid (TCA), the tubes were centrifuged at 5000xg f o r 10 min. The p r e c i p i t a t e was twice resuspended i n 5 ml of 12% TCA and centrifuged to remove 2-mercaptoethanol. The p r e c i p i t a t e was dissolved i n 10 ml of Ellman's reagent. Absorption of the solutions was measured i n a 1-cm c e l l at I4.I2 nm. The amount of SH- groups was calculated as follows: 7 3 . 5 3 A W 2 D M SH/g = • C where A ^ 2 ~ absorbance at ij.12 nm; C = the sample concen-t r a t i o n i n mg solids/ml; D = the d i l u t i o n f a c t o r which i s 10 fo r gluten. Gel Electrophoresis Polyacrylamide gels were made i n glass tubes, 6 cm long and 0.6 cm i n t e r n a l diameter, according to the procedure of Davis modified by Chen and Bushuk . Buffer solutions f o r upper and lower e l e c t r o l y t e s were made with the following reagents: Upper buffer, pH I4..O Glycine 28.1 g Acetic acid 3.05 ml Add water to a volume of 1 l i t r e . Lower buffer, pH I4..3 Acetic acid i^3 ml 1M KOH solution 120 ml Add water to a volume of 1 l i t r e . Each protein solution f o r disc electrophoresis was made up to 1% (W/V) i n AUC (0.1 M acetic acid, 3M urea and 0.01M hexadecyltrimethyl ammonium bromide) solution. Sucrose was added to the solution at a concentration of 15-20% to increase the density. Approximately 50 pi of the sample solution were applied to the top of the upper gel. Electrophoretic separa-tion was carried out for approximately 120 min under a current of I I mA per column. Crystal violet was used as a marker dye. The gels were removed from the glass tubes and stained in 1% aqueous coomassie b r i l l i a n t blue R 250 diluted 1 to 20 with 10% trichloroacetic acid. After fixing the gels for 30 min in 12.5% trichloroacetic acid, the gels were destained in 1% trichloro-acetic acid. Gel F i l t r a t i o n Two kinds of gel columns were used: A. Sephadex G-100 column (lpc72 cm). Sephadex G-100 (Pharmacia) was allowed to swell for IL.8 hr in excess AUC solution and then packed into the column which was subsequently washed with 1 l i t r e of AUC solution. B. Sepharose 6B column (2 .5x80 cm). Sepharose 6B (Pharmacia) was diluted approximately 1 to 5 with AUC solution and packed into column and the column was then eluted with 500 ml AUC solution. Five ml of a modified or untreated gluten solution were applied to the top of the column. When the sample was absorbed,CCthfe^'c^^ with AUC solution at the rate of 15-20 ml/hr. The elution profile was obtained by reading the absorbance at 280 nm of each fraction. Fractions were 16 c o l l e c t e d i n an Iaco Golden Retrieve f r a c t i o n c o l l e c t o r equip-ped with a drop counting system. The f r a c t i o n size was 5 ml f o r Sephadex G-100 column and 3*5 ml f o r Sepharose 6B column. Methods of U l t r a c e n t r l f u g a l Analysis 1. Determination of Sedimentation C o e f f i c i e n t A Beckman model L2-65B ul t r a c e n t r i f u g e with schlieren optics was used to determine the sedimentation c o e f f i c i e n t . The speed was set at 59,000 rpra and four or more photographs were taken with an i n t e r v a l time of 30 min. The r a d i a l distan-ces of the sample meniscus and of the boundary (highest point of the peak) from the center of r o t a t i o n were measured and the 18 c a l c u l a t i o n was done according to the following equation : 1 dr 2.303 d log x CO2!* dt 60 t o 2 dt where s « sedimentation c o e f f i c i e n t 27cppm u> s angular v e l o c i t y i n radians/sec * 1 1  60 = 0.10^72 rpm r » the distance between the sc h l i e r e n peak and the center of r o t a t i o n t = time i n minutes x = scale reading 2. The Trautman p l o t by Sedimentation Equilibrium Technique The molecular weight d i s t r i b u t i o n of gluten was determined by using the approach to sedimentation equilibrium 17 technique of Archibald** as extended by Klainer and K e g e l e s ^ , cn and data was handled by using the Trautman plot"^ as modified 22 "M. by Erlander and Poster * J . In t h i s method the centrifuge i s operated at various speeds and time i n t e r v a l s . A function of the concentration gradient at the meniscus i s plotted against a function of the area under the schlieren diagram f o r d i f f e r e n t speeds and times. In accordance with the conventions of 22 * Erlander and Poster , the ordinate i s Y.i and the abscissa i s X , where A Ya tane Xa£>y * 7.3737 tan8Az£Ay. Xa2 ^Ya = the height of the schlieren pattern at the meniscus, 0 = the angle between the schlieren diaphragm and the l i g h t source* Xa s the distance from the center of r o t a t i o n to the meniscus, A z ^ ^ y - the area under the schlieren pattern corrected f o r r a d i a l d i l u t i o n using Trautman 1s Z-scale, 7.3737 i s the o p t i c a l constant f o r the centrifuge and enlarger. In t h i s study of sedimentation equilibrium,. the Beckman model L2-65B ultracentrifuge equipped with the UV scanner was also used. A t y p i c a l UV scanner chart i s shown i n Figure 1. In the UV patterns, the area under schlieren peak i s equal to Co-Cm i n t h i s chart, the angle between the schlieren diaphram. and the l i g h t source, and the o p t i c a l constant were not involved, therefore the above equation can be s i m p l i f i e d as follows: Inner. Reference Edge Solvent Region Boundary Region Plateau Region F i g u r e 1 T y p i c a l UV scanner c h a r t f o r u l t r a c e n t r i f u g a t i o n where Co * i n i t i a l concentration of the s o l u t i o n , Cm = the concentration of the solution at the meniscus when measuring. The value of y, which i s the concentration gradient at the meniscus, was calculated by extrapolation of the slope of UV scanning pattern towards the solution meniscus. Amino Acid Analysis The gluten samples were f i r s t treated with 14-vinyl pyridine to block cysteine as described by Gavins et al*'*, and then the samples were hydrolyzed with p-toluene sulfonic acid according the method of L i n and Chang 3®. One hundred mg of untreated or modified gluten were dissolved i n 10 ml of urea t r i s buffer, reduced by 0.1 ml 2-mercaptoethanol under nitrogen while s t i r r i n g f o r 16 hr and then 0 . 1 5 ml lj.-vlnyl pyridine was added. After s t i r r i n g under nitrogen f o r another 2 hr, g l a c i a l acetic acid was added to adjust the pH to 3 . The sample was then dialyzed against 0.01N acetic acid f o r 36 hr, followed by d i a l y s i s against water overnight. The sample was then freeze dried. A 2 . 5 - 3 * 5 mg protein sample treated with i j - v i n y l pyridine was dissolved i n 1 ml of p-toluene sulphonic acid solution prepared according to L i n and Chang3**. Tubes were evacuated to a l e v e l of 2 0 - 3 0 .^m of Hg and sealed. The protein was hydrolyzed at 110 C f o r 2l\. hr. After hydrolysis, 2 ml of IN NaOH were added and the volume was made up to 5 ml with water. After the sample was f i l t e r e d through a sintered glass funnel (UP), i t was subjected to amino acid analysis. A Phoenix micro amino acid analyzer model M 6500 with a single column system of Durrum Chem. Corp., Palo Alto, C a l i f , was used. Small, medium and large amounts of a prepared sample were injected to the column f o r amino acid analysis and each analy-s i s was repeated three times to confirm the r e s u l t s . Standard amino acid solutions were also injected several times during the analysis period to v e r i f y the r e s u l t s . Determination of Functional Properties 1. Emulsifying Capacity The emulsifying capacity of the untreated and modified gluten samples was determined by using an S o r v a l l homogenizer (Omni-mixer), type 0M. 225 mg of freeze-dried modified or untreated gluten sample were dissolved i n 30 ml of water. 10 ml of corn o i l (Pisher Chemical Company) were added and mixed with the protein solution at a no load speed of 8600 rpm. Additional corn o i l was continually added into the jar at a rate of 5 ml per min from a graduated funnel through Tygon tubing. An o i l -in-water emulsion was formed and became increasingly more . ^ viscous as o i l was added up to the point when the emulsion broke whereupon the v i s c o s i t y decreased abruptly. The t o t a l amount of added corn o i l was recorded and c a l c u l a t i o n was made of the amount of corn o i l needed to' break the emulsion contain-ing 100 mg gluten or modified gluten samples. 2. Emulsion S t a b i l i t y Rating Emulsion s t a b i l i t y r a t i n g was performed following the method of Acton and S a f f l e * , which i s a modification of the method of Titus et &V* . Two 10-ml samples of each homo-genized emulsion mixture (by using high speed blender at 8600 rpm) were held at 5 C f o r 1-3 hr and then placed i n a 37 C incubator f o r 2i|. hr. Two additional 5>-ral aliquots of the emulsion were immediately analyzed f o r moisture using the A.O. A.C. standard procedure**. Following the 37 C incubation period, the bottom 5-ml of the tube samples were removed with a 5-ml pipette and analyzed f o r the moisture percentage. A s t a b i l i t y r a t i n g f o r each sample was determined on the basis of the percentage change i n moisture. The following equation was used: 100-IL.^ SR = x 100 1 0 0 - M o r i g i n a l where SR i s the s t a b i l i t y r a t i n g , Mtes^. the percent moisture of the bottom 5 ml of the sample incubated at 37 C f o r 2lj. hr, and M Q r^g^ n a^ the i n i t i a l percent moisture of the sample. 3« Foamability and Foam S t a b i l i t y F i f t y ml of a 0,5% gluten or modified gluten solution was beaten i n a bowl by a Sunbeam 12-speed mix master beater at highest speed f o r 6 min. The foam mixture was trans-f e r r e d to a volumetric f l a s k and the volume was measured. The volume increase calculated i n percentage was used to express the foamability. The drain volume and the speed of draining were used as a measure of foam s t a b i l i t y . RESULTS AND DISCUSSION Preparation L. Recovery of S o l u b i l i z e d Gluten Commercial v i t a l gluten has an appreciable amount of entrapped starch and some bound and free l i p i d s . In 20 electron micrographs of v i t a l gluten prepared by Cumming , intac t starch granules were found within the stranded structure of hydrated gluten. The analysis of the sample of v i t a l gluten f o r t h i s study i s presented below: Components Content Protein 79 .3 % Moisture 6 .2 % Extraetable f a t 0 .98 % Ash 1.03 % Carbohydrate and bound l i p i d s 12.1+9 % (by difference) Conditions f o r the preparation of acid-modified gluten (described i n Materials and Methods section) were decided by preliminary experiments. Before n e u t r a l i z a t i o n , the acid-modified gluten solutions were l i g h t tellow to brown in colour and ranged from clear to somewhat turbid depending 2k on the acid concentration, temperature of heating and heating time. Complete recovery of s o l u b i l i z e d gluten was ca r r i e d out by tran s f e r r i n g an acid-modified gluten solution to a cel l u l o s e d i a l y s i s tubing, d i a l y z i n g against water at J4. C f o r 2 days and subsequently freeze drying the centrifuged solution. D i a l y s i s was car r i e d out to remove the sugars, s a l t s and polypeptides formed during the acid treatment and the residual hydrochloric acid. Maximum p r e c i p i t a t i o n of protein i n acid-treated gluten solutions was obtained by adjusting the pH to ii.2 to It. 9 depending on the concentration of acid used f o r hydrolysis (see Table 1 ) . This maximum p r e c i p i t a t i o n was determined by measuring the absorbance of the supernatant of the hydrolysate at 280 nm at various pH yval'ues'The pHV:"wi^thulGWest A2QQ value was selected as the condition t h a t maximum p r e c i p i t a t i o n was formed. After centrifugation, the supernatant was decanted and the p r e c i p i t a t e was dissolved i n same volume of water by adding IN NaOH solution to pH 7.5. The solution was centrifuged at 5000 xg f o r 10 min and the supernatant was freeze dried (Figure 2 ) . When gluten was hydrolyzed w i t h acetic acid, the i s o e l e c t r i c point was broad (pH 3.0 to 9.0). After d i a l y s i s of the modified protein solution, the pr e c i p i t a t e d protein could be s o l u b i l i z e d at pH 7.5. Table 2 contains the percentage yie l d s of s o l u b i l i z e d gluten prepared by the d i a l y s i s - f r e e z e drying method after autoclaving acid-gluten mixtures at 121 C. The re s u l t s show Table 1 Protein content, pH f o r maximum p r e c i p i t a t i o n and colour of the acid-modified gluten „ _ A .. Time of heating Cone, of Acid a ^ ^gj. c Protein content1 % pH f o r Max. ppt Moisture % Colour 0.02N HCl 30 rain 89.4 4.9 6.1 pale yellow 0.05N HCl 15 min 87.6 4.7 6.3 pale yellow 0.1 N HCl 15 min 86.2 4.5 7.1 pale yellow 0.5 N HCl 15 min 77.4 4.2 7.2 brown 1.75N acetic 30 min 85.5 — 6.2 pale yellow 3.5 N acetic 15 min 85.4 — 6.3 pale yellow 8.75N acetic 15 min 6.5 pale yellow 0.02N HCl (pH 5 fraction) 30 min 99.5 4.9 6.3 white 0.05N HCl (pH 5 fraction) 15 min 99.3 4.7 6.3 white 1 Dry weight basis 5% gluten suspension acid autoclave Hydrolysate adjust pH to 4.2-4.9 •< Centrifuge ppt supernatant (whey) resuspend i n water adjust pH to 7 .5 Centrifuge supernatant (freeze dried) Solubilized gluten Procedure for the preparation of s o l u b i l i z e d gluten by p r e c i p i t a t i o n method Table 2 Yield of solubilized gluten (dry weight b a s i s ) * a f t e r autoclaving gluten at 121 C as recovered by d i a l y s i s method Solubilized Gluten (% of t o t a l gluten protein) Time ; 8.75N 575N 1.75N of a c e t i c acetic a c e t i c heating 0.5N HC1 0.1N HC1 0.0?N HC1 0.02N HC1 acid acid acid 5 min - 88.5 92.1 89.8 - 89.8 60.2 15 min 66.1 89.8 88.5 92.1 90.9 93.3 83.9 30 min 44.9 87.4 85.1 90.9 86.2 92.1 88.5 60 min 41.3 85.1 86.2 82.6 85.1 88.5 8?.4 * -average of 4uplibate;.samples , .t: --i? 28 that the y i e l d of gluten i n the s o l u b i l i z e d form was between 85 to 92 percent except f o r the treatments with 0.5N HCl and 1.75N acetic acid. The recovery of protein from the 0.5N HCl-treated gluten when heated at 121 C f o r 15 min was only 66%. When the heating time was extended to 60 min, the recovery was further decreased to ij.1%. At a concentration of 0.5N HCl, the gluten molecules undergo extensive hydrolysis with longer heating times. In the case of the 1.75N acetic acid treatment, with a heating period of 5 min, the recovery of pr o t e i n was only 60%. As the heating time was extended to 30 min, the recovery of protein was Increased because the s o l u b i l i t y of the gluten was increased. Data shows that with lower heating temperatures, heating time had to be increased to reach the equivalent degree of s o l u b i l i z a t i o n . For example, six hr were needed to r e f l u x the 5% gluten suspension containing 0.1N HCl at 100 C to obtain a y i e l d of about 85% ( d i a l y s i s method). The g e l f i l t r a -t i o n r e s u l t s showed that the protein composition of a 6-hr refluxed sample was si m i l a r to the 15-min autoclaved sample (page 5 5 ) . Although the y i e l d s of s o l u b i l i z e d gluten from the 0.02N HCl-treated gluten with heating times of 5 and 15 min were 89.8 and 92.1 % respectively, the s o l u b i l i t y of the freeze-dried samples i n water was not complete. The 1.75N acetic a c i d -treated gluten with 15 min heating time showed the same r e s u l t . However, a l l other acid-treated gluten samples i n the freeze-dried form were completely water soluble. Considering both the y i e l d and the s o l u b i l i t y charac-t e r i s t i c s of the freeze-dried samples, gluten was acid-treated at 121 C for 15 min f o r further studies but 0.02N HCl and 1.75N acetic acid treated gluten were heated f o r 30 min. The y i e l d s of proteins p r e c i p i t a t e d from acid treated gluten solutions were between 70-78% of the o r i g i n a l gluten protein. 2. Carbon Column Treatment of Acid-modified gluten ^ The acid-modified gluten samples were almost odorless and pale yellow i n colour, however, when i t i s dissolved i n water to make a concentrated solution, a wheaty odor appeared. A p o s s i b i l i t y of eliminating the wheaty odor by activated carbon treatments was examined. Granular activated carbon was f i r s t washed to eliminate the f l o a t i n g f i n e p a r t i -cles and then paoked into a column (1.2x8 cm). Deodorization was c a r r i e d out by passing a 5% acid-modified gluten solution through t h i s column. Treatment was ca r r i e d out at two d i f f e r e n t conditions (a) The 5% acid-modified gluten solution was passed through the column at room temperature, (b) The column was warmed by passing a large amount of b o i l i n g water through the column, then the b o i l i n g s o l u b i l i z e d gluten was passed through the column. The gluten solution eluted from the column was then subjected to an organoleptic test by a six-member taste panel. Table 3 Taste panel score s of the wheaty flavour of the acid-modified gluten ftftereJaciti Sample 1 2 3 Panelists Control Room temp, treatment High temp, treatment 1 2.5 1.5 1 2 3 1 2 3 3 2 1 4 3 2 1 5 2 3 1 6 3 2 1 Rank sum 16.5 11.5 7 Rank 1 # weakest wheaty flavour, rank 2 = strongest wheaty flavour 3 1 There were three samples served to the pa n e l i s t s f o r t h e i r judgement. 1 . The s o l u b i l i z e d gluten solution without activated carbon treatment. 2 . The gluten solution eluted from the room temperature column. 3 . The gluten solution eluted from the hot column. Five of the s i x panel members judged sample No. 3 having the mildest wheaty flavour, f i v e of the members agreed that sample No. 2 had a milder flavour than sample No. 1 (Table 3 ) . This preliminary test c l e a r l y showed that the hot activated carbon treatment eliminated most of the wheaty flavour present i n the s o l u b i l i z e d gluten solution; the room temperature carbon column was less e f f e c t i v e f o r t h i s purpose. Characterization 1 . Amide Nitrogen Content The main purpose of the mild acid treatment of gluten was deamidizing the gluten so that the amount of intermolecular hydrogen bonding would be decreased and the s o l u b i l i t y of the gluten would be increased. The amide nitrogen content i n protein of untreated gluten was found to be 2 7 9 . 3 ± 7 « 2 mM/100 g protein. Table k shows the amount of amide nitrogen which remained i n the acid-modified gluten a f t e r s p e c i f i c acid t r e a t -ments. It i s apparent from the results that only a small amount of amide nitrogen remained i n the acid-modified gluten samples which were treated with 0 . 5 N H C 1 f o r 1 5 to 6 0 min. In general, Table ij. Amide nitrogen i n acid-modified gluten p r o t e i n * Amide nitrogen, mM per 100 g protein Time 8.75N 3.5N 1.75N of a c e t i c acetic a c e t i c heating 0.5N HCl 0.1N HCl 0.07N HCl 0.Q2N HCl -acid acid acid 5 min — 163.5 223.3 249.1 — 270.3 262.0 15 min 23.2 160.9 205.7 249.1 262.8 265.2 264.4 ,.;30 min 17.3 147.2 213.3 246.8 255.1 262.5 — 60 min 14.5 120.7 181.5 246.9 252.6 249.7 261.9 * Untreated gluten contained 280 mM amide per 100 g protein 1 t r i p l i c a t e d samples the lower the HG1 concentration, the larger the amount of amide nitrogen remaining i n the protein. Gluten treated with 8.75N, 3.5H and 1.75N acetic acid solutions had higher amide nitrogen values than HC1 treated gluten regardless of the heating time between 5 and 60 min. Gluten samples were solubi-l i z e d when the amount of amidex i^trogeni^was ^ e i d ^ ^ ^ by^as 1 ^ as f o r the acetic aeid-modified gluten and by 10% f o r the HCl-modified gluten. These facts indicate that acetic acid i s a milder acid f o r the deamidation of gluten. 2. Disc Gel Electrophoresis The untreated and acid-modified gluten1samples were examined by disc gel electrophoresis according to the procedure •*>&, 21 To of Davis and Chen and Bushuk . The disc gel electrophoretic pattern of untreated gluten showed more than twelve clear bands as indicated i n Figure 3« Acid modification of gluten brought about consider-able changes i n the electrophoretogram. With 0.02N and 0.05N HC1, 1.75N and 3»5N acetic acid-treated gluten samples, bands s i m i l a r to those of the untreated gluten were obtained but with s l i g h t l y lower r e s o l u t i o n . When the acid concentration was increased to 0.1N HC1, a few bands near the o r i g i n disappeared and the rest of the bands became i n d i s t i n c t . The 0.5N HC1 modified gluten showed only few poorly resolved bands l e f t at the front end. A soluble protein f r a c t i o n with high foamability was obtained by dispersing the p r e c i p i t a t e from 35 0 . 0 2 N and 0.05N HCl-treated gluten i n water to a 5% l e v e l ( f i n a l pH 5 .2 -5 . I j . ) , centrifuging f o r 1 0 rain at 5000 xg and freeze-drying of the supernatant. This pH SlU protein f r a c t i o n represented 30-35 % of the o r i g i n a l gluten protein. The majori-ty of proteins i n t h i s f r a c t i o n migrated at a slow rate but four minor f a s t moving bands were noted i n the disc gel electrophore-togram (Figure Chen and Bushuk 1^ compared the gel f i l t r a t i o n and disc gel electrophoresis pattern of glutenin, g l i a d i n and water-soluble protein f r a c t i o n s . They reported that the components with higher molecular weights revealed slow moving bands and the water-soluble protein appeared as bands with r e l a t i v e l y high m o b i l i t i e s on gel electrophoretogram. The r e s u l t s shown i n Figure 3 suggest that the gluten samples treated with higher concentrations of hydrochloric acid contained more smaller molecular weight components, and those treated with d i l u t e hydrochloric acid or acetic acid contained more higher molecular weight components. In general, the pH 5 f r a c t i o n s contained more higher molecular weight components than the remainder of the 0 . 0 2 N and 0.05N HCl treated gluten samples. 3. Determination of Molecular Weight D i s t r i b u t i o n A. Gel F i l t r a t i o n To study the properties of proteins i t i s important to know t h e i r molecular weight or the molecular weight d i s t r i b u t i o n 1 ( i ) (2) (3) (4) Figure k Disc electrophoresis pattern of (1) untreated gluten (2) 0.02N HCl pH 5 f r a c t i o n (3) 0.05N HCl PH 5 soluble f r a c t i o n (4) 0.02N HCl treated gluten ON when they are polydisperse. Gel f i l t r a t i o n , a chromatographic procedure which separates materials on the basis of molecular s i z e , has become a standard laboratory technique f o r the determination of these parameters. Large molecules are completely excluded from the gel bed at the void volume (Vo), while smaller molecules i n a s p e c i f i c size range are able to penetrate the gel of p a r t i c u l a r pore sizes and emerge l a t e r with the p o s i t i o n depending the size of the molecules. In t h i s study, two d i f f e r e n t gel columns were prepar-ed, Sephadex G-100 column with a size of 4. x 72 cm, and Sepharose 6B column with a size of 2.5 x 80 cm. The untreated gluten was dissolved i n the AUC solution to make a 2.5% solution arid applied to these two columns. Sample sizes were 7.5 ml and 5 ml respectively, and the e l u t i o n p r o f i l e s are shown i n Figures 5 and 6. Comparing the e l u t i o n patterns on Sephadex G-100 (column I) and on Sepharose 6B (column I I ) , three major peaks and one minor peak were found i n both p r o f i l e s . As expected, the f i r s t peak was eluted at void volume on column I but eluted a f t e r void volume from column I I . Gel f i l t r a t i o n has been used f o r f l o u r proteins or f l o u r protein f r a c t i o n s . Wright et a l ^ reported that the gel f i l t r a t i o n on Sephadex G-100 of the f l o u r extract eluted with an aluminium lactate buffer resulted i n the separation of four main u l t r a - v i o l e t absorbing peaks. The f i r s t peak represents protein or;protein; complexes of molecular weight> 4 0 8 0 TUBE NUMBER 1 2 0 Figure 5 Gel f i l t r a t i o n pattern on Sephadex G - 1 0 0 f o r untreated gluten. Column size 4 x 7 2 cm. Buffer : , AUC. Fraction size 5 ml. Sample size 5 ml. Vo=void volumet was determined with blue dextran. 1.0 .8 •8 - 6 < .4 .2 Figure 6 4 0 III 8 0 1 2 0 TUBE NUMBER Gel f i l t r a t i o n pattern on Sepharose 6B for untreated gluten. Column size 2.5x80 cm. Buffer ilL AUC. Fraction s i z e : : 3.5 ml. Sample size = 4 ml. Vo=void volume, was determined with blue dextran. V*) 100,000, probably, the glutenin complexes of molecular weight 2-3 x 10°. Peak 2 and 3 may represent the g l i a d i n s and the water-soluble proteins of molecular weight,<1100,000, and peak k represents small non-protein molecular with some absorption of u l t r a - v i o l e t r a d i a t i o n . Meredith and Wren**0 reported that using gel f i l t r a t i o n on Sephadex G-200 of wheat f l o u r proteins eluted with AUC solution, four peaks were again obtained. 51 Others reported similar r e s u l t s . The e l u t i o n pattern from column I i n t h i s study i s sim i l a r to the re s u l t s of Wright et a l and Meredith and Wren**0. Sepharose 6B column; however, was f o r the f i r s t time i n the l i t e r a t u r e to be used i n t h i s study to determine the molecular weight d i s t r i b u t i o n of wheat protein. There have been several relationships proposed c o r r e l a t i n g the molecular weight of protein with i t s e l u t i o n volume from the column. Laurent and Killander^' introduced a convenient measure of the degree of retention of a molecule, which i s independent of the size of the column. They explained the gel-matrix as three dimensional with a random network of straight polymer f i b e r s and suggested the equation: K a v = e x P lTy L ( r s + r r 0 where K o w i s the f r a c t i o n of gel volume available f o r the substance and calculable by K „ = ( V - V )/( V.-VV. ) from av e o z u e l u t i o n diagrams. L i s the concentration of gel rods i n the solution, r f l the radius of the spherical solute, r w the radius of the gel rod, the t o t a l volume of the gel bed and V q i s the elution volume of the peak. To estimate the molecular weight d i s t r i b u t i o n of gluten proteins and to compare the correspondent peaks of the two e l u t i o n patterns, the approximate molecular weights of the peaks were calculated. L value of 2 . 9 was used f o r Sephadex G - 1 0 0 according to Laurent and K i l l a n d e r 3 7 and a value of 1 . 0 was calculated f o r Sepharose 68 from a water regain value analyzed. The calculated r e s u l t s of the approximate molecular weights of the peaks are as follows: Calculated Molecular Weight Sephadex G - 1 0 0 Sepharose 6B Peak I Peak II Peak I I I 1 0 0 , 0 0 0 2 ^ , 0 0 0 1 2 , 0 0 0 5 3 , 0 0 0 2 3 , 0 0 0 9 , 0 0 0 The Sepharose 6B peak I, which should include the glutenin f r a c t i o n had a calculated molecular weight of 5 3 , 0 0 0 . This value i s smaller than most values i n the l i t e r a t u r e f o r g l u t e n i n 7 T h e powerful d i s s o c i a t i n g reagent AUC must have dissociated the protein complexes to monomers and the better separation c a p a b i l i t y of Sepharose 6B f o r larger molecular components rendered the glutenin components spread i n a wide range on the el u t i o n pattern, thus suggesting the great d i s p e r s i t y of these proteins. Figures 7 and 8 show the gel f i l t r a t i o n pattern of the 0.02N HCl and 0.05N HCl-treated gluten on the Sephadex 6-100 column. The r e s u l t s of Figures 5» 1 and 8 showed that the 0.02N HCl treated gluten contained lower molecular weight components than the untreated gluten and that the 0.05K HCl treated gluten contained lower molecular weight components than the 0>02N HCl treated gluten. Figures 9 and 10 show a comparison of gel f i l t r a t i o n patterns of the pH 5-soluble f r a c t i o n and the unfractionated s o l u b i l i z e d protein of the 0.02N and 0.0$B HCl-treated gluten. These r e s u l t s are i n agreement with the disc gel electrophoresis patterns implying that the pH 5 soluble f r a c t i o n s contained higher molecular weight components. Although Sephadex G-100 column showed the a b i l i t y of distinguishing the molecular d i s t r i b u t i o n of various gluten samples (Figures 7-10), only steepness of the peaks were varied i n the e l u t i o n p r o f i l e s while a l l the patterns had the same general shape. Since Sepharose 6B column showed better separa-t i o n c a p a b i l i t y (Figure 6) than Sephadex G-100 f o r larger molecular components, i t was selected to be used f o r the gel f i l t r a t i o n experiments i n further studies. Figures 11 to 16 show the gel f i l t r a t i o n patterns on Sepharose 6B column f o r the 0.02N, 0.05N, 0.1N and 0.5N HCl-treated gluten and the 1.75N and 3«5N acetic acid treated gluten. TUBE NUMBER Figure 7 Gel f i l t r a t i o n pattern on Sephadex G - 1 0 0 f o r 0 . 0 2 N HCl-modified gluten (heated at 1 2 1 C for 30 min). Column size 4 x 7 2 cm. Buffer: AUC. Fraction s i z e : 5 ml. Sample s i z e : 5 ml. Vo=void volume, was determined with blue dextran. 1.0 4 0 8 0 12 0 T U B E NUMBER Figure 8 Gel f i l t r a t i o n pattern on Sephadex G-10G f o r 0.05N HCl-modified gluten (heated at 121 C f o r 15 min). Column s i z e 4x?2 cm. Buffer AUC. Fraction size : ~ 5 ml. Sample size : 5 ml. Vo=void volume, was determined with blue dextran. 5 0 1 0 0 TUBE NUMBER Figure 9 Gel f i l t r a t i o n patterns on Sephadex G-100 f o r upper» 0.02N HCl-modified gluten (heated at 121 C f o r 30 min), lower.» pH 5 soluble f r a c t i o n of 0.02N HCl-treated gluten. Column size : . 4x72 cm. Buffer? AUC. Fraction size :J 5 ml. Sample s i z e : ' 5 ml. I 0 1 0 0 TUBE NUMBER Gel f i l t r a t i o n patterns on Sephadex G - 1 0 0 f o r upper; 0.Q5N HCl-modified gluten (heated at 1 2 1 C for 1 5 min), lower » pH 5 soluble f r a c t i o n of 0 . 0 5 N HCl-treated gluten. Column size : 4 x 7 2 cm. B u f f e r : AUC. Fraction s i z e : 5 ml. Sample s i z e : 5 ml. 4 0 8 0 1 2 0 T U B E NUMBER Figure 11 Gel f i l t r a t i o n pattern on Sepharose 6B for 0.02N HCl-modified gluten (heated at 121 C for 3 0 min). Column size : : 2.5x80 cm. Buffer : AUC. Fraction size s v 3 . 5 ; m i . Sample size 1 4 ml. TUBE NUMBER Figure 1 2 Gel f i l t r a t i o n pattern on Sepharose 6B for 0.05N HCl-modified gluten (heated at 1 2 1 C f o r 1 5 min). Column size ij 2.5x80 cm. Buffer:1s AUC. Fraction size?; 3 . 5 ml. Sample s i z e . i - 4 ml. 4 0 80 T U B E NUMBER 12 0 Figure 13 Gel f i l t r a t i o n pattern on Sepharose 6B for Q.1N HCl-modified gluten (heated at 121 C for 15 min). Column size :t 2.5x80 cm. Buffer *p AUC. Fraction size 3.5 ml. Sample s i z e :l k ml. TUBE NU M BER Figure 14 Gel f i l t r a t i o n pattern on Sepharose 6B for 0.5N HCl-modified gluten (heated at 121 C for 15 min). Column siz e :J 2.5x80 cm. Buffer : AUC. Fraction s i z e : 3.5 ml. Sample s i z e : 4 ml. 8 0 12 0 T U B E N U M B E R Gel f i l t r a t i o n pattern on Sepharose 6B for 1.75N acetic acid-modified gluten (heated at 121 C for 30 min). Column size r ; 2.5x80 cm. Buffer AUC. Fraction s i z e : 3.5 ml. Sample s i z e : : 4:;ml. TUBE NUMBER Gel f i l t r a t i o n pattern on Sepharose 6B f o r 3.5N acetic acid-modified gluten (heated at 1 2 1 C for 1 5 min). Column size : 2 . 5 x 8 0 cm. Buffer : AUC. Fraction size : 3 . 5 ml. Sample size : 4 ml. 53 Comparing the gel f i l t r a t i o n patterns i n Figures 1 1 to Ik-, i t i s obvious that the el u t i o n volume of the peaks increased with the concentration of hydrochloric acid used to treat gluten. * This r e s u l t indicates the breakdown of polypeptide chains during treatment. The extent of breakdown of polypeptide chains increased with the increasing concentrations of hydrochloric acid. Figures 1 5 and 16 show the ef f e c t of acetic acid i s much weaker than that f o r hydrochloric acid. The change i n gel f i l t r a t i o n volumes of the peaks from d i f f e r e n t samples i s l i s t e d i n Table 5 . Gluten treated with the same concentration of acid but using d i f f e r e n t heating temperature and time gave sim i l a r r e s u l t s on gel f i l t r a t i o n as shown i n Figures 1 7 , 18 and 19. Figure 1 7 indicates that the gluten hydrolyzed with 0.1N HC1 by re f l u x i n g the sample at b o i l i n g point f o r 6 hr and by autoclaving the sample at 1 2 1 G ( 1 5 psig) f o r 15 min revealed'% s i m i l a r molecular weight d i s t r i b u t i o n . The difference i n heating times used f o r autoclaving gluten solutions either with 0 . 0 7 N HC1 or with 0.5N HCl did not af f e c t the e l u t i o n p r o f i l e s (Figures 1 8 , 1 9 ) . These r e s u l t s are i n agreement with the r e s u l t s of the amide nitrogen content i n the acid modified gluten samples. Both the degree of deamidation and the extent of the breakdown of peptide chains were controlled mainly by the concentration of acid and the kind of acid used, whereas, the heating period was less important. Breakdown of peptide chains resulted i n the formation 5V/ Table 5 The el u t i o n volume of the peaks from gel f i l t r a t i o n of d i f f e r e n t acid-modified gluten samples 2 Treatment E l u t i o n volume (tube number) 1 peak I peak II peak III peak IV Untreated gluten 35-55 70 90 105-115 0.02N HG1 40-55 75 92 105-115 0.05N HG1 60-70 84 102 110-120 0.1 N HCl 70-80 96 102 110-120 0.5 N HCl 80-90 100 108 110-120 1.75N ace t i c acid 40-55 70 92 105-115 3.5 N acetic acid 50-60 76 92 105-115 1 Sepharose 6B column, 3.5 ml per tube 2 autoclaved samples 1 the heating time f o r 0.02N HCl and 1.75N acetic acid was 30 min, for the other acid concentrations was 15 min. 1.0 8 O - 6 CO < 2 4 0 8 0 T U B E N U M B E R 1 2 0 Figure 17 Gel f i l t r a t i o n patterns on Sepharose 6B f o r — — 0 . I N HCl-modified gluten (refluxed at 100 C for 6 h r ) , < - — — 0 . I N HCl-modified gluten (heated at 121 C for 15 min). Column s i z e : 2.5x80 cm. B u f f e r : AUC. Fraction sizes 3.5 ml. Sample s i z e : 4ml. TUBE NUMBER Figure 18 Gel f i l t r a t i o n patterns on Sepharose 6B for 0.0?N HCl-modified gluten, sample heated at 121 C for 30 min, — — — — — sample heated at 121 C for 5 min. Column size s 2.5x80 cm. Buffer * AUC. Fraction size 3.5 ml. Sample size U- ml. 8 O oo 4 0 T U B E 8 0 NUMBER 1 2 0 Figure 1 9 Gel f i l t r a t i o n patterns on Sepharose 6B f o r 0.5N HCl-modified gluten, heated at 121 C for 15 min, — heated at 121 C f o r 30 min. Column size:- 2.5x80 cm. B u f f e r : AUC. Fraction size:. 3.5 ml. Sample s i z e : . ' k ml. 5 8 of smaller molecular weight components including small peptide chains and free amino acids. In order to obtain more informa-t i o n about the changes during acid modification, a c h l o r a n i l test was used, Birks and S l i f k i n ^ " have reported that some amino acids form n-rv charge transfer complexes with c h l o r a n i l ( 2 , 3 , 5 , 6 tetrachloroquinone) i n aqueous 5 0 % ethanol buffered at pH 8 and over. As a r e s u l t of complex formation, the c h l o r a n i l absorbance (>>___. - 2 9 5 nm) decreases and a new band ( s 3 5 0 nm) develops, which i s attributed to the formation of the molecular complex. 2 According to Al-Sulimany and Townshend , the amino ac i d - c h l o r a n i l complex forms slowly, the reaction rates increas-ed markedly when the solution was heated, and a temperature of 6 5 C was selected f o r heating. They also found c h l o r a n i l to react weakly with ammonium chloride. Tyrosinase, which was chosen as a p u r i f i e d protein, gave a s i m i l a r spectral peak to the amino acids as well as to other proteins as observed by Birks and S l i f k i n ^ . Urea gave no response to a c h l o r a n i l - i n -5 0 % ethanol solution. 2 Al-Sulimany and Townshend suggested t h e i r method for determining amino acids since i t i s sensitive and reproduci-ble. This method was applied i n t h i s study f o r detecting the small peptides and amino acids i n acid modified gluten. The ninhydrin reaction which i s commonly used f o r t h i s purpose was not applicable i n t h i s study because of the interference by a high concentration of urea which was included i n the e l u t i n g 5? buffer AUC f o r gel f i l t r a t i o n . Figure 20, 21 show the c h l o r a n i l test patterns of 0.02N and 0.05N HCl-treated gluten, i n both cases, a peak occur-red at V 115-130 tubes, the peak of 0.05H HCl treated gluten v i s a l i t t l e bigger than 0.02N HCl treated gluten. These are evidences that small peptides and/or free amino acids have been produced during acid modifications; the amount of the small peptides and amino acids i n 0.05N HCl-treated gluten was more than 0.02N HCl-treated gluten. In the graphs of c h l o r a n i l test of 0.1N and 0.5N HCl-treated gluten (Figures 2 2 , 2 3 ) , two peaks are observed at the V ^ 1 1 5 tubes. The f i r s t peak probably © indicates small peptides and the next one free amino acids or NH^. A l l these r e s u l t s suggested the breakdown of peptide chains of gluten proteins due to the acid-modification. In the el u t i o n p r o f i l e s analyzed by the c h l o r a n i l test f o r the determination of small peptides and amino acids, i t i s notable that the absorbance at 2ti0 nm of the effluents were usually lower than the absorbance at 3 5 0 nm of the chlora.-; n i l test except f o r the f r a c t i o n s between 90 to 110 tubes. This p o s i t i o n represents the low molecular weight proteins derived from gluten during the heating process of acid modifications. The extremely high absorbance at 280 nm f o r the effluents may implicate the existence of decomposed components containing exposed tyrosine and tryptophan residues. 4 0 8 0 1 2 0 TUBE NUMBER Chloranil test of 0.02N HCl-modified gluten (heated at 121 C f o r 30 min). - gel f i l t r a t i o n pattern on Sepharose 6B with absorbance at 280 nm; c h l o r a n i l test of the eluate from gel f i l t r a t i o n , absorbance at 3 5 0 nm. 1 2 O T U B E NUMBER Figure 21 C h l o r a n i l test of 0.05N HCl-modified (heated at 121 C f o r 15 min) gluten. gel f i l t r a t i o n pattern on Sepharose 6B with absorbance at 280 nmj c h l o r a n i l test of the eluate from g e l f i l t r a t i o n with absorbance at 3 5 0 nm. p 40 80 I 2 0 TUBE NUMBER Figure 2 2 Chloranil test of 0.1N HCl-modified (heated at 1 2 1 C for 1 5 min) gluten. - gel f i l t r a t i o n pattern on Sepharose 6B with absorbance at 280 nm» - — — — — • c h l o r a n i l test of the eluate from gel f i l t r a t i o n with absorbance at 3 5 0 nm. G> TUBE NUMBER Chloranil teat of 0.5N HCl-modified (heated at 1 2 1 C f o r 1 5 min) gluten. •gel f i l t r a t i o n pattern on Sepharose 6B with absorbance at 280 nm; - — — — c h l o r a n i l test of the eluate from gel f i l t r a t i o n with absorbance at 3 5 0 nm. OS B. U l t r a c e n t r i f u g a l Sedimentation Study 61; U l t r a c e n t r i f u g a l analysis by the sedimentation v e l o c i t y technique i s a useful method f o r the characterization of protein since i t i s capable of resolving protein mixtures into t h e i r various components based on t h e i r molecular s i z e . Gluten i s a protein mixture of complex patterns. According to Ewart 2 3' 2^, more than t h i r t y components have been shown i n g l i a d i n by starch gel electrophoresis. W r i g l e y ^ ' ^ has found at l e a s t f o r t y g l i a d i n components using a two-dimensional electrophoresis technique. These components may be divided into at least three major groups with regard to t h e i r molecular weight. It was expected that the sedimentation v e l o c i t y method of u l t r a c e n t r i f u g a l analysis would provide a rough estimate of the molecular weight d i s t r i b u t i o n of d i f f e -rent gluten samples. A Beckman ultracentrifuge model L2-65B equipped with schlieren optics was used f o r t h i s purpose. A double sector c e l l was f i l l e d with 0 . 4 ml of 1% gluten solution i n 0 . 0 5 M phosphate buffer pH 7 * 5 . The running speed of u l t r a c e n t r i f u g a -t i o n was set at 5 9 , 0 0 0 rpm, and four or more pictures were taken at an i n t e r v a l period of 3 0 min. Under the conditions described above, only one peak was observed f o r each acid-modified gluten samples. Calculation of the sedimentation c o e f f i c i e n t of these peaks yielded a value of 1 . 9 4 S f o r the 0 . 0 2 N HCl-treated gluten and 2 . 0 7 S f o r the 1 . 7 5 N acetic acid-Typical schlieren pattern of 1% acid-modified gluten solution a f t e r reaching 59,000 rpm f o r 120 min. Temperature 20 C, phase plate angle treated gluten. These values are exceedingly small compared to the estimated molecular weight from gel f i l t r a t i o n . I t was l a t e r found that at the very high speed (59,000 rpm) of c e n t r i -fugation, the high molecular component r a p i d l y sedimented to the bottom of the centrifuge c e l l without forming d e f i n i t e boundaries. At a slower speed of centrifugation (38,000 rpm), two peaks appeared i n the schlieren pattern f o r a short period of time. Jones et a l ^ 1 determined the molecular weight of wheat gluten proteins i n an ultracentrifuge using the approach to the sedimentation equilibrium technique of Archibald** as extended by Klainer and Keg e l e s ^ . Data were handled by using 22 the Trautman p l o t modified by Erlander and Poster . This method was used to study the molecular d i s t r i b u -t i o n of acid-modified gluten samples. Both the schlieren optic accessory and the UV scanner were attached to a Beckman ult r a c e n t r i f u g e model L2-65B. When schlieren optics were used, the pictures were usually taken within the f i r s t one hour afte r changing the speed of the run. As a r e s u l t , the schlieren peak: were very steep and narrow loc a t i n g very close to the meniscus (Figure 25). Therefore i t was d i f f i c u l t to obtain accurate values i n measurement f o r the height of the schlieren pattern at the meniscus and the area under schlieren peak. When the UV scanner was used, the area under schlieren peak was calcu-l a t e d by d i r e c t measurement of Co, the i n i t i a l concentration of the solution, and Cm, the concentration of the solution at the Typical schlieren pictures of acid-mod i f i e d gluten solu t i o n (1%) for Trautman p l o t . Speed 59,000 rpm Time 1. 15 min, 2. 30 rain. meniscus when measuring. The concentration gradient at the meniscus was calculated by extrapolating, of the slope of UV scanning pattern towards the solution meniscus. Compared to the method using schlieren optics, the UV scanning method was more accurate and r a p i d , c although i n some cases there were d i f f i c u l t i e s i n the extrapolation of the concentration gradient to the meniscus. With the UV scanning accessory, there was no need to know the angle between the schlieren diaphragm and the l i g h t source and the o p t i c a l constant, and the area under schlieren peak i s simply measured as Co-Cm. Therefore, the equation f o r the c a l c u l a t i o n of X and Y can be s i m p l i f i e d as follows: Y* = —5 C J 2 Xa „ Co-Cm Examples of the UV scanning pattern are shown i n Figure 26 and 27 f o r the 0.02N HCl-treated gluten and i n Figures 28 and 29 f o r the G.5N HCl-treated gluten. From these patterns, i t i s c l e a r that the concentration at meniscus decreased very r a p i d l y f o r the 0.02N HCl-treated gluten compared to that f o r the G.5K HCl-treated gluten. The higher the speed of centrifuga-t i o n , the more rapid the decrease i n protein concentration at ^ 1 the solution meniscus. The data obtained from the UV scanning patterns were used f o r c a l c u l a t i o n using the equations given above f o r the F i g . 2 6 UV scanning patterns of 0 . 0 2 N HCl-modified gluten(heated at 1 2 1 C for 3 0 min). Left column speed at 2 8 , 0 0 0 rpm, rig h t column speed at 3 8 . 0 0 0 rpm. Time i n t e r v a l 1 0 min. 7P — — t — — — - — - — — — I - - -- - - - - - - - --- - - - - --- - -r T 1 i 4 J ] -_ i' i — — D — 2 F - * T ~4 r u '~r ... ... — r ._. — j...... i j — — _ " T " ~I r _ ... -y- 1 ! 1 1 — I . / I " i — ~ i | _ | ._ " 7 ~ - - ---- -f -- - - - - - -- - - H---z --- - -- _ _ -- - _ _ — — i =P - 1 i - - 'J ...j -i ~ !•"" j 1 ...I _j 1 i —i— 1 1 i i i i ] . j.... — I — 1 i ~ ! — + ! 1 i i —!— i i 1 i ! T i ~T" ~\ ~ x: - j — j i • -j" 1 i H— i "i I- 1 ~r "i" n .1 1 _ j _ i j 1 1 i 1 1 •; i • 1 "I " ~_T_ f- " -I—!— r V ~ "i I l 1 i i • ~n <—!—i , F i g . 27 UV scanning patterns of 0.02N HCl-modified gluten (heated at 121 C f o r 30 min). Left column speed at 48,000 rpm, rig h t column speed at 59.000 rpm. Time i n t e r v a l 7 min. 71' 1 — ... — -Z - - ... -- - - - - ... — — — -_ — --- - j - ' ---- - L - - - --- ... ... - - - ... -; - --- - -- - -- - - --- - .... ... - -- -- - - -- - - — f / i _ z> -_ --... .... -- ---- - -- ... ... - ... -- i ... - - -- - - --- - --— — --- - ... — — ... z - ----— --— - — -d it - f _ i-- — - — --— - - -- - ... - - -ti, -- - - - - -— ----- - ----— r, 1 j ;' - - ----'- -- - - - _ | _ ' i T -ir -L - -... I t; i -- -- -- . . . - ... 1 - ---- .... - - i • _ L . . . -- - - • \ - - -- ... — -- - -- -- - ... -- -- --- i | - -- - - 1 --... - -- - - - ... --- - - - -. . . . — ..... - - ... -- — - ---- ... - — -— i ' "I )z ---- -- -- - - - - ... -... -: - - ---- - -- — - - f - — ... -- - - - — : - - -- - ... -- — - - -— -... --- - - . . . - -- -- — Z .!.. i t ~ i \) 1 - - ---- -- - -- - ... --• --— — — zi -- - - - - — ---- - - - - -5!-- - - - -[ i \ _ -- -... _ ------ ... ----- - .... ------- - - _ -... -... -- - - -_ --- -- - ... - r - ... --- ----— 1 i .......... -1 "1"" --- - --- T - - - - — — — - — — -... - --- - -- - _ | . „ -- - - - •- - - -— - - - - -TL - ... - - - - - — - - - ---- ----... -• ---— ... - — \~ - — — — - - . . . ... -- - - - --— - - - - -_ - -} i - — -- J+ -- - ---V -- -- ... - - -- - -r i - — -- ... -- --- -- i r - H4-I-- i ! - . ( - - - - - -- - - - --- • - .... - .... ----- -- - r r i - - -I i r ... . . . L L . 1 i - - - — - ~ r j: -- ...... -- . . . .... ... - r i ~ r ---- -1 .... -- - i ... L. 1— i ! " T " 7 i... -- - - - -... - --- ... -- - -- - . . . -... -- .... -- -- - - - ... - --. . . -F 1 1 Z'\Z. --- j - -- -- - — 1 - - — - i - — t~-4r: ... i 1 — - ---i ... -- r r -- - — - ... ... --- - 1 . - - - - 1 - - - 1 -- 1 --1 I 1 ! 1 ... --ZZ-l _ — : zr 1 - - : -- ----- - -- - _ _ - ! -- - ! 1 1 " 1 " _.|... -- --— - - - - - 1 : r - -- -& -- -- -- 1 ~1~ i ! • — n • A I F i g . 28 UV scanning patterns of 0.5N HCl-modified gluten (heated at 121 C for 15 min). Left column speed at 28,000 rpm, ri g h t column speed at 38,000 rpm. Time i n t e r v a l 10 min. 72 i 1 f T | J J - 1 — ... ... _ | _ . . _ _ - _. _ lz j — _ r ._ _ _ j - • i • i -•-- _ ~_ t i If -i V - u — f ... 1 — — 7 1 - - - - - ._ - - - - -. , ( . . . _ — ._ -. _. ... .•/ - ... ])„- — ._ ... ._ _ - -4 V — — t - _ ' ' 1 f 1 ! • _ . ._ ._ ... ..I _ ... _. „ - — __ _ __ _ -' „_ _ — _ _ — • ... _ ... ... . _ _ 7 ... - A • _: — — — : i — — — — - - -- •-— _ •— - - - - - - ... __ / .... ._. r 44-i — / — - „ _ I ... — Jl-P _ .... _ V r " — _ _ 2_ ~ — _ . p . J . j - - — __ - - — .__ __ . ... "* ~ ' — — — .... — — - — - — — — -- - - ... - - - - - ~ - - - -- — - -- -_ : . / - 4 -_ ... -I _ . T . T 7 JZ ._ — ._ — ... .._ > 2 - — — — — ... — — — — — -/ _.. f... — — — - \ - \ r ri -±: _ - - _ - - - • 3 - - — -- L 1 ... ..... J - - -L 1 ! - — i —1 - I i - ±:_ - -- - -f - - - T - ... - H II — . . — ! . „ — _!._ „_!.„_! i ! ... t ; ... _ . 1 " ~ r • 1 1 i r r 1 t i 1 ! ! r ~ i i _ . " i i . . 1 i 1 i r i 1 ! I — ... _ __ ... _ — I j l ~ T ~ i 1 1 I ! / | 1 T" _ _ / ... =F —i—i. I j 1 — / l r 1 1 I -... - i 4 - - - = 1 1 — i — i i • 1 1 i -1 i i i ^ J -- — - - J - - — _ — — A A ... _ - i j — ! 1 i i i | p i p 1, t I - J— '~\ 1 ! - - - - ... M ••"!•"' j p| F i g . 29 UV scanning patterns of 0.5N HGl-modified gluten (heated at 121 C f o r 15 min). L e f t column speed at 48,000 rpm, rig h t column speed at 59.000 rpm. Time i n t e r v a l 7 min. value of X and Y . An example of the c a l c u l a t i o n f o r the 0.G5N HCl-treated gluten i s indicated i n Table 6. The data of X and Y obtained at d i f f e r e n t speeds and times were then subjected to the least squares method to obtainfthe best f i t curve. In order to seek a proper equation, least squares method was applied to f i t n pairs of x, y (y>G) data points to the logarithmic equations such as In y = a + b In x, In y•= a + bx, y = a + b In x and polynomial equations up to 5th power. Although the c o r r e l a t i o n c o e f f i c i e n t of several equations were high, i t was found that the equation In y s* a + bx was the only equation which best f i t s the data obtained from the experiments. However, t h i s equation ^ s i i l (notpdefine^'a-valueK^^x^hShc^ y = 0, therefore the equation was modified to: In (y • c ) -Ca j j * bx The best value of c was determined by substituting values of 2 1.5» 1.0, 0.8, 0,5 and 0.2 f o r c, then selecting the value of which derived the highest c o r r e l a t i o n c o e f f i c i e n t . Pairs of x y value of the best f i t curve were then calculated from the regression equation, and the y values obtained were plotted r against the x values to draw the best f i t curve of the Trautman p l o t . The r e s u l t s f o r the 0.02N-, 0 . 0 5 N - , 0.1H-, and 0.5N HCl-treated gluten samples are shown i n Figures 30-33. A l l of the best f i t curves were c o l l e c t i v e l y superimposed i n Figure 3$ f o r easier comparison. Table 6/ Data from UV scanning pattern and calculated X*, Y* for 0.05N HCl-treated gluten Co • 64.6/74.6 = 0.8660 Cm Xa X* (Co-Gm)/Xa* ( y/u)Xa) I. speed« 18,720 rpm 39.2/74.6 3 2 . 3 / 7 4 . 9 2 9 . 8 / 7 4 . 8 2 7 . 0 / 7 5 . 0 13/74.6 8/74.9 5/74.8 4/75.0 25 .6/75.2 3.8/75.2 24.8/75.3 3 .5/75.3 23.8/75.2 3 . 0 / 7 5 . 2 I I . speedt 28,034 rpm 37.5/74.5 35.2/74.5 27 .5/74.8 2 5 . 0 / 7 4 . 8 23 .6/74.6 22 .6/74.6 25/74.5 18/74.5 12/74.8 8/74.8 6/74.6 4/74.6 21.0/74.6 3.5/74.6 I I I . speedt 3 8 , 0 9 2 rpm 26.0/73.0 I5/73.O 21.0/73.0 8/73.0 15.2/72.6 5/72.6 14.4/72.8 4/72.8 13.0/73.0 2.5/73.0 12.6/73.0 2.0/73.0 IV. speed« 48 ,055 rpm 26.5/73.6 21/73.6 18.8/73.4 9/73.4 16.2/73.2 4/73.2 14.3/73.0 3.5/73.0 12.8/73.1 2.5/73.1 11.7/73.4 2.0/73.4 V. speedi 59.341 rpm 24.6/73.8 21/73.8 17.1/73.8 10/73.8 13.6/73.8 7/73.8 11.6/73.8 4.5/73.8 10.2/73.7 4.0/73.7 9.0/74.1 3.0/74.1 7 5 . 8 / 8 5 . 0 75.6/84.8 6.67X10"3 6.35x10 8.52 3.89 75.6/84.7 9.16 2.43 76.1/85.0 9.90 1.94 75.5/84.6 IO.29 1.84 75.5/84.6 10.51 I . 6 9 75.6/84.6 10.76 1.45 -9 75.0/84.1 75.5/84.5 75.5/85.0 75.0/84.2 75.2/84.4 75.5/84.5 75.5/84.5 75.5/84.6 75.7/84.8 75.5/84.5 75.5/84.8 75.0/84.5 75.7/84.8 75.5/85.0 75.6/85.5 75.0/85.0 74.9/85.0 74.5/84.5 74.2/84.5 7 4 . 5 / 8 5 . 5 74 .5/85.5 74 .5/85.5 7 4 . 5 / 8 5 . 4 74 .5/85.5 7 4 . 3 / 8 5 . 4 7.10 7.70 9.78 10.42 10.77 11.02 11.44 9.98 11.33 12.85 13.07 13.50 13.58 9.90 11.96 12.65 13.56 13.59 13.89 10.47 12.47 13.40 13.93 14.30 14.65 5.45 3.92 3.26 1.74 1.31 0.87 0.76 1.81 CO. 9.6 0.61 0.48 0.30 0.24 1.58 0.68 0.30 0.27 0.19 0.15 1 .03 O.49 0.34 0.22 0.19 0.15 75. 9 1 6 1 4 1 2 1 0 8 6 4 2 X - 1 0 3 F i g . 30 Trautman's plot of 0.02N HCl-modified gluten (heated at 121 C for 30 min). c o - u r n 9 y 2 / 2 , \ x = . _ (mg/ml cm ) Y = — ^ (mg sec /R ml) Xa<? 6JTXa 76 A • 18,720 rpm 28,034 rpm 38,092 rpm 48,055 rpm 59.3^1 rpm 1 6 X - 1 0 Y-1CT 1 F i g . 31 Trautman's plot of 0.05N HCl/-mod i f i e d gluten (heated at 121 C for 15 min). Co - Cm X = Xa' (mg/ml cm J Y U) 2Xa 2 2 (mg sec /R ml) tr-ie 14 12 10 8 6 4 2 X -10 3 F i g . 32 Trautman's p l o t of 0.IN HCl-modified gluten (heated at 121 C for 15 min). Co - Cm y x = . (mg/ml cm2!)) y » (mg sec 2/R 2 ml) Xa u) Xa 78' o 18,875 rpm • 28,178 rpm A 37.404 rpm • 48,072 rpm • 59,046 rpm 46 J 5 Y.101 2 16 14 12 10 8 6 X*10 3 T F i g . 33 Trautman's plot of 0.5N HCl-modified gluten (heated at 121 C for 15 min). Co - Cm Xa 2~— (mg/ml cm ) Y = U) 2Xa ^ — (mg sec 2/R 2 ml) 16 14 1 2 1 0 8 6 4 2 X * 1 0 3 F i g . 3 4 Trautman's plo t s of 1 . 0 . 0 2 N HCl-modified gluten 2 . O . 0 5 N HCl-modified gluten 3. G.1N HCl-modified gluten k. 0.5N HCl-modified gluten The heating time for 0 . 0 2 N HCl-modified gluten was 3 0 min at 1 2 1 C, for the other acid concentration was 15 min at 1 2 1 C. 80 The Trautman p l o t was used by Jones et al"*"1 to determine the molecular weight of gluten f r a c t i o n s . Information about the molecular weight d i s t r i b u t i o n which can be obtained from such p l o t s , assuming there i s no concentration dependence, are i l l u s t r a t e d i n Figure 35* I f a polymer i s monodisperse, a straight l i n e i s obtained (35a). The slope of the l i n e Y*/X* is equal to s/D, where s i s the sedimentation c o e f f i c i e n t and D the d i f f u s i o n constant. Molecular weight, Mw i s calculated by substituting i n the Svedberg equation Mw = RTs/D ( 1 - V J ) where R i s the gas constant, T the absolute temperature, V the p a r t i a l s p e c i f i c volume, and $ the density of the solution. A break i n the straight l i n e i s observed i f the solu-t i o n contains two polymers of d i f f e r e n t molecular weights (35b), weight average molecular weight, Mw i s calculated from the slope of the straight l i n e drawn between intercepts a and c. A pbly-disperse solution r e s u l t s i n a curve l i k e 35c again Mw can be calculated from the slope of the straight l i n e ac. As shown i n Figures 30 to 34, the Trautman plots f o r a l l the acid modified gluten samples revealed great heterogenei-ty and indicated the wide d i s t r i b u t i o n of d i f f e r e n t molecular weight components. Extrapolation of the curves to f i n d the intercepts then to calculate Mw i s d i f f i c u l t because a s l i g h t deviation i n the d i r e c t i o n of the extrapolated curves causes a great error i n c a l c u l a t i o n of Mw. The Y intercept at i t s high c x A B C Mono-component Two-component Multi-component Figure 35 Typical Trautman plots for various polymeric system with no concentration dependence (from Jones et a l ^ ). CO 82 coordinate p o s i t i o n which i s essential f o r the c a l c u l a t i o n can be obtained only from the run at very low centrifugation speed fo r very short time a f t e r reaching that speed. Under these conditions, the error i n reading the concentration curve i s usually high. 22 Erlander and Poster derived following two equations from the theory of A r c h i b a l d 0 , Klainer and K e g e l e s 3 3 and 50 Trautman ' s X (s/DL. c, r (S/DL c. ( S / D ) W = ^ 1 1 * * — ± - (1) SL G o Y* ( S / D ) = (2) (G y/G x)a»b» >£-X* where (S/D) = weight average value of S/D of the multicompo-nent system x = t o t a l r a d i c a l magnification G = magnification of the c y l i n d r i c a l lens times the magnification of the projector a' = o p t i c a l path of the c e l l b f =5 o p t i c a l lever arm = i n i t i a l number of moles per m i l l i l i t e r of solute at the meniscus C 0 = the i n i t i a l concentration at the meniscus i n grams per m i l l i l i t e r Y* = Aja tan©/cJ 2Xa a X* = [(X 3/3000G x 2$(tan6 ) A z ^ y ] / X f t 2 83 V GLf G » a', b', a l l are constant tinder c e r t a i n x y experimental condition, and ^ = « k C Q /. equation (2) can be rewritten as Y* (S/D) 8 8 „ — where A i s a constant (3) AC Q - X Prom equation (1) and (3), the following r e l a t i o n s h i p can be es t a b i l i s h e d : £ ( S / D ) . G, Y* (S/D) w » = 5 ( 4 ) C i A C 0 * X As we know.-i5:1, 1 M ; » k(S/D) w ^/V^ * Mj^&tt,, I M i = k i ( S / D ) i (S/D) i « = K±K± k J L i therefore Y* = K 1^ ( A C Q - X* ) K Z M ; i - C. = A K L H w C. =—± • X (5) C 0 In equation (5) » * K51M.C. when Y • 0, X = A K I H i C . / = AC r t when X* » 0, Y* = A K S M ^ = AK (M a C f t + + M c C c +-") The above t h e o r e t i c a l r e l a t i o n s h i p can be applied to explain the experimental r e s u l t s . When the Trautman p l o t i s plotted from the experimental r e s u l t s , (S/D) w and G (= G^ W C 2 + + ) are known as Y*l/X* and X*, the unknowns such as Clt C 2, Qy , M 1C 1, M 2C 2, M^C^,---- can be calculated from the p l o t . For (example, the Trautman plot of two components (a and b) system i s as ACB i n Figure (a) I F G Figure (b) Y*(AO) \ = — X (BO) B(M aC a • M bG b) Draw CD//BO intersects AO at D CE//AO intersects BO at E From D draw DF//BC intersects BO at F From E draw EG//AC intersects AO at G Then BF = AC a OF « AC, OD B * b C b AD s B M C a a and BF : OF = C : C,_ a b 85 In the case of a system of three component (a, b and c) mixture, Trautman plot i s as ADBC i n Figure (b). Y * U 0 ) , B ( \ c a * M b G b + M c C c ) X*(BO) A(G a + C b + C c) F i r s t connect AB and consider ABC as two components system, use the method stated i n the above example, draw EG and FH so that CG : GO = C . : C and AH : HO = M C : M.-. Cxl_. ab c L ; c c ao aD Then in^ABE, consider ADB as another two components system, use same method to draw E'G' and F'H', and also G',IFf CO. Then CI : IG : GO = G j G. : 0 a b c AH' : H»E : EO = MKC. : M C : M C b b a a c c In a system containing more components than three, the concentration r a t i o of components c a s C D : c c s C^: can be ~ obtained i n s i m i l a r way. Acid modified gluten samples are very complicated multicomponent systems. For the purpose of a rough comparison f o r the weight concentration r a t i o of components i n d i f f e r e n t molecular weight range, three slopes were drawn (to represent three d i f f e r e n t molecular weights a r b i t r a r y assigned) to the Trautman plots of 0.02N HC1 and 0.5N HC1 treated gluten samples. By using the method i l l u s t r a t e d i n Figures (a) and (b), the weight concentration r a t i o of three molecular Weight range were obtained (Figures 3° and 37) I r A, B, C represent large, medium and small molecu-l a r weight components respectively contained i n acid treated samples, from Figures 36 and 37, the weight concentration r a t i o 86 Figure 36 Weight concentration r a t i o analysis of the Trautman p l o t of 0.02N HC1-treated gluten, a, b, c are three slopes representing large, medium and small molecular weight a r b i t r a r i l y assigned. C a : C b : C c = AB : BC : CO = 3I.5 : 9.2 : 8.3 = 38 : 11 : 10 87 Figure 37 Weight concentration r a t i o analysis of the Trautman p l o t of 0.5N HC1-treated gluten, a, b, e are three slopes representing large, medium and small molecular weight a r b i t r a r i l y assigned. C a : C b : C e = AB : BC : CO = 13.9 : 9.7 : 10.6 = 13 : 9 : 10 88 of these two samples are as follows: a b c 0.02N HC1 treated gluten 38 11 10 0.5 N HC1 treated gluten 13 9 10 This weight concentration r a t i o f o r 0.05N and 0.1N HCl-treated gluten samples should be between the above two samples. The o r e t i c a l l y , the concentration r a t i o obtained from the Trautman p l o t should correlate to the concentration r a t i o observed from gel f i l t r a t i o n . As the AUC buffer which possesses a strong d i s s o c i a t i n g c a p a b i l i t y was used f o r gel f i l t r a t i o n , and a phosphate buffer was used f o r Trautman p l o t i n t h i s study, the two r e s u l t s are not d i r e c t l y comparable, however, the trend was sim i l a r between them implicating that 0.02N HC1 treated gluten contained larger molecular weight components than 0.5N HCl-treated gluten. The Trautman pl o t s of d i f f e r e n t acid-modified gluten and the analysis of the weight concentration r a t i o of Trautman plot s supplied adequate information about the molecular weight d i s t r i b u t i o n of these gluten samples. Among the four samples 0.02N HCl-treated gluten had the highest Y*/X* or S/D value 89 and highest high molecular weight component content, therefore i t has the largest weight average molecular weight. The 0.5N HCl-treated gluten, on the other hand, has the smallest weight average molecular weight among the four samples. This Y /X '•->, ^ or S/D value seems to be a function of the concentration of hydrochloric acid used f o r hydrolyzing the gluten. These results conformed to the r e s u l t s from gel f i l t r a t i o n i n d i c a t i n g that the higher the concentration of acid used to hydrolyze the gluten, the more extensive was the peptide linkage breakdown caused by the acid treatment. k* Determination of the Amino Groups Exposed by Acid- modification A. TUBS Method The technique of Fi e l d s ^ f o r measuring amino groups i n proteins and peptides was applied to d i r e c t l y prove that the breakdown of peptide chains of gluten occurred during acid-modification. The breakdown of peptide linkages i n gluten proteins should increase the free amino groups i n the hydrolysis products. per F i e l d s reported the molar absorptivity at 1+20 nm of c< - and(5-amino groups as 22,000 and 19,000/M cm, respective-l y . Using these f i g u r e s , the content of released amino groups was calculated and shown i n the Table 7. Determination of amino groups i n the untreated gluten was attempted without 90 Table 7 . Amino groups exposed by peptide hydrolysis i n acid modified gluten samples (autoclaved at 121 C) as determined by the TNBS method 1 2 Treatment Amino group content(moles/g. of protein) 0.02N HC1 0.1607 x 10" 3 0.05W HC1 0 . 1 6 6 1 ; 0.1 N HC1 0.1775 0.5 M HC1 0.2102 1 . 7 5 H acetic O.I63O 1 the heating time f o r 0.02N HC1 and 1.75N acetic acid was 30 min, f o r the other acid concentrations was 15 min. 2 average of t r i p l i c a t e samples 91 success since a l l of the solvents used to dissolve gluten (e.g. AUC, sodium deoxycholate) interfered with the TNBS colour production. As seen i n Table 6, there was a tendency that as the concentration of acid was increased f o r acid s o l u b i l i z a t i o n of gluten more amino groups to be exposed from gluten. This C. P supplies other evidence to prove the breakdown of the peptide linkages i n gluten during acid treatments. The e f f e c t of 1.75N acetic acid on gluten was s i m i l a r to that of 0.02N HG1, t h i s f i n d i n g agrees with the r e s u l t s obtained from gel f i l t r a t i o n . B. Ghloranil Test The mechanism of the c h l o r a n i l test i s not well under-stood. Al-Sulimany and Townshend used tyrosinase as an example of a protein which gave a s i m i l a r spectral peak to the amino acids. They reported that a solution containing lj.2 ppm of tyrosinase gave a maximal absorbance of O.lj.6 when i t was subjected to the recommended procedure. I f 130 i s used as the average molecular weight of an amino acid, the molar a b s o r p t i v i -ty at 350 nm f o r amino acids i s about ten to twenty times as much as the absorbance of the same amount of tyrosinase. This fa c t indicates that the colour complex developed by c h l o r a n i l i n 50% ethanol solution with amino acids and proteins i s probably due to the reaction of c h l o r a n i l with free o4- or £-amino groups. Therefore, the c h l o r a n i l test may be able to perform the same function as the TNBS method and also to detect the exposed amino 92 groups i n acid-modified gluten, thus providing a test of the breakdown of peptide chains during the heating process. Untreat-ed gluten can be dissolved i n the AUC solvent, and the c h l o r a n i l test would not be int e r f e r e d with the urea contained i n the AUC solution, t h i s i s a major advantage of t h i s method as compared to the TNBS method. Ten milligrams of the 0.02H-, 0.05N-, 0.1N-, 0.5N-HC1 and 1 .75N-acetic acid treated gluten samples were dissolved i n 15 mljof the AUC solvent. An aliquot of 0.5 ml of each sample solution was subjected to the c h l o r a n i l t e s t , and absorbance was recorded at 350 nm. The re s u l t s are presented i n Table 8. The r e s u l t s showed that the absorbance at 350 nm of the untreated and the 0.02N-, 0.05N- HCl and 1.75M acetic acid treated gluten* were close, but the absorbance of 0.1N and 0.5N HCl-treated gluten had considerably higher values. These r e s u l t s were i n agreement with the re s u l t s of the TNBS method, £hus providing:>";/\£\more evidence f o r extensive exposure of free amino groups from gluten as the concentration of acid i s increased f o r acid s o l u b i l i z a t i o n of gluten. 5. SS- and SH- Group Determination Pence and O l c o t t ^ observed that the v i s c o s i t y of solutions of gluten proteins decreased when the SS- bonds of the proteins were cleaved by the addition of reducing agents. Molecular weight studies on is o l a t e d wheat proteins have suggested that SS- bonds are responsible f o r the high molecular Table 8 The absorbance at 350 nm of 0.1% untreated and acid modified gluten solutions (the c h l o r a n i l test) 2 1 Treatment absorbance at 350 nm untreated gluten 2.91/' \.^C_:J1p^ 0.02N HC1 3.00 0.05N HC1 3.06 0.1 N HC1 3.5^ 0.5 N HC1 3.75 1.75N a c e t i c 2.9^ 1 Average of duplicate samples 2 Autoclaved samples; the heating time for 0.02N HG1 and 1.75N a c e t i c acid was 30 min, f o r the other acid concentrations was 15 min. weight and v i s c o s i t y of glutenin. When the SS- bonds of wheat glutenin are chemically cleaved by reduction with mereaptans, the molecular weight drops sharply from 2 5 0 , 0 0 0 to 2 0 , 0 0 0 and the cohesive-elastic nature of the protein i s destroyed. In contrast, the molecular weight of p u r i f i e d g l i a d i n i s l i t t l e affected by SS- bond disruption, because the molecules contain intramolecular SS- bonds which unite portions of the same poly-peptide chains. Since acid-modification of gluten dramatically reduced i t s v i s c o s i t y , increased i t s s o l u b i l i t y and i t s molecular weight, i t i s of inte r e s t to f i n d out i f these changes are re l a t e d to SS- bond disruption. The determination of SS- and 1 2 SH- groups was c a r r i e d out by the method of Beveridge et a l . Using a guanidine H 0 1 solution as a solvent to dissolve gluten or acid-modified gluten samples, the colour was developed with Ellman's reagent. The r e s u l t s are l i s t e d i n T a b l e d . There were no s i g n i f i c a n t changes i n the content of SS- groups of gluten as a r e s u l t of acid modification. No SH-groups were detectable i n any sample. Thus, i t i s u n l i k e l y that the loss of the cohesive-elastic nature, the increase of \} the s o l u b i l i t y and the decrease i n the molecular weight of the acid-modified gluten samples were due to the SS- bond disrup-t i o n . However these data are i n s u f f i c i e n t f o r denying the p o s s i b i l i t y of s p l i t t i n g of i n t e r SS- groups and formation of i n t r a SS- groups through SS-interchange. .95 Table 7 Sulfhydryl and d i s u l f i d e values of acid modified gluten samples Product SH (M/G dry weight) SS (M/G dry weight) untreated gluten undetectable 82 . 7 5 x 1° 0.02N HCl " 80.14 0.05N HCl " 79.08 0.1 N HCl " 78.04 0.5 N HCl " 81.96 1 average of duplicated samples , M/G = moles/gram of protein. 96 6. Amino Acid Composition The amino acid composition of acid modified gluten samples and untreated gluten are l i s t e d i n Table 10. No great damage occurred i n amino acids by the acid treatment. Under the severe condition of 0.5N HCl hydrolysis, a s l i g h t decrease i n glycine and tryptophan was observed. Wheat gluten has a E/T value (the r a t i o of es s e n t i a l amino acids to the amount of t o t a l amino acids) of 2.0 (grams essen t i a l amino acid per gram t o t a l nitrogen), which i s lower than the value of egg, 3.2, which i s about twice as much as needed f o r most e f f i c i e n t use of i t s es s e n t i a l amino acids . Amoung esse n t i a l amino acids of gluten, lysine i s the f i r s t l i m i t i n g amino acid with a low A/E value (the r a t i o of s p e c i f i c e s s e n t i a l amino acid to the sum of the es s e n t i a l amino acids). This n u t r i t i o n a l weakness can be improved by either combining other proteins to form a mixture with n u t r i t i o n a l value si m i l a r to animal protein or to supplement iifith free amino acids, whiohC. usually r e s u l t i n s i g n i f i c a n t improvement i n qual i t y . S o l u b i l i -zation of gluten enable these processes when necessary. Functional Properties The functional properties of proteins are the charac-t e r i s t i c s which allow them to perform special tasks when used i n the preparation of food products. The broad functional attributes provide food c h a r a c t e r i s t i c s such as s t a b i l i t y , texture, flavour, appearance and other factors that affect the Table 10 Amino acid composition of untreated and a c i d -modified gluten Amino acid content, g per lOOg Amino acids Un-treated gluten Ac id-modified gluten 1 0 . 0 2 N H C 1 0 . 0 5 N H C 1 0 . 5N H C 1 Aspartic acid 2 . 8 9 2 . 2 6 2 . 5 8 2.28 * Threonine 2 . 2 9 2.14 2.31 2 . 1 9 Serine 4 . 6 0 4 . 5 8 4 . 6 0 ^ . 3 3 Glutamic acid 35.81 35.^6 35. W 35.28 Proline 12.08 12.41 1 2 . 3 0 1 2 . 9 3 Glycine 2 . 9 6 2 . 7 2 2 . 7 8 1 . 7 2 Alanine 2 . 6 1 2.08 2.40 2.15 *Valine 3.08 2 . 9 2 3 . 0 7 3.19 * Methionine 1.25 1 . 1 0 1.33 1.04 Isoleucine 3 . 0 1 3.28 2 . 9 8 3 . 8 3 # Leucine 6.51 6 . 6 0 6 . 8 5 7.35 Tyrosine 3 . 1 7 3 . 0 9 3 . 1 9 2.58 * Phenylalanine 5.48 5.65 5 . 0 8 5 . 6 9 # Lysine 1 . 6 0 1 . 2 7 I . 3 6 1.18 Histidine 2 . 2 3 2.80 1 . 9 1 1 . 9 1 Tryptophan O .83 0 . 6 0 0.59 0.48 Arginine 3 . 6 8 3.35 3.^ 5 3 . 0 1 Cystine 2 . 2 1 2 . 1 7 2 . 3 4 2 . 2 9 1 the heating time for 0.02N HC1 was 30 min, for the other ac concentrations was 15 min at 121 C. essen t i a l amino acids. 98 properties of food. Functional properties of protein and protein i s o l a t e s i n food processing i s a major concern f o r s e l e c t i o n of a new food protein. Proteins i n various forms can serve as binders, thickeners, s t a b i l i z e r s , emulsifiers, g e l l i n g agents and moisture conditioners. The protein molecules are highly complex and can be processed into a wide range of materials with desirable functional properties. Wheat protein has s p e c i f i c functional properties required by the baking industry. Gluten adds strength to yeast raised baked goods and to paste products. The gas retention and water absorption properties can be used to advantage i n providing the required textural properties. However, as an emulsifier, gluten i s poor i n i t s performance. Due to the increasing cost of soyisolate and sodium caseinate, commonly used as emulsifiers, i t i s hopedV that the s o l u b i l i z e d gluten may improve the a b i l i t y of gluten as an emulsifier and therefore provide" a new p o t e n t i a l low cost emulsifying agent f o r the food industry. In t h i s study, emulsification capacity and s t a b i l i t y , foamability and foam s t a b i l i t y of acid-modified glutenCsamples were measured. 1. Emulsifying Capacity and the S t a b i l i t y Rating Table 11 l i s t e d the emulsifying capacity of the acid modified gluten samples, where soy i s o l a t e was used f o r compara-son. The emulsifying capacity was determined using a high speed cutting blender. Corn o i l was added continuously to the 0.75% gluten solution u n t i l the v i s c o s i t y increased and then decreased sharply because of the collapse of the emulsion. The t o t a l volume of o i l needed per 100 mg of gluten sample was recorded as emulsifying capacity. The s t a b i l i t y r a t i n g of o i l i n water emulsion formed by gluten and corn o i l was determined (Figure 12) by Acton and S a f f l e method 1, which modified the •- .' ; method of Titus et a l ^ . As seen i n Tables 11 and 12, untreated gluten had a very low emulsifying capacity and s t a b i l i t y r a t i n g . Both of these properties were remarkably improved by acid modifications. The gluten samples modified by 0.02K, 0.05N and 0.1N HC1 and 1.75N acetic acid demonstrated an emulsifying capacity equal to or even better than that of soy i s o l a t e s . The 0.5N HC1 and 3.5N acetic acid modified gluten samples showed an emulsifying capacity s l i g h t l y i n f e r i o r to soy protein i s o l a t e s , although f a r better than the untreated gluten. The acid modifications considerably improved the emulsion s t a b i l i t y as compared to the untreated gluten, and the 0.02N HC1 treated gluten showed almost the same emulsion s t a b i l i t y as soy i s o l a t e s (Table 12). 2. Foamability and Foam S t a b i l i t y The foamability of acid-modified gluten samples was measured as the increase i n volume of a 0.5% gluten solution lGt) Table 11 Emulsifying capacity of acid-modified gluten samples (autoclaved samples) Treatment ml of corn oil / 1 0 0 mg of protein' Soy i s o l a t e 31.85±.25 Untreated gluten 15.92±.46 0 . 0 2 N HC1 34.96±.67 0 . 0 5 N HC1 3 3 . 2 ^ . 5 § 0 . 1 N HC1 32.15±.51 0.5N HC1 2 9 . 9 0 1 . 6 7 acetic acid 32.15±.63 3.5N a c e t i c acid 30 .07± .5 l 1 the heating time for 0.02N HC1 and 1 . 7 5 N a c e t i c acid was 30 min, f o r other acid concentrations was 15 min. 2 at room temperature* average of t r i p l i c a t e samples and standard deviation. Table lij S t a b i l i t y r a t i n g of oil-in-water emulsions contain-ing 1% of various acid-modified gluten (autoclaved samples.) Type of protein Corn o i l l e v e l , • % Soy i s o l a t e Untreated gluten 0.02N HCl G . 0 5 N HCl 0.1N HCl 0.5N HCl 1.75N acetic acid 3.5N a c e t i c acid 16.7 33.3 S t a b i l i t y ^ Rating 2> 10.20 5.22 10.26 9.32 8.23 7.^9 24.19 15.31 24.83 21.37 19.83 18.52 50.0 70.53 47.32 70.84 67.59 54.21 50.30 61.59 59.35 1 the heating time f o r 0.02N HCl and 1.75N acetic acid was 30 min, f o r the other acid concentrations was 15 min. 2 Duplicated samples 'Q1Q2 upon the introduction of a i r bubbles by high speed beating. The drain volume and the speed of draining were used as a measure of foam s t a b i l i t y . A foam with small i n i t i a l drain volume and slow draining speed i s high i n i t s foam s t a b i l i t y . Table 13 shows the foamability and the foam s t a b i l i t y of untreated gluten and acid-modified gluten samples. The untreated gluten showed a better foamability than the acid-modified gluten with the exception of the pH 5 soluble f r a c t i o n s from 0.02N and 0.05N HG1 treated gluten. I t was found that the lower the concentration of the acid used f o r the modification the higher the foamability of the sample. Acetic acid-modified gluten had greater foamability than HCl-modified gluten. After introducing a i r bubbles Into the protein solution by high speed beating, the foam was immediately trans-ferred to a volumetric cylinder; the drain volume was recorded at G min and every 5> 10 or 20 min. With a s t a r t i n g volume of 50 ml, i t was found that the untreated gluten had 10 ml drain volume at 0 time and the drain volume reached the f i n a l volume af t e r 1+0 min, while the pH 5 soluble f r a c t i o n s of 0.02N and 0.05N HCl-treated gluten had no drain at 0 time and the drain volume reached the f i n a l volume afte r 80 min. Prom the r e s u l t s , i t i s clear that the pH 5 soluble protein f r a c t i o n of 0.02N and 0.05N HC1 treated gluten had an excellent foamability and foam s t a b i l i t y . McDonald and Pence-*7 reported that although the unmodified g l i a d i n had a better foamability than deamidized g l i a d i n ; as a foam agent, unmodified g l i a d i n performed poorly Table 13 Foamability and foam s t a b i l i t y of acid-modified gluten (autoclaved samples) Type of protein Foamability. % of volume increase Foam s t a b i l i t y , drain volume ( m l ) 1 0 min 5 10 20 30 40 50 60 80 Untreated gluten 460 10 25 30 40 44 46 46 46 46 0.02N HC1 400 10 25 30 38 42 44 *5 46 46 0.05N HC1 300 15 30 40 46 46 46 46 46 46 0.1N HC1 180 30 46 46 46 46 46 46 46 46 0.5N HC1 80 40 *5 46 46 46 46 46 46 46 0.02N HG1 pH 5 f r a c t i o n 560 0 7 15 25 30 35 38 40 46 G.G5N HC1 pH 5 f r a c t i o n 560 0 7 15 25 32 38 40 42 46 1.75 N acet i c acid 420 5 10 20 30 40 42 42 44 46 3.5N acetic acid 380 5 15 25 32 40 44 46 46 46 8.75N acet i c acid 340 10 20 36 40 46 46 46 46 46 1 s t a r t i n g volume 50 ml i n the food products such as cakes, dessert toppings, meringue and dessert s h e l l s , whereas deamidized g l i a d i n performed as good as egg white or even better. The same may be true f o r gluten used i n t h i s study, however, further studies are needed to c l a r i f y t h i s point. 1 0 5 GENERAL DISCUSSION In t h i s study, acid modification of gluten was carr i e d out with d i l u t e hydrochloric acid or acetic acid solutions. The r e s u l t s of the amide nitrogen analysis and gel f i l t r a t i o n revealed that acetic acid i s much milder than hydrochloric acid f o r deamidation. In spite of the mildness as a modifying agent which can be an advantage i n some cases, acetic acid had some disadvantages. F i r s t , acetic acid-modified gluten f a i l e d to show a sharp i s o e l e c t r i c point or a maximum p r e c i p i t a t i o n pH. It was necessary, therefore, to recover the soluble protein by d i a l y s i s or u l t r a f i l t r a t i o n which may be too c o s t l y f o r commer-c i a l a p p l i c a t i o n . E a r l i e r , i t was thought that the d i f f i c u l t y i n obtaining a sharp i s o e l e c t r i c point was due to the high concentration of acetic acid (1.75N, 3.5N, 8.75N) i n the solution. However, f l a s h evaporation to decrease the l e v e l of the acetic acid did not help the appearence of a d i s t i n c t i s o e l e c t r i c point. Secondly, acetic acid i s f a r more expensive than hydrochloric acid, and a much higher concentration i s needed f o r the modifica-t i o n process. I f acetic acid i s used f o r the modification of gluten i n industry, a rec y c l i n g system must be established to reduce the cost. Due to the s i m p l i c i t y i n processing, i . e. heating with d i l u t e hydrochloric acid followed by separation of protein by pH adjustment, i t seems to apply the HCl modifica-t i o n method to i n d u s t r i a l manufacturing of soluble gluten i s economically f e a s i b l e . After acid modification, 70-78% of the gluten protein can be recovered by a p r e c i p i t a t i o n method, and 15-20% goes to the whey. The whey protein can be recovered by a separate treatment, which i s being studied, or i t can be included i n the f i n a l product by the following procedure. After coolong of the autoclaved acid-gluten mixture, the pH of the mixture i s adjusted d i r e c t l y to 7«5» then the supernatant aft e r c e n t r i f u -gation i s freeze dried. The product contains most of the s o l u b i l i z e d protein with about 3-8% of sodium chloride. Although t h i s high s a l t content i n the product may r e s t r i c t i t s u t i l i z a t i o n i n food processing, no drawback of th i s procedure f o r recovering whole protein can be expected. The molecular weight d i s t r i b u t i o n has been studied by gel f i l t r a t i o n and Trautraan p l o t ; the c h l o r a n i l test was used to detect the small peptides and free amino acids formed during the modification process; the TNBS method and the c h l o r a n i l test were used to determine the amount of amino groups exposed by peptide hydrolysis. A l l the resu l t s showed a consistent trend related to the concentration of the acid used f o r the modification of gluten. These trends are shown i n Table I4. A l l of the re s u l t s from gel f i l t r a t i o n , Trautman p l o t , the c h l o r a n i l test and the TNBS method showed the break-down of peptide linkages of gluten proteins during the heating process with the degree of breakdown dependent on the concentra-tions of acid used i n the process. In the case of the 0.02N HCl treated gluten, which was autoclaved at 121 C f o r 30 min, the Table l : k Trends in molecular weight distribution, weight average molecular weight, released small peptides and free amino acids, and released amino groups of the HCl-modified gluten (autoclaved samples) 0.02N HCl 0.05N HCl 0.1N HCl 0.5N HCl Higher molecular weight components more Weight average molecular weight high Released small peptides, amino acids less Released amino groups less less low more more 1 the heating time for 0.02N HCl was JO min, for the other acid concentrations was 15 min. 108 amide content was reduced to around 10%, however, the r e s u l t s of gel f i l t r a t i o n and the c h l o r a n i l test indicated that the breakdown of peptide linkage had occurred. According to Holme 30 and Briggs^ , when the degree of deamidation was greater than 10%, the deamidized g l i a d i n became insoluble i n water at pH 3.8 and 0.01 io n i c strength, the p r e c i p i t a t e d p r o t e i n was no longer sticky and hydrated, and the protein became r e a d i l y soluble i n water or buffer solution at pH 7 and above. This i s probably due to the decrease i n the number of amide groups which are responsible f o r the intermolecular hydrogen bonding of the protein molecules as well as the increase i n charged groups (free carboxyl) i n protein. The same may be true i n t h i s study, that i s , the increased s o l u b i l i t y of the acid modified gluten i s mainly due to the deamidation of the protein, and the breakdown of the peptide linkages i s enhancing t h i s e f f e c t . New f u n c t i o n a l properties created by acid modification may help to expand the u t i l i z a t i o n of wheat p r o t e i n i n food industry. The s o l u b i l i z e d gluten may open up a new area f o r food processing such as food emulsification, food foam forma-t i o n and manufacturing of inexpensive and n u t r i t i o u s drink and coffee whitener. In some cases, high v i s c o s i t y of s o l u b i l i z e d gluten solution may be undesirable f o r u t i l i z a t i o n i n food manufacturing. More concentrated hydrochloric acid decreases the v i s c o s i t y , however, 0.1N i s the highest l i m i t f o r the acid treatment since browning starts when higher acid concentrations are used. 1'09 CONCLUSION i The purpose of th i s study was to search f o r the best condition f o r modifying gluten by a mild acid hydrolysis. The product should be soluble i n water with a bland flavour, the process causes the minimun change i n molecular size and the least damage i n n u t r i t i o n a l value, and with the economical f e a s i b i l i t y of application to the i n d u s t r i a l manufacturing. To meet these requirements, d i f f e r e n t heating conditions with d i f f e r e n t concentrations of hydrochloric acid and acetic acid were compared. The best condition was: a 5% gluten solution containing 0.02N HC1 i s autoclaved at 121 G f o r 30 min, or with 0.05N HC1 autoclaved f o r 15 min. The conclusion drawn from the study i s as follows: 1. After autoclaving with 0.02N HC1 at 121 C f o r 30 min or with 0.05N HC1 f o r 15 min, 78% of the o r i g i n a l gluten protein was recovered as s o l u b i l i z e d gluten by i s o e l e c t r i c p r e c i p i t a t i o n , and the o r i g i n a l cohesive and e l a s t i c properties of gluten disappeared. 2. Gel f i l t r a t i o n chromatography on a Sepharose 6B column showed that the concentration of acid used f o r hydrolysis affected the molecular size of the product, the higher the concentration of the acid the smaller the amount of higher molecular weight components i n the product. 3. The degradation of peptide linkage during the treatment was detected by increase i n free amino groups i n i i o protein, using the TNBS and the c h l o r a n i l t e s t . The TNBS method;and the c h l o r a n i l test showed the lowest free amino i groups i n the 0.02N HCl-treated gluten i n d i c a t i n g that t h i s gluten had longer peptide chains than other treated gluten samples. Trautraan p l o t supported the r e s u l t s of gel f i l t r a t i o n ; the 0.02N HCl-treated gluten showed the largest r a t i o of Y*/X*, which means the largest weight average molecular weight among a l l acid treated gluten samples. 5. The emulsifying capacity of gluten has been greatly improved by these acid treatments, 2.2 and 2.0 times that of the untreated by the 0.02N and 0.05N HCl treatments, respectively. 6. The foamability of gluten was deteriorated due to the acid treatments but only s l i g h t l y by the 0.02N and 0.05N HCl treatment. 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