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UBC Theses and Dissertations

Preparation and characterization of acid-solubilized wheat flour for use as a dairy substitute base Fung, Chi-Pun 1976

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PREPARATION AND CHARACTERIZATION OF ACID-SOLUBILIZED WHEAT FLOUR FOR USE AS A DAIRY SUBSTITUTE BASE by CHI-PUN FUNG B.Sc. (Hon.), University of Manitoba, 1971 M.P.H., University of C a l i f o r n i a , Los Angeles, 1972 A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of Master of Science i n the Department of FOOD SCIENCE Faculty of A g r i c u l t u r a l Sciences We accept t h i s thesis as conforming to the required standard ' THE UNIVERSITY OF BRITISH COLUMBIA June 1976 (c) Chi-Pun Fung, 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my writ ten permission. Department of SOENCB The Un ivers i ty of B r i t i s h Columbia 20 75 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date |5' J O i - / , i i ABSTRACT A main problem in preparing dairy substitute bases from wheat flour i s the insolubility of wheat gluten and starch. In this study, wheat flour was solubilized by acid hydrolysis. More than 90 percent of the dry weight of wheat flour solids were solubilized by autoclaving a 10 percent flour suspension in 0.1N hydrochloric acid at 121:*C for 15 minutes. The dried hydrolysate was similar in colour to the original flour and had excellent sol u b i l i t y . The apparent viscosity at 4*C of a 5 percent wheat flour hydrolysate was 4.9 centipoises as compared to 5*7 centipoises for pasteurized skimmilk. Proximate analysis of the dried hydrolysate showed the total carbohydrate and protein contents similar to those of the original flour. Gel f i l t r a t i o n of the wheat proteins eluted with AUC (an aqueous mixture of acetic acid, urea and cetyltrimethyl-ammonium bromide) revealed an extensive degradation of most of the glutenin and the high molecular weight gliadin into a protein fraction of 24,000 daltons during hydrolysis. The chloranil test did not indicate a significant increase in amino acids or in small molecular weight peptides. According to a Kjeldahl analysis subsequent to dialysis, ammonia cations accounted for 22$ of the total nitrogen, indicating almost complete deamidation during acid hydrolysis. Other nonprotein nitrogen was not detacted in the hydrolysate. ' i i i SDS-polyacrylamide gel electrophoresis showed disappearance of the t y p i c a l wheat protein "bands and appear-ance of 3 diffused bands of molecular weights 5 3 , 5 0 0 , 3 0 , 0 0 0 and 1 1 , 7 0 0 upon hydrolysis. The 24 , 0 0 0 daltons f r a c t i o n from Sephadex G -150 column appeared as 2 diffused "bands of molecular weights 3 0 , 0 0 0 and 1 1 , 7 0 0 . A high temperature gel f i l t r a t i o n chromatography on Biogel P-2 exhibited the following saccharide composition: 18 . 7 percent glucose, 1 1 . 3 percent maltose, 37 percent o l i g o -saccharides ( 3 - 7 glucose units) and 3 3 percent higher saccharide (more than 7 glucose u n i t s ) . A ge l f i l t r a t i o n chromatography on Sephadex G - £ 0 indicated only a trace amount of polysaccharides with more than 2 0 glucose u n i t s . No s i g n i f i c a n t destruction of ess e n t i a l amino acids during hydrolysis was observed. Using 0 . 5 percent protein solutions, the foamability of the hydrolysed wheat f l o u r was found to be s l i g h t l y better than that of casein, although the foam s t a b i l i t y was i n f e r i o r . A strong wheaty of f - f l a v o u r appeared a f t e r hydro-l y s i s . The most e f f e c t i v e method f o r decreasing t h i s o f f -flavour was a treatment with granular activated carbon at 90*C. A m i l k - l i k e beverage made by adding butter was acceptable i n taste, appearance and s t a b i l i t y . The protein to carbohydrate r a t i o of the product was lower than that of cow's milk. Add-i t i o n of other proteins or decreasing wheat starch by a sel e c t i v e washing of wheat f l o u r was suggested to improve the r a t i o . iv TABLE OF CONTENTS PAGE INTRODUCTION 1 LITERATURE REVIEW 4 A. Chemistry of wheat protein and starch 4 1. Soluble proteins 4 2. Gluten proteins 5 3. Carbohydrates 9 4. Lipids 12 B. Hydrolysis of wheat flour 12 MATERIALS AND METHODS 16 A. Materials 16 B. Preparation of acid-solubilized wheat flour 17 C. Viscosity measurement 17 D. Methods of proximate analysis 17 1. Carbohydrates 17 2. Protein 18 3. Moisture 19 4. Ash 19 E. Chloranil test 19 F. Ninhydrin test i 19 i G. Gel f i l t r a t i o n analysis 20 1. Sepharose 4B column 20 2. Sephadex G-150 superfine column 21 3. Sephadex G-150 column 22 4. Sephadex G-200 column 23 5. Sephadex G-100 column 23 V PAGE 6. Biogel P-2 column 24 7. Sephadex G-50 column 26 H. SDS-polyacrylamide gel electrophoresis 27 I. Analysis of ammonia, protein-nitrogen and nonprotein nitrogen 28 J . Amino acid analysis 29 K. Foamability and foam s t a b i l i t y 29 L. Flavour improvement of the s o l u b i l i z e d wheat f l o u r . ^ : 30 1. Solvent washing 30 2. Heated activated carbon treatment 3Q RESULTS AND DISCUSSION 31 A. Preparation of acid-hydrolysed wheat f l o u r 31 B. Proximate analysis 32 C. Apparent v i s c o s i t y 34 D. Gel f i l t r a t i o n analysis of the hydrolysate 34 E. G e l , f i l t r a t i o n analysis of protein 36 F. Analysis of ammonia, nonprotein and protein nitrogen 52 G. Gel f i l t r a t i o n analysis of carbohydrate 55 H. SDS-PAGE analysis 62 I. Amino acid analysis 67 J . Foamability and foam s t a b i l i t y 69 K. Flavour improvement 69 L. Imitation milk product 72 VI PAGE CONCLUSION 74 LITERATURE CITED 76 v i i LIST OF FIGURES FIGURE PAGE 1. Elution curve of proteins of hydrolysed wheat 35 flour eluted from Sepharose 4B column with phosphate buffer. 2. Elution curve of proteins of wheat flour 37 hydrolysed with higher acid concentration (0.15 N HC1).eluted from Sepharose 4B column with phosphate buffer. 3. Elution curve of unhydrolysed wheat starch 38 eluted from Sepharose 4B column with phosphate buffer. 4. Elution curve of carbohydrates of hydrolysed 39 wheat flour eluted from Sepharose 4B column with phosphate buffer. 5. Calibration curve of Sephadex G-150 (superfine) 40 column eluted with phosphate buffer. 6. Elution curve of proteins of hydrolysed wheat 43 flour eluted from Sephadex G-150 (superfine) column with phosphate buffer. 7. Calibration curve of Sephadex G-150 column 44 eluted with AUC. 8. Elution curve of wheat flour proteins eluted 47 from Sephadex G-150 column with AUC. 9. Elution curve of proteins of hydrolysed wheat 48 flour eluted from Sephadex G-150 column with AUC. 10. Cnloranil test of hydrolysed wheat flour 49 proteins eluted from Sephadex G-150 column in AUC. • v i i i FIGURE 11. 12. 13. 14. 15. 16. 17. 18. 19. PAGE Elution curve of proteins of hydrolysed wheat 51 flour eluted from Sephadex G-200 column with phosphate buffer. Ninhydrin test and Choranil test of hydrolysed 53 wheat flour proteins eluted from Sephadex G-200 column i n phosphate buffer. Gel f i l t r a t i o n elution curve on Sephadex G-100 56 column for the major carbohydrate peak of hydrolysed wheat flour collected from Sepharose 4B column. Eluant: phosphate buffer. Elution curve of carbohydrates of hydrolysed 58 wheat flour eluted from Biogel P-2 column with d i s t i l l e d water. > " Calibration curve for Biogel P-2 column using 60 corn syrup. i Elution curve of higher saccharides (G8 and 61 above) of hydrolysed wheat flour eluted from Sephadex G-50 (fine) column with Phosphate buffer. Calibration curve for proteins in SDS-polyacry- 63 lamide gel electrophoresis. SDS-Polyacrylamide Gel Electrophoretic Pattern 64 of the major peak of acid-hydrolysed wheat flour protein, whole acid-hydrolysed wheat flour protein and whole wheat flour protein. SDS-Polyacrylamide Gel Electrophoretic Pattern 66 of the major peak of acid-hydrolysed wheat flour protein, whole acid-hydrolysed wheat flour protein and whole wheat flour protein, with or without reduction prior to electro-phoresis. ix LIST OF TABLES TABLE PAGE 1. Proximate analysis of acid hydrolysed wheat 33 flour. 2. Proteins used in calibration of Sephadex 42 G-150 superfine column eluted with 0.01M phosphate buffer, pH 7. 3. Proteins used in calibration of Sephadex 45 G-150 column eluted with AUC. 4. Total-nitrogen, protein-nitrogen and ammonia- 5^ nitrogen content of the freeze-dried hydrolysed wheat flour. 5. Comparison of amino acid content of untreated 68 wheat flour and hydrolysed wheat flour. 6. Foamability and foam s t a b i l i t y of hydrolysed 70 wheat flour, casein and soy protein isolate solutions. X ACKNOWLEDGEMENT I would l i k e to express my deepest gratitude to Dr. S. Nakai of the Department of Food Science f o r h i s invaluable advice, constructive c r i t i s m and appropiate guidance during the course of t h i s study. I would also l i k e to thank the f a c u l t y members and graduate students i n the Department of Food Science 1 ; f o r t h e i r suggestions and assistance. 1 INTRODUCTION World average per capita protein i s expected to exceed average human nutritional requirement in 1980. Projections indicate there w i l l be a net positive world supply-demand balance of about 5»7 million metric tons of protein in I98O. Plant seed protein w i l l be a positive supply-demand balance of about 8.2 million metric tons and animal proteins a negative balance of about 2 .5 million metric tons. Wheat, coarse grains and oilseeds w i l l a l l be in general under supply (1). The short supply of animal proteins has pointed towards the direction of more rational use of vegetable proteins as supplement or partial replacements of animal proteins i n food. Since the tastes of people for their protein-rich food of animal origin i s not going to change radically, the preference w i l l s t i l l be for meats, dairy products, eggs, etc. Therefore, i f food demands are to be met by food producers, traditional products must be made available essentially in their commonly accepted form at a high level of nutritional quality and organoleptic appeal. Vegetable proteins, like soy protein has been successfully used to extend various types of red meats and poultry meat. In the dairy area, a number of substitute pro-ducts have been developed and marketed during the past 2 decades. Most of these products like f i l l e d milk, mellorine, 2 coffee whitener, simulated whipped toppings, simulated sour cream and simulated dressing or dip base, however, have proteins of dairy origin ( 2 ) . Recent realization of surplus plant protein and milk protein d e f i c i t has resulted in a worldwide research trend directed towards creation of dairy product analogues based f u l l y or partially on vegetable protein raw materials. Soy (3,4,5,6,7*8,9,10,11,12), peanut (13»14) and coconut (15»16,17) proteins are the centre of investigation for the development of a milk-like beverage. Among these three, soy protein has been most thoroughly studied. Indeed the f i r s t successful commercial venture in producing a vegetable-based, milk-like beverage is a sweetened soy milk under the name •Vitasoy* in Hong Kong (18). In the research of preparing cheese analogues from vegetable source, soy protein i s again the center of investigation with minor attempts i n using peanut protein to make cheese spread analogues. A study on the Canadian protein supply and demand showed that by 1980, Canada w i l l have a projected 3.2 million metric tons of plant protein surplus ( 1 ) . Over the last 14 years the domestic consumption of wheat was only 27% of the total production. The surplus wheat was exported and amounted to between 20 -25$ of the total world trade of wheat (19,20, 21 ,22) . Since this cereal i s rather well accepted by most people and i s readily available: in this country, inexpensive conversion of the bland floury endosperm to high quality 3 m i l k - l i k e beverage or other dairy substitutes would s i g n i f i -cantly broaden the u t i l i z a t i o n of t h i s cereal grain both at home and abroad. LITERATURE. REVIEW A. Chemistry of wheat protein and starch The wheat grain i s divided roughly into 3 parts i n the following proportions (a) germ 2?o, (b) endosperm 85# and (c) husk 13$. During m i l l i n g , the endosperm i s separated from the outer husks and germs together with the reduction of the endosperm into f l o u r which i s made up of agglomerates of the broken endosperm, free starch granules, broken starch and a broken proteinaceous matrix. There are 4 main classes of protein i n wheat flours albumin, globulin, g l i a d i n and glutenin. The water soluble albumin and s a l t soluble g l o b u l i n constitute about 15 - 20$ of the t o t a l f l o u r protein whereas the remaining 80 - 85$ i s the gluten protein consisting of g l i a d i n and glutenin. G l i a d i n i s soluble i n 70$ ethanol whereas glutenin i s not. These 2 gluten proteins are present i n roughly the same proportion. 1. Soluble Proteins Soluble proteins are f l o u r proteins soluble i n d i l u t e s a l t s o l u t i o n . They are composed of what i s known c l a s s i c a l l y as albumins and globulins together with minor quantities of glycoproteins, nucleoproteins and many of the l i p i d - p r o t e i n complexes found i n wheat f l o u r . S a l t soluble proteins contain about h a l f of a l l the f l o u r SH and 20$ of the SS groups (23) . The s a l t soluble proteins i n wheat f l o u r 5 are very heterogeneous. In a gel electrophoretic analysis, three different protein groups, with over 20 constituents were found in saline extract of wheat flour with distinct differences in distribution between Triticum vulgare and T. durum (24). 2. Gluten Proteins The gluten proteins are prepared traditionally as a wet elastic mass by kneading a dough in water to remove starch. Gluten can be separated into gliadin and glutenin fractions by the so l u b i l i t y of gliadin in 70% ethyl alcohol. The unusally high glutamic acid content and high proline content are distinctive features of a l l gluten proteins. About one of every three amino acids residues i s glutamine and about one of every seven residues is proline. Relatively large number of non-polar side chains contribute possibilities for apolar bonding. Few of the carboxyl groups of glutamic acid and aspartic acid side chains are free to ionize (25»26) and the low content of lysine, histidine and arginine results i n a low ionic character for the gluten proteins. Certain essential amino acids such as lysine, methionine and trypto-phan are present i n relatively small amount resulting in low nutritional quality of the proteins. The gluten proteins have strong aggregation tendencies resulting from the hydrogen-bonding potential of the unusually large number of glutamine side chains, the potential for apolar bonding of the many nonpolar side chains and their low ionic character. Conse-6 quently they are usually insoluble near t h e i r i s o e l e c t r i c points i n most aqueous solvents. The i s o e l e c t r i c point of the gluten proteins generally f a l l i n the pH range 6 - 9 (27,28). Gl i a d i n and glutenin d i f f e r i n t h e i r physical properties, most notably i n t h e i r v i s c o e l a s t i c i t y . G l i a d i n i s cohesive with low e l a s t i c i t y , whereas glutenin i s both cohesive and e l a s t i c (29). G l i a d i n i s composed of proteins of r e l a t i v e l y low molecular weight i n coraparision with the high molecular weight proteins of glutenin f r a c t i o n . These f r a c t i o n s also d i f f e r s l i g h t l y i n amino acid compositions g l i a d i n tends to have larger amount of proline, glutamic a c i d plus glutamine, cystine, isoleucine, phenylalanine and amide nitrogen than glutenin whereas glutenin has larger amount of glycine, l y s i n e and tryptophan (30). Molecular weight reported f o r various g l i a d i n preparations ranges from about 30,000 to 80,000. G l i a d i n molecules are made up of single polypeptide chains held i n a folded conformation by intramolecular d i s u l f i d e bonds (31). The glutenin i s often considered to be made up of two or more polypeptide chains joined by intermolecular and intramolecular d i s u l f i d e bonds. The molecular weights of glutenin components extend over a wide range and i n general are much higher than those of the g l i a d i n . Average molecular weight reported ranges from 150,000 to 3 m i l l i o n s (32). 7 Moving boundary electrophoresis of wheat gluten i n aluminum lactate buffer at pH 3.1 revealed 3 major peaks, ot, y8 , T and a fourth minor peak, Od (33). Electrophoresis i n starch g e l showed that the OL-component of gluten was a c t u a l l y made up of the ot-gliadins and the glutenin which was too large to migrate into the starch g e l (34)• From eight to t h i r t y g l i a d i n components were detected using starch gel e l e c t r o -phoresis (34,35). The g l i a d i n components were eluted as a single peak i n gel f i l t r a t i o n . Evidently the components which were shown to have d i f f e r e n t m o b i l i t i e s upon gel electropho-r e s i s had approximately the same size (36). Attempts to determine molecular weights of g l i a d i n components on columns c a l i b r a t e d with proteins of known molecular weight yielded a range from 25,000 to 50,000 (37,38,39). The molecular weight of the d i f f e r e n t g l i a d i n components has been extensively studied by various techniques. Molecular weight of the ot-gliadin f r a c t i o n was found to be 49,000 by gel f i l t r a t i o n and 55,000 from l i g h t s c a t t e r i n g (38). SDS-PAGE analysis of the ot-gliadin f r a c t i o n revealed 6 bands ranging i n molecular weight from about 30,000 to 75,000 with 2 major bands at 32,000 and 36,000 (40), Molecular weight of the ^ - g l i a d i n f r a c t i o n was found to be 31,000 by ul t r a c e n t r i f u g a t i o n (41). SDS-PAGE analysis of the j8-gliadin f r a c t i o n showed 3 major species and several minor ones. The major species l i e d i n the molecular weight range of 36,000 and 40,000 (40). Minimum molecular weights of T , - , 7k-, and . 8 % -gliadins were estimated to be 16,00, 17»000 and 18,000 respectively from amino acid analysis (42). Using ul t r a -centrifugation, different molecular weights have been reported for r gliadin in different solvents. Values of 26,000 (43), 31,000 (44) and 37.000 (41) were reported for r gliadin in 4M guanidine hydrochloride, i n 6M guanidine hydrochloride and in aluminum lactate buffer respectively. Using sedimentation in a variety of solvents, Sexson and Wu assigned molecular weight of 30,300 and 34,700 to 7[- and % -gliadins respectively (45). SDS-PAGE analysis showed that the main constituents of the T-gliadin fraction were within a molecular weight range of 38,000 to 44,000 (40). Booth and Ewart (46) separated three 00 -protein components from Wichita flour and one from Cappelle flour by ion exchange chromatography on carboxylmethylcellulose. These proteins had very high content of proline and phenyla-lanine but no cystine or methionine. Molecular weight of the 2 Wichita proteins was found to be 73»000 and 7^.000 by ultra-cent r i f ugation. These values were consistent with the subunit molecular weight of 69,300 and 78,100 for 0)-gliadin as deter-mined by SDS-PAGE analysis (47). The gliadin peak separated by gel f i l t r a t i o n gave rise to 3 bands i n SDS polyacrylamide gel electrophoresis corresponding to molecular weights 46,000, 38,000 and 33,000 (48). SDS-polyacrylamide gel electrophoresis of whole gliadin prepared by alcohol extraction revealed that most gliadin proteins have molecular weight near 36,500 (47). The whole gliadin fraction also contained 11,400 molecular 9 weight polypeptides, a major.polypeptide of molecular weight 44,200 and W-gliadins. Glutenin constituted approximately 30 - 40$ of the protein in wheat flour and about half of that in gluten. Glutenin can be extracted from wheat flour with dilute acetic acid after previous extraction with saline and 70% alcohol. Some glutenin remained as gel protein and could be extracted with solution of mercuric chloride, dissociating agent (urea or guanidine hydrochloride), detergents, a l k a l i or reducing agents (49). Although glutenin migrated as a single component in moving boundary electrophoresis with a mobility equivalent to the fastest moving gliadin component (33), i"t was eluted f i r s t when a solution of gluten protein was applied to the gel column (36). SDS-polyacrylamide gel electrophoresis of reduced glutenin prepared by extracting gluten in 70% ethanol which was 0.01N in acetic acid and precipitating at pH 6 .5 , showed that glutenin was composed of subunits of at least 15 distinct molecular weights ranging from 11,600 to 133,000 (47). In another study, SDS-polyacrylamide gel electrophoresis of reduced glutenin prepared by gel f i l t r a t i o n on Sephadex G-100 column showed at least 8 bands (48). 3. Carbohydrates The carbohydrates of wheat flour are made up of starch, pentosan, hemicellulose, cellulose and sugars. Analysis of a low extraction flour with 73•&% carbohydrate 10 showed that there were 70.4$ starch, 2$ pentosan, 0.9$ sugars and 0.5$ c e l l u l o s e (49). The sugars i n wheat f l o u r are composed of d i f f e r e n t low molecular weight saccharides, glucose, fructose, sucrose, maltose, r a f f i n o s e and a series of oligosaccharides composed of D-fructose and D-glucose referred to levosine or gluco-fructans (50,51,52,53). Pentosan and hemicellulose belong to a group of non-starchy polysaccharide. Pentosan i s usually referred to as the water soluble and hemicellulose as the water insoluble pentose-containing polysaccharide. Upon hydrolysis, the hemi-ce l l u l o s e s and pentosans y i e l d derivatives of pentoses and hexoses. The monomeric units most frequently found i n the 2 polysaccharides are the pentose sugars D-xylose and L-arabinose. The major portion of the soluble pentosans i s a straight chain of anhydro-D-xylopyranosyl residues linked beta-1, 4 to which are attached anhydro L-arabinofuranosyl residues at the 2 or 3 positions of i n d i v i d u a l anhydro-xylose units (54). The b u i l d -ing blocks of insoluble pentosan or hemicellulose are s i m i l a r to those of soluble pentosan (55)« The water-soluble and water insoluble.pentosans were s t r u c t u r a l l y s i m i l a r and the main difference was the greater degree of branching i n the l a t t e r (56). The unique branching structure of water soluble pentosan polymers i s responsible f o r t h e i r a b i l i t y to form solutions of high v i s c o s i t y and t h e i r a b i l i t y to imbibe consi-derable quantities of water. 11 Starch i s the major component of wheat endosperm and hence, of the flour prepared from i t . Starch i s made up of 2 types of molecules, amylose, a linear or straight chain poly-saccharides and amylopectin, a branch polysaccharide. Both polysaccharides are polymers of D-glucose. The glucose units i n amylose are joined by ot-1, 4 glucosidic bonds. In amylo-pectin, ot-l, 4 bonds also predominate, but branching i s intro-duced by ot-1, 6 linkages (57) . There i s a branch, on the average for every 20 to 25 glucose units of the amylopectin molecule (58). Both amylose and amylopectin contain molecules with a wide range of sizes and values obtained from molecular weight determination are averages only. Molecular weights of wheat amylose and wheat amylopectin as determined by osmotic pressure measurement has been reported to be 140,000 and 4,000,000 respectively (59)• Amylose content of wheat starch ranges from 23.4$ to 27.5$ (60). Wheat starch exists in i t s native state as discrete microscopic granules. The starch granules i s composed of amylose and amylopectin molecules associated by hydrogen bonding either directly or through water hydrate bridges to form radially oriented, micelles or crystalline area of various degrees of order. An intercon-nected three dimensional micellar l a t t i c e i s formed by the participitation of segments of individual molecules in several micellar areas. As a result, wheat starch i s insoluble in cold water although the starch molecule i s highly hydroxylated and therefore very hygroscopic. Starch granules however 12 exhibit a l i m i t e d capacity f o r absorbing water and swell r e v e r s i b l y . 4. L i p i d s Commercial straight grade f l o u r contains less than 2$ t o t a l l i p i d s . Only a part of the l i p i d s , the free l i p i d s can be extracted from wheat with the usual nonpolar f a t solvents such as petroleum ether. Polar solvent l i k e water-saturated n-butanol can extract the more d i f f i c u l t l y extracted bound l i p i d s as w e l l . About 1.5$ f l o u r has been removed by water-saturated butanol (6l,62). Of t h i s 1.5$ t o t a l l i p i d , about two-thirds was free and one-third bound. About h a l f of the t o t a l l i p i d was polar and almost a l l the bound l i p i d s f e l l i n t o t h i s category. Thin layer chromatography resolved the t o t a l l i p i d f r a c t i o n into 23 l i p i d classes (63). Wheat f l o u r l i p i d consisted of a spectrum of compounds ranging from very nonpolar substances, such as s t e r o l s and t r i g l y c e r i d e s , to neutral polar l i p i d s , such as galactoglycerides and charged phospholipids (64). B. Hydrolysis of wheat f l o u r Wheat f l o u r i s unsuitable f o r d i r e c t use as a dairy substitute base because of the i n s o l u b i l i t y , the dough-forming e l a s t i c properties and the i s o e l e c t r i c point of 6.6 of the gluten proteins (65)• In addition, wheat starch exists as 13 granules i n wheat f l o u r and i s insoluble i n cold water. The starch s o l u b i l i z e d by heating or chemical treatment s t i l l ex-h i b i t s - high v i s c o s i t y at low s o l i d content. Wheat f l o u r can be s o l u b i l i z e d by enzyme or acid h y d r o l y s i s . In the enzymatic process, a p r o t e o l y t i c enzyme i s required to break down the gluten and a carbohydrase to break down the wheat starch. Wheat gluten can be rendered e a s i l y d i s p e r s i b l e by a v a r i e t y of p r o t e o l y t i c enzymes, but broad spectrum enzymes l i k e papain y i e l d an undesirable increase i n small fragments and free amino groups (65). The l a t t e r potentiates toward flavour changes, such as from glutamate, and to storage i n s t a b i l i t y by way of the M a i l l a r d browning reaction. Pepsin treatment produced larger fragments and thus r e l a t i v e l y small amount of free amino groups (66). A scheme i n preparing a m i l k - l i k e product from enzymatic hydrolysis of wheat f l o u r has been proposed (65). In t h i s scheme, a 50% f l o u r suspension i n 0.04-5N HC1 was incubated with 10 ppm pepsin f o r 2 to 4 hours at 30*C. The supernatant contained most of the protein. The starch containing p r e c i - . pi t a t e was washed and centrifuged. About 30% of the c e n t r i -fuged starch was treated with ot.-amylase, heated at 70 *C f o r 30 minutes and f i n a l l y heated to 90 *C f o r another 30 minutes to destroy the enzyme. The neutralized pepsin-hydrochloric a c i d extract of wheat f l o u r was mixed with the OL-amylase treated starch f r a c t i o n and a s t a b i l i z e r , polysaccharide gum B-14-59 was added at a l e v e l of 0.7 to 1%. The enzyme pepsin 14 produces an excellent type and degree of hydrolysis but suffers from the disadvantages of restricted supply and the low pH condition required for effective action. Low pH during pro-cessing i s undesirable because i t may lead to a bitter flavour and a somewhat astringent mouthful in the f i n a l product (67). A neutral protease prepared from Bacillus subtilis has been used for the desirable type of limited hydrolysis of flour protein without recourse to low pH conditions. Up to 85$ of flour protein was rendered dispersible and dialyzable nitrogen was as low as 10$ (67). Solubilization of wheat flour for milk-like products using acid hydrolysis has not been reported. The amide groups on wheat protein are more readily susceptible to acid hydro-l y s i s than the peptide linkages. Mild hydrolyzing conditions can break off ammonia from the amide to a considerable degree without appreciable hydrolysis of the peptide chain. Such mild and limited hydrolysis of the amide group would increase the so l u b i l i t y and decrease relative interacting tendencies of the protein (68). Deamidation of a 1$ gliadin solution was carried out at 100'C in 0.008N to 0.04N hydrochloric ac id468) . N6 hydrolysis of the peptide bonds was observed even when over 50$ of'*the amide groups were hydrolysed. When the degree of hydrolysis of the amide was greater than 10$, gliadin protein became readily soluble in water at pH ?. Deamidation of gliadin was also carried by heating a 5.5$ gliadin solution in 0.07N hydrochloric acid for 3 hours at 96*-98,G (69). 15 Mild acid hydrolysis has also "been used to s o l u b i l i z e wheat gluten (70). Gluten was s o l u b i l i z e d when the amide content was reduced by approximately 10$. Soluble gluten was recovered at 85 - 92% y i e l d . Acetic acid was found to be milder than hydrochloric a c i d f o r the gluten modification. Increased free amino groups were detected i n the s o l u b i l i z e d gluten as the concentration of acid increased showing an increased breakage of the polypeptide chain. The acid hydrolysis of starch i s we l l established i n the glucose syrup and sugar industry. When starch i s hydro-lysed with acid as the ca t a l y s t , a random cleavage of the -C-O-C linkage occurs with the production of glucose and many of i t s polymers. Upon continuing the hydrolysis there i s an increase i n the number of the lower molecular weight sugars. The extent of conversion depends on acid concentration, time, temperature and pressure during the reaction. Acid conversion has a p r a c t i c a l top l i m i t of 55 Dextrose Equivalence with 32$ dextrose because above t h i s value there i s much colour develop-ment and appearance of a b i t t e r taste element (71) . Starch hydrolysis i s complicated by secondary reversion reaction. When breakdown has progressed to a certa i n l e v e l , some of the sugars formed are themselves converted to unwanted substances which cause colour and b i t t e r t a s t e . Use of enzymes i n the hydrolysis eliminate the production of reversion and thus high D. E. syrup can be produced. 16 MATERIALS. AND METHODS A. Materials Wheat f l o u r was a commercial brand of Robin Hood A l l -Purpose Enriched Flour. Wheat starch was a p u r i f i e d product from MCB. Casein was a s o l u b i l i z e d and freeze-dried sample prepared i n the laboratory. Soy protein i s o l a t e was a product of General M i l l Inc. L i l y White com syrup was a commercial product from Best Foods. The standard proteins used i n t h i s study and t h e i r sources were: ovalbumin (5x c r y s t a l l i z e d ) was a product of f»; • Calbiochem} ovomucoid and lysozyme were from Worthington Bio-chemical Corporation; conalbumin and y S-lactoglobulin (bovine) were from N u t r i t i o n a l Biochemicals Corporation. Chymotryp-sinogen A (bovine pancreas, 6x c r y s t a l l i z e d ) , cytochrome C (horse heart) and myoglobin (sperm whale) were from Schwartz-Mann Biochemical. r-Globulin (human, Cohn f r a c t i o n I I ) , serum albumin (bovine, f r a c t i o n V), t r y p s i n (bovine, type XI), t r y p s i n i n h i b i t o r (soybean, type 1-S), ribonuclease A (bovine pancreas, type 1-A, 5x c r y s t a l l i z e d ) , oi-chymotrypsin (bovine pancreas, type I I , 3x c r y s t a l l i z e d ) , catalase (bovine l i v e r ) and thyroglobulin (bovine, type 1) were purchased from Sigma Chemical Co. The Blue Dextran 2000, the Sepharose 4B gels and the Sephadex G-series gels, G-50 f i n e , G-100, G-150, G-150 super-f i n e and G-200 were the products of Pharmacia Fine Chemicals. 17 Biogel P-2 minus 400 mesh, was from Bio-Rad Laboratories. B. Preparation of acid-solubilized wheat flour Ten grams of wheat flour was suspended in 100 ml of 0.1N HCI. in a 1000 ml erlenmeyer flask. The suspension was heated to 121*C for 15 minutes i n a Barnstead Autoclave. The coming up time of the autoclave was about 15 minutes and the coming down time was about 2 minutes (using vent dry instead of liquid cool at the end of autoclaving). The autoclaved sample was cooled rapidly, adjusted to pH 7 with concentrated NaOH solution and centrifuged at 8000 x g for 15 minutes. The supernatant was freeze-dried. The extent of solubilization was determined from the weight of the freeze dried residue. C. Viscosity measurement The viscosity of a 5$ solution of the hydrolysed wheat flour and a commercial skimmilk sample was measured with a Haake Rotovisko Viscometer. The viscosity was measured at 5 different shear rates ranging from 152 to 1370 sec" 1 using an MV1 spindle. The sample temperature was held at 4*C by a thermostated water jacket surrounding the measuring head. D. Methods of proximate analysis (1) Carbohydrates The phenol-sulfuric acid method of Dubois (72) was used for carbohydrate determination. To a test tube containing 18 2 ml of carbohydrate, 0.1 ml of 80$ phenol was added followed by a rapid addition of 5 ml of concentrated s u l f u r i c a c i d from a 50 ml burette, with the stream of acid directed against the l i q u i d surface. The tes t tube was allowed to stand f o r 10 minutes before the content was thoroughly mixed. Upon cooling down, the absorbance of the reaction mixture was measured at 4-90 nm against a blank (without carbohydrate) i n a 1 cm l i g h t path cuvette with a Beckman DB spectrophotometer. The amount of carbohydrate (glucose or glucose polymers) was determined by r e f e r r i n g to a glucose standard curve and with adjustment based on the degree of polymerization of the glucose polymers. A l l samples were prepared and determined i n t r i p l i c a t e . To prepare:a wheat f l o u r s o l u t i o n f o r carbohydrate determination, about 0.4 gram of wheat f l o u r was exactly weighed out and dissolved i n a minimum amount of IN NaOH. The al k a l i n e s o l u t i o n was neu t r a l i z e d with concentrated s u l f u r i c a c i d and was c a r e f u l l y made up to 2 l i t r e s i n a volumetric f l a s k with d i s t i l l e d water. (2) Protein Nitrogen content of the untreated wheat f l o u r and the hydrolysed wheat f l o u r was determined by a rapid micro-Kjeldahl procedure (73)• The protein content was calculated by multiplying the amount of nitrogen by a fact o r of 5«7. 19 (3) Moisture Moisture content of the untreated wheat f l o u r and the hydrolysed wheat f l o u r was determined by heating a 10 gram sample at 90'G i n a i r oven u n t i l constant weight was obtained. (4) Ash Ash content was determined by ashing 1 gram sample i n a type 1300 Thermolyne E l e c t r i c Furnace at 600*C f o r 6 hours or u n t i l constant weight reached. E. C h l o r a n i l test The c h o l o r a n i l test (74) was used to determine micro-gram amount of amino acids and small peptides eluted from gel f i l t r a t i o n column. To 1 ml of each f r a c t i o n , 2 ml of c h l o r a n i l reagent (ethanol saturated with c h l o r a n i l ) , 2 ml of borate buffer (0.05M sodium borate solution, pH 9) and 5 ml of d i s t i l l e d water were added. The solution was thoroughly mixed and incubated at 65 1*C f o r 1 hour. Absorbance was measured at 350 nm i n a 1 cm l i g h t path cuvette against blank prepared i n the same way without sample. F. Ninhvdrin test The ninhydrin test was used to detect ammonia, amino acids and small peptides which emerged as the nonprotein nitrogen peak i n gel f i l t r a t i o n . Ninhydrin reagent was pre-pared by d i s s o l v i n g 1 gram of ninhydrin and 0.15 gram of 20 hydrindatin in 37.5 nil methyl cellosolve with pH adjusted to 5.5 by adding 12.5 ml of 4 M sodium acetate buffer. One ml of ninhydrin reagent was mixed with 1 ml of sample solution and was heated in a boiling water bath for 15 minutes. The mixture was diluted with 3 ml of 5$ ethanol, mixed and absorbance measured at 570 nm in a 1 cm light path cuvette with a Beckman DB spectrophotometer against blank prepared in the same way without sample. G. Gel f i l t r a t i o n analysis (1) Sepharose 4B column (4.0 x 70.0 cm) The column was prepacked and was equilibrated with phosphate buffer before sample application. A 4 ml aliquot was applied and the column was eluted with phosphate buffer at a flow rate of 30 ml per hour. Fract-ions of 4 ml were collected. The elution profile of protein was obtained by measuring absorbance of each fraction at 280 nm. The elution profile of carbohydrate was obtained by determining the carbohydrate content of each fraction by the phenol-sul-furic acid method. # Eluants used in gel f i l t r a t i o n analysis were d i s t i l l e d water, 0.01 M phosphate buffer at pH 7 with 0.02$ sodium azide and an aqeous dissociating medium of 0.1 M acetic acid, 3 M urea and 0.01 M cetyltrimethylammoniura bromide (AUC). The frac-tions were collected in a Gilson Medical Electronics fraction collector. Absorbance was measured-in a 1 cm light path cuv-ette with a Beckman DB spectrophotometer. 21 A soluble wheat starch sample was prepared by d i s -s o l v i n g 20 mg of wheat starch i n 8 ml of IN NaOH. The a l k a l i n e s o l u t i o n was neutralized with concentrated s u l f u r i c acid and was made up to 10 ml with d i s t i l l e d water. A standardization run was c a r r i e d out using a sample of 10 mg Blue Dextran 2000, 2 mg tryptophan and 5 mg thyro-g l o b u l i n d i s s o l v i n g i n 4 ml of phosphate buffer. Void volume and t o t a l bed volume were estimated from the e l u t i o n volume of the Blue Dextran 2000 and tryptophan r e s p e c t i v e l y . (2) Sephadex G-150 superfine column (2.5 x 45.0 cm) F i f t e e n grams of dry Sephadex-G-150 superfine sus-pended i n 1 l i t r e of d i s t i l l e d water was allowed to swell i n a b o i l i n g water bath f o r 5 hours. The swollen gel suspension was cooled, deaerated and packed i n a 2.5 x 45.0 cm Pharmacia Column using an extension tube and following the procedure recommended by the manufacturer. An operating pressure of 15 cm of water was employed during packing. The packed column was eq u i l i b r a t e d f o r 2 days with phosphate buffer i n an upward flow d i r e c t i o n . The operating pressure was adjusted to give a flow rate of, about 10 ml per hour during e q u i l i b r a t i o n and i n subsequent runs. The column was operated i n an upward flow d i r e c t i o n . The i n l e t tubing was joined to a 3-way valve which was connec-ted to a one l i t r e mariotte f l a s k f i l l e d with phosphate buffer and to a 5 ml syringe tube. Sample of 3 ml was applied to the column v i a the syringe. The eluant was c o l l e c t e d i n 2 ml 22 fractions immediately after the sample drained clear of the syringe. The column was eluted with another 3 ml of phosphate buffer from the syringe before started eluting from the mariotte flask. A total of 130 fractions were collected. The elution profile of protein was obtained by measuring the absorbance of each fraction at 280 nm. The column was calibrated with standard proteins. Void volume was estimated from elution volume of Blue Dextran 2000. (3) Sephadex G-150 column (2.5 x 45.0 cm) Fifteen grams of dry Sephadex G-150 was suspended in 1 l i t r e of AUC and was allowed to swell at room temperature for 3 days with occasional s t i r r i n g . The swollen gel was deaerated and packed as in the previous column. The packed column was equilibrated for 2 days with AUC in an upward flow direction. A flow rate of about 20 ml per hour was maintained during equilibration and in subsequent runs. The column set up, sample application, fraction collection and protein detec-tion were the same as in the previous column except that AUC was used both as eluant and as sample solvent. An AUC wheat flour extract was prepared by blending 4 grams of wheat flour with 69 ml of AUC for 2 minutes in a Sorvall omnimixer operated at the top speed. After standing 1 at 25*C for 2 hours, the blended mixture was centrifuged at 35,000 x g for 30 minutes. A 3 ml aliquot of the deaerated supernatant was applied to the column. 23 An AUC extract of the freeze-dried hydrolysed wheat flour was prepared similarly. Again a 3 ml aliquot of the deaerated supernatant was applied to the column. Protein eluted from this run was monitored both by absorbance at 280 nra and by the chloranil test. The column was calibrated with standard proteins. Void Volume was estimated from the elution volume of Blue Dextran 2000. The*Blue Dextran 2000 dissolved very slowly in AUC. Thus i n preparing this sample, the compound was dissolved i n water f i r s t , followed by the addition of calculated amount of acetic acid, urea and cetyltrimethylammonium bromide. (4) Sephadex G-200 column (2.5 x 100,0 cm) The upward flow column was packed and calibrated.** A sample containing 0.3 gram of the freeze-dried hydrolysed wheat flour in 3 ml of phosphate buffer was applied to the column. The column was eluted with phosphate buffer from a reservoir at a flow rate of 25 ml per hour. A total of 120 four-ml fractions were collected. The absorbance of each fraction was measured at 280 nm. One m i l l i l i t r e from each fraction was used for the ninhydrin test. Another m i l l i l i t r e from each of the fractions within the absorbance peak was used for the chloranil test. (5) Sephadex G-100 column (2.5 x 100.0 cm) The upward flow column was packed and calibrated.** ** The column was prepared and calibrated by Mr. C. Y. Ma, Dept. of Food Science, U. B. C. 24 The major carbohydrate peak of hydrolysed wheat f l o u r eluted from the Sepharose 4B column was pooled and freeze-dried. A sample containing 5 mg of the freeze-dried product dissolved i n 5 ml of phosphate buffer was applied to the column. The column was eluted with phosphate buffer from a r e s e r v o i r . A t o t a l of one hundred 5-ml f r a c t i o n s were c o l l e c t e d . Two m i l l i -l i t r e s from each f r a c t i o n was transferred to a te s t tube f o r carbohydrate determination by phenol-sulfuric acid method. (6) Biogel P-2 Column About 200 grams of dry g e l was shaken through 2 sieves, 200 mesh and 325 mesh, such that 160 grams of dry gel of p a r t i c l e size between 43 - 74/*. were obtained. The gel was allowed to swell over night i n excess d i s t i l l e d water. After hydration, the gel was fractionated by repeated s e t t l i n g and decanting the fin e s u n t i l a sharp zone of s e t t l i n g g e l p a r t i -cles was attained. The gel s l u r r y was deaerated before packing. The chromatographic column was made up of 2 jacketed pyrex glass tubes (1.5 x 100.0 cm) pulled out at the bottom and plugged on the top by rubber stoppers. There were 2 d r i l l e d openings on the rubber stopper of the f i r s t tube, one f o r sample a p p l i c a t i o n and the other was the i n l e t f o r eluant from the r e s e r v o i r . The lower end of the f i r s t tube was joined to the top of the second, tube by narrow p l a s t i c tubing: so as to give an e f f e c t i v e column length of 200 cm. The 2 chromatographic tubes were jacketed by 2 larger tubes (4.4 x. 95.0 cm) f i t t e d with d r i l l e d rubber stoppers at both ends. 25 The 2 tubes were packed i n d i v i d u a l l y before connect-ing. Each tube was f i r s t f i l l e d to h a l f i t s length with deaerated d i s t i l l e d water. Deaerated gel suspension was then poured into the tube... When a ge l layer of 2 - 4 cm was formed at the bottom of the tube, a slow stream of water was allowed to flow-out. More gel suspension was poured into the tube as the water l e v e l f e l l u n t i l the tube was packed to about 1 cm from the top. The 2.packed tubes were connected. Tight and close packing was achieved by pumping a solut i o n of 0.1$ NaCI with a Milton Roy Minipump at a flow rate of 40 ml per hour. Af t e r compacting, further addition of the ge l suspension was necessary to bring the l e v e l of the packed gel to about 1.5 cm from the top. The packed and connected column was eluted with bo i l e d d i s t i l l e d water from a re s e r v o i r maintained at 80*C i n a constant temperature water bath with a regulated flow rate of 28 ml per hour by a Milton Roy Minipump. A f t e r e l u t i n g f o r 5 hours to eliminate dissolved oxygen, the pumping and the flow were stopped. The column was heated by a constant temp-erature c i r c u l a t o r which c i r c u l a t e d heated water i n the water jackets and maintained a temperature of 65 *C. Afte r heating up to the required temperature, the pump was turned on again to s t a r t the flow. The column was eluted f o r 10 hours f o r e q u i l i b r a t i o n . In a t y p i c a l run, the column was f i r s t heated up to 65*C. No eluant was pumped through the ge l bed during heat-ing up. The column was eluted with b o i l e d d i s t i l l e d water 26 from the reservoir maintained at 80*C. The flow rate was maintained at 28 ml per hour by the Milton Roy Minipump. Sample ( 0 . 2 - 0 . 3 ml) was applied to the f i r s t tube with a 1-ml syringe through a rubber septum. For column calibration, 0 . 3 ml of 5 0 $ com syrup solution was injected into the column. The eluant was moni-tored for carbohydrates using an E-C column monitor which measures the refractive index. For analysis of carbohydrate composition of the hydrolysed wheat flour, 0 . 2 ml of a 2 0 $ hydrolysed wheat flour solution was injected into the column. The eluant was collec-ted i n 2 ml fractions and 1 5 0 fractions were collected. The elution pattern of the carbohydrates was determined by taking 0 . 4 ml from each fraction, diluting to 2 ml with d i s t i l l e d water and performing the phenol-sulfuric acid test. The amount of carbohydrate in each peak was obtained by pooling the fractions of individual peak and determine the carbohy-drate by the phenol-sulfuric acid method?. ( 7 ) Sephadex G-50 column (2 x 9 5 cm) The column operated in a downward flow direction was packed and calibrated ( 7 5 ) - The fractions (from Biogel P-2 column) of the hydrolysed wheat flour with glucose units greater than 8< were pooled. A sample of 5 ml of the pooled fractions was applied to the column and eluted with phosphate buffer from a reservoir. A total of two hundred; 2-ml frac-tions were collected. The carbohydrate of each fraction was 27 determined by the phenol-sulfuric acid method. H. SDS-polyacrvlamide gel electrophoresis Both the wheat f l o u r and hydrolysed wheat f l o u r were defatted by several extractions with dry n-butanol. AUC extracts of defatted samples were prepared as described pre-v i o u s l y . The two AUC extracts were dialysed against 500 volumes of d i s t i l l e d water at V C f o r 3 days with 2 changes of water. The dialysed samples were freeze-dried. The 24,000 molecular weight protein peak of the hydrolysed wheat f l o u r eluted from the Sephadex G-150 column with AUC was prepared by pooling the peak f r a c t i o n s . The pooled peaks from several runs were dialysed against 500 volumes of d i s t i l l e d water at 4*C f o r 3 days with 2 changes of water. The dialysed sample was freeze-dried. Sodium dodecyl s u l f a t e was r e c r y s t a l l i z e d from b o i l i n g ethanol before use. SDS-polyacrylamide gel electrophoresis was c a r r i e d out according to the method of Sharpiro et a l (76) modified by Weber and Osborn (77). Bovine serum albumin, ovalbumin, tr y p s i n i n h i b i t o r , £-lactoglobulin, lysozyme and cytochrome C were used as molecular weight standards i n each electropho-r e t i c run. A s l i g h t l y modified procedure was adopted f o r reducing the proteins. Instead of incubating at 37*C for 2 hours, the protein sample i n 0.01M phosphate buffer (pH 7» Ifo SDS, 1% p-mercaptoethanol) was incubated at 95"-100*C 28 f o r 5 minutes. /S-Mercaptoethanol was omitted from Samples not r e q u i r i n g reduction. Amount of protein used i n each gel ranged from 15 to 40 jag. The concentration of acrylamide i n the separating gel was 7.5$. Electrophoresis was carried out at 7mA per gel f o r 3-4 hours at 15'C i n a Pharmacia gel electrophoresis apparatus. Bromophenol blue was used as an i n t e r n a l standard. The length of the gel and the distance moved by the dye before s t a i n i n g were measured. After e l e c -t r o p h o r e t i c a l l y destained i n a Pharmacia gel destainer, photographs of the gels were taken. The mobility of a protein band was calculated as distance of protein migration length before (in photograph) staining M o b i l i t y = : X — length a f t e r destaining distance of dye (in photograph) migration I. Analysis of ammonia, protein-nitrogen and nonprotein  nitrogen Five grams of the hydrolysed wheat f l o u r were d i s -solved i n 20 ml of d i s t i l l e d water. The solution was dialysed i n d i a l y s i s casing against 100 volumes of d i s t i l l e d water at 4"C f o r 3 days with 2 changes of d i s t i l l e d water. After d i a l y s i s , the dialysed sample was c a r e f u l l y made up to 50 ml with d i s t i l l e d water. The t o t a l nitrogen content of the hydrolysed wheat f l o u r was determined by a rapid Micro-Kjeldahl procedure (73) using 1 ml sample of 10fo hydrolysed wheat f l o u r s o l u t i o n . 29 The protein-nitrogen content was determined by the same pro-cedure of nitrogen determination of 1 ml of the dialysed sample. The ammonia-nitrogen was determined by analysing the nitrogen content of 1 ml of 10% hydrolysed wheat flour solution with the same procedure but omitting the digestion step. The nonprotein nitrogen was determined by subtraction. J. Amino acid analysis Freeze-dried hydrolysed wheat flour and wheat flour were hydrolysed with p-toluenesulfonic acid and 3 - (2 - amino ethyl) indole at 110*C for 24 hours (78). Amino acids were determined on a single column system (Durrum Chem. Corp., Palo Alto, Calif.) attached to a Phoenix Amino Acids Analyzer -Model M 6800. K. Foamability and foam s t a b i l i t y F i f t y m i l l i l i t r e s of pH 7 protein solution were whipped at 25*C for 5 minutes i n a Sunbeam Mixmaster at a speed setting of 10. The foam was quickly transferred to a 500 ml measuring cylinder and the foam volume was measured. Foamability was expressed as percentage volume increase whereas foam s t a b i l i t y was expressed as the time required for a certain volume of drainage to be collected. 30 L. Flavour improvement of the solubilized wheat flour (1) Solvent washing '' One hundred grams of wheat flour were suspended in 1000 ml of a solvent. The suspension was beaten in a kitchen aid mixer for 30 minutes at 58 rpm. The beaten suspension was centrifuged at 16,000 xg for 20 minutes. The supernatant was discarded and the residue was washed in 1000 ml of water before resuspended i n 1000 ml 0.1 N HC1 and autoclaved at 121*C for 15 minutes. The solvents used were water, water saturated butanol, ethanol (95$) and 0.05 M sodium hexameta-phosphate solution at pH 3. (2) Heated activated carbon treatment Granular activated carbon from Whitco Chemical Co. was f i r s t washed to eliminate the floating fine particles and then packed into a jacketed column (2 x 40 cm) to a level of 2/3 of the column length. The column was heated up to and maintained at 90 *C with a constant temperature circulator. A 10$ solution of the hydrolysed wheat flour was boiled and passed through the heated column at a rate of about 30 ml per minute. 31 RESULTS AND DISCUSSION (A) Preparation of acid-hydrolysed wheat f l o u r Wheat f l o u r was s o l u b i l i z e d by acid hydrolysis i n the present i n v e s t i g a t i o n . Two groups of acids could be used, organic acids and mineral acids. Acetic acid was found to be s i g n i f i c a n t l y milder than hydrochloric acid i n the s o l u b i l i z a -t i o n of wheat gluten (70). A much higher concentration was required f o r phosphoric acid than hydrochloric acid to solu-b i l i z e wheat f l o u r to a s i m i l a r extent (79). Consequently, hydrochloric acid was employed f o r s o l u b i l i z i n g wheat f l o u r . N i t r i c acid was not considered because of i t s possible damage on the n u t r i t i o n a l q u a l i t y of proteins. S u l f u r i c acid was found to impart a b i t t e r taste to the hydrolysate. The degree of wheat f l o u r s o l u b i l i z a t i o n was depen-dent on the acid concentration, the f l o u r concentration and the extent of heat treatment. Increased s o l u b i l i z a t i o n was observed with increasing acid concentration, increasing heat treatment and decreasing f l o u r concentration. An acceptable s o l u b i l i z e d product was obtained by autoclaving a 10 percent f l o u r suspension i n 0.1N hydrochloric acid at 121*C f o r 15 minutes. Over 90 percent of f l o u r s o l i d s was s o l u b i l i z e d under t h i s condition. Higher acid concentration and heat treatment increased the y i e l d but resulted i n an undesirable brown-coloured product. Higher a c i d concentration also 32 increased the s a l t i n e s s of the taste of the hydrolysed product a f t e r n e u t r a l i z a t i o n with sodium hydroxide. Using f l o u r con-centrations greater than 10 percent increased the browning and decreased the y i e l d , however, using concentrations less than 10 percent would be uneconomical. The freeze-dried powder of the s o l u b i l i z e d wheat f l o u r had colour s i m i l a r to that of the untreated f l o u r . It was r e a d i l y soluble i n cold water. The aqueous solution was l i g h t l y straw coloured and possessed a s l i g h t l y sweet and s a l t y taste. A strong wheaty of f - f l a v o u r was also detected i n the s o l u t i o n . B. Proximate analysis Table 1 shows the r e s u l t of proximate analysis of the hydrolysed wheat f l o u r as compared to the o r i g i n a l wheat f l o u r . Hydrolysed wheat f l o u r I and II were two separate preparations hydrolysed under the same conditions. The y i e l d s of the hydrolysed products I and II were 95.3$ and 93.3$, r e s p e c t i v e l y . The s l i g h t l y higher y i e l d i n product I was probably, due to a longer temperature-rising time during auto-c l a v i n g . The carbohydrate and protein content of the treated and untreated samples were quite s i m i l a r . Apparently there was s i m i l a r degree of s o l u b i l i z a t i o n of wheat protein and wheat starch during acid hydrolysis. The s i g n i f i c a n t l y higher ash content i n the hydrolysed samples was due to the presence 33 Table 1 Proximate analysis of acid hydrolysed wheat f l o u r . Proximate composition Sample • Protei n * Carbohydrate** Moisture* Ash* Untreated Wheat Flour Hydrolysed Wheat Flour (I) Hydrolysed Wheat Flour (II) 11.3$ 73.7$ 10.6$ 74.0$ 10.7$ 76.9$ 11.1$ 0.3$ 6.7$ 6.0$ 5.4$ 6.1$ * Average of duplicate a n a l y s i s . ** Average of t r i p l i c a t e a n a l y s i s . The values from the glucose standard curve had been adjusted by a correc-t i o n f a c t o r of 0.9 f o r the wheat starch and a correc-t i o n f a c t o r of 666/720 f o r the saccharides of the hydrolysed wheat f l o u r (assuming an average degree of polymerization of 4 f o r the saccharides). 34 of sodium chloride resulted from n e u t r a l i z a t i o n with sodium hydroxide. C. Apparent v i s c o s i t y The average apparent v i s c o s i t y of a 5f° solution of the hydrolysed wheat f l o u r , as calculated by averaging the apparent v i s c o s i t i e s at d i f f e r e n t shear rates, was 4.9 c e n t i -poises at 4'C. A commercial skimmilk sample measured and calculated s i m i l a r l y gave a value of 5.7 centipoises. Apparently, during a c i d hydrolysis, starch granules, molecules of amylose and amylopectin and wheat gluten were extensively degraded or modified, thus r e s u l t i n g i n a s o l u b i l i z e d wheat f l o u r s o l u t i o n of low v i s c o s i t y . D. Gel f i l t r a t i o n analysis of the hvdrolvsate Gel f i l t r a t i o n chromatography was used to study the extent of degradation of the wheat proteins and the wheat starch, caused by acid hydrolysis. This technique has been used to fractionate (36,80) and to determine the molecular weight d i s t r i b u t i o n of the wheat proteins (39). F i g . 1 shows the e l u t i o n p r o f i l e of hydrolysed wheat f l o u r protein eluted with phosphate buffer from a Sepharose 4B column. Three absorbance peaks were observed. A major peak was eluted at the void volume corresponding to molecular weights of 3 m i l l i o n s or over. One peak was observed at the e l u t i o n 2.0 F i g . 1 E l u t i o n curve of proteim of hydrolysed wheat f l o u r eluted from Sepharose 4B column with phosphate buffer. 36 volume of tryptophan corresponding to a molecular weight of 3,00,000 or under. The third broad peak was eluted between the above two peaks. Fig. 2 shows the elution profile of wheat flour proteins hydrolysed at a higher acid concentra-tion. A considerable increase i n the size was observed for the peak at the trytophan position suggesting either more extensive degradation of proteins or the appearance of more browned or conjugated small compounds. Fig. 3 shows the elution profile of untreated wheat starch eluted from Sepharose 4-B column. Most of the carbohy-drate appeared at the void volume indicating molecular weight ••I of 3 millions or over. Fig. 4- shows the elution profile of carbohydrates of hydrolysed wheat flour eluted from the same column. The major carbohydrate peak shifted from the void volume to the elution volume of tryptophan. This clearly shows degradation of high molecular weight starch molecules to lower molecular weight carbohydrates during acid hydrolysis. E. Gel f i l t r a t i o n analysis of protein For determining the molecular weight of protein by gel f i l t r a t i o n , the standard proteins were used. A linear standard curve was obtained by plotting the logarithm of the molecular weights of the standard proteins against the ratios of their elution volumes to the void volume of the column (81). Fig. 5 i s the calibration curve of a Sephadex G-150 superfine o 00 CN 03 CO c= ca CO E lu t ion vo lume (m l . ) F i g . 2 E l u t i o n curve of proteins of wheat f l o u r hydrolysed with higher acid concentra-t i o n (0.15 N HCI) eluted from Sepharose 4B column with phosphate buffer. -N3 F i g . :3 Elution curve of unhydrolysed wheat starch eluted from Sepharose 4-B column with phosphate "buffer. CO 2 . 0 - 1 1 . 5 4 cn c= CO 1.0-1 - C 2 0.54 6 7 ) ,000 / ' 1 160 """" ' -m ^ 3 2 0 : — i 4 8 0 Elut ion volume ( m l . ) F i g . E l u tion curve of carbohydrates of hydrolysed wheat f l o u r eluted from Sepharose 4-B column with phosphate buffer. 40 3 . 0 - | 2 6 H B> c y t o c h r o m e C 8 r i b o n u c l e a s e A 2.2-1 \ 1.8 • c h y m o t r y p s i n /3- lactog l o b u l i n o v o m u c o i d i ^ o v a l b u m i n 1.4-c o n a l b u m i n . J i o v i n e serum a l b u m i n (monomer ) ( d ime r ) bovine serum a lbumin » c a t a l a s e I I I I I I I I 3 4 5 6 7 8 9 1 0 .8 .9 1 2 0 3 0 M o l e c u l a r w e i g h t ( x i c r 4 ) F i g . 5 Ca l i b r a t i o n curve of Sephadex G-150 (superfine) column eluted with phosphate buffer. 41 column operated i n phosphate buffer. The standard proteins used were l i s t e d i n Table 2. The curve i s a straight l i n e between molecular weights 11,700 and 100,000 and l e v e l s o f f beyond 100,000. F i g . 6 shows the e l u t i o n p r o f i l e of hydro-lysed wheat f l o u r proteins from the Sephadex G-150 superfine column. The major peak again appeared at the void volume suggesting a molecular weight of 400,000 or higher. The. other absorbance peak had molecular weight less than 10,000 and could be nonprotein nitrogen compounds due to protein degradation during acid h y d r o l y s i s . Gel f i l t r a t i o n patterns of the hydrolysed wheat proteins i n phosphate buffer might not give a true picture of the extent of protein degradation during acid hydrolysis. Hydrolysed wheat proteins might aggregate i n a neutral phos-phate buffer s o l u t i o n . The huge absorbance peak at the void volume from both Sepharose 4B and Sephadex G-150 superfine g e l f i l t r a t i o n studies could be due to aggregates of hydro-lysed wheat proteins. In addition, as gluten i s insoluble i n phosphate buffer, a d i r e c t comparision of gel f i l t r a t i o n patterns between the untreated and the hydrolysed wheat f l o u r proteins were impossible. A highly d i s s o c i a t i n g solvent con-t a i n i n g 0.1M acetic acid, 3M urea and 0.01M cetyltrimethy-lammonium bromide (AUC) was used f o r t h i s purpose. This solvent could dissolve about 95f° of f l o u r protein which was dissociated to the monomers (39) • F i g . 7 shows the c a l i b r a -t i o n curve f o r the standard proteins (Table 3), eluted from a 42 Table 2 Proteins used in calibration of Sephadex G-150 superfine column eluted with 0.01M phosphate buffer, pH 7. Proteins Ve/Vo Mol. wt. (Reference) Catalase 1.11 244,000 ( 89 Bovine serum albumin (Dimer) 1.12 134,000 ( 90 Conalbumin 1.47 76,600 ( 81 Bovine serum albumin (monomer) 1.41 ,67,000 ( 90 Ovalbumin 1.65 45,000 ( 81 /3-Lactoglobulin 1.83 36,800 ( 77 Ovomucoid 1.66 27,000 ( 81 od -Chymo trypsin 2.04 22,500 ( 81 Ribonuclease A 2.38 13.600 ( 81 Cytochrome C 2.43 11,700 ( 77 Ve: Elution volume Vo: Void volume F i g . 6 E l u t i o n curve of proteins of hydrolysed wheat f l o u r eluted from Sephadex G-150 (superfine) column with phosphate buffer. 44 3.0-2.6-t • r i b o n u c l e a s e A 2.2-^myog lob i n • t r y p s i n 1.8-1 1.4-c h y m o t r y p s i n o g e n A • • o v o m u c o i d T T T .8 9 1 o v a l b u m i n • bovine serum a l bum in (monomer) • . c o n a l b u m i n T 2 3 bov ine serum a l b u m i n ( d i m e r ? * * — • " " m a n 7 globulin | [ 1 1 1 1 1 | ? 4 5 6 7 8 910 20 3 0 Molecular weight ( x i c r 4 ) F i g . 7 C a l i b r a t i o n curve of Sephadex G-150 column eluted with AUC. 45 Table 3 Proteins used in calibration of Sephadex G-150 column eluted with AUC. Proteins Average YeA© No. of run Mol. wt. (Ref.) Human- nC -globulin 1.04 2 156,000 ( 81 ) Bovine serum albumin (Dimer) 1.02 3 134,000 ( 90 ) Conalbumin 1.25 3 76,600 ( 81 ) Bovine serum albumin (Monomer) 1.32 4 67,000 ( 90 ) Ovalbumin 1.40 6 45,000 ( 81 ) Ovomucoid 2.04 2 27,000 81 ) Chymotrypsinogen A 2.04 2 25,000 ( 90 ) Trypsin 2.23 4 23,800 ( 81 ) Sperm whale myoglobin 2.34 2 17,800 ( « ) Ribonuclease A 2.65 2 13l,-600 ( 81 ) Tryptophan 3.38 2 240 N-Ethylraaleimide 3.34 4 125 Vet Elution volume Vos Void volume *Mol. wt. provided by supplier. 46 Sephadex G-150 column with AUC. Significant deviation from the calibration curve was observed for ovalbumin. Fig. 8 shows an elution profile of untreated wheat flour protein from the AUC eluted Sephadex G-15Q column. Unlike an elution profile of wheat flour proteins on an AUC eluted Sephadex G-200 column which separated into distinct glutenin and gliadin peaks (39)» no clear separation of glutenin and gliadin peaks was observed. Possible explanations for this difference are in poorer resolution of Sephadex G-150 gel at a higher mole-cular weight range and in variety difference. Elution profiles of wheat proteins similar to Fig. 8 were also reported in another study using a Sephadex G-150 gel column (82). Both the albumin and the nonprotein nitrogen peaks in Fig. 8 were quite small and most of the gliadins were found between mole-cular weights of 40,000 and 100,000. Fig. 9 is an elution curve of hydrolysed wheat flour proteins from the AUC eluted Sephadex G-150 column. Comparision between Fig. 8 and Fig. 9 shows that almost a l l the glutenins and higher molecular weight gliadins were degraded to 2 groups of lower molecular weight compounds, one with molecular weight around 24,000 and the other, at the position with a molecular weight less than 10,000. The existence of a huge peak near suggested an extensive degradation of wheat proteins yielding a large amount of,nonprotein nitrogen during acid hydrolysis. Fig. 10 shows an elution profile of the hydrolysed wheat flour proteins eluted from the same Sephadex G-150 column but detected by the 0.5 80 10,000 120 160 E l u t i o n vo lume ( m l . ) 200 240 F i g . 8 E l u t i o n curve of wheat f l o u r proteins eluted from Sephadex G - 1 5 0 column with AUC. 100,000 25,000 10,000 200 240 80 120 160 Elution volume (ml.) F i g . 9 E l u t i o n curve of proteins of hydrolysed wheat f l o u r eluted from Sephadex G-150 column with AUC. 100,000 25,000 10,000 80 120 160 200 240 Elution volume (ml.) F i g . 10 Chloranil test of hydrolysed wheat f l o u r proteins eluted from Sephadex G-150 column i n AUG. 50 chloranil test. The two elution profiles are quite similar except for the relatively smaller nonprotein nitrogen peak detacted by the chloranil test. In the chloranil reaction, the amino groups i n amino acid and protein form n-ir charge transfer complex with chloranil, resulting i n a red shift of the chloranil band on complexing (83)• The test i s capable of measuring microgram amounts of amino acids with no inter-ference from urea and claimed to be more sensitive than the ninhydrin method (7b). Proteins e.g. tyrosinase, gave a spectral peak similar to amino acids but the response was about one-tenth of the absorbance produced by the same weight of amino acids. Ammonium chloride also responded to a similar extent as proteins (74). The chloranil test was a more specific test for amines, amino acids and peptides than the absorbance at 280 nm. The huge nonprotein nitrogen peaked detected by measuring absorbance at 280 nm could be due to the presence of conjugated or browning compounds produced during acid hydrolysis. The nature of the absorbance peak of the nonprotein nitrogen compounds was further investigated by eluting the hydrolysed wheat flour with a neutral phosphate buffer from a Sephadex G-200 column. The elution profile monitored by measuring absorbance at 280 nm (Fig. 11) was similar to that from the Sephadex G-150 superfine column (Fig. 6) with a high sharp peak at the void volume and a lower and broader peak of nonprotein nitrogen compounds. Although the chloranil test F i g . 11 E l u t i o n curve of proteins of hydrolysed wheat f l o u r eluted from Sephadex G-200 column with phosphate buffer. 52 demonstrated only milder reactions with the nonprotein nitrogen compounds, aihuge peak was detacted "by the ninhydrin test (Fig. 12). During mild acid hydrolysis of gluten, ammonia was released by selective hydrolysis of the amide groups of glut-amines and asparagines (68). The ninhydrin reagent, unlike the chloranil reagent i s sensitive to both free amino acids and ammonia. The huge peak detected by the ninhydrin reagent i n the region of nonprotein nitrogen compounds may be due to the presence of ammonia released during acid hydrolysis. The small ninhydrin peak at the void volume could be due to the presence of high molecular weight protein aggregates in the hydrolysate. F. Analysis of ammonia, nonprotein and protein nitrogen Gel f i l t r a t i o n chromatography of the hydrolysed wheat flour protein did not provide conclusive quantitative results for the proportion of ammonia-nitrogen, other non-protein nitrogen and protein nitrogen, Nonprotein nitrogen i n wheat flour may be separated from protein nitrogen by heat coagulation, TCA precipitation and dialysis. Of these, dialysis was found to be the most reproducible method on the basis of molecular size (84). A combination of dialysis and a rapid micro-Kjeldahl procedure was. used to study the quan-ti t a t i v e distribution of nitrogen in the hydrolysed wheat flour. The result i s shown in Table 4. Ammonia constituted 160 3 2 0 4 8 0 Elution volume (ml.) F i g . 12 Ninhydrin t e s t ( ) and Choranil test (. ••• ) of hydrolysed wheat f l o u r proteins eluted from Sephadex G-200 column i n phosphate buffer. A-0 Table 4 Total-nitrogen, protein-nitrogen and ammonia-nitrogen content of the freeze-dried hydrolysed wheat flour. Nitrogen content ($ by weight of 1 freeze-dried % Total nitrogen hydrolysate) Total-nitrogen 1.827 ± 0.041- ' 100.0 Protein-nitrogen 1.464 ± 0.041 80.1 Ammonia-nitrogen 0.394 ± 0.010 21.6 •a-Average of 4 determinations and standard deviation. 55 21.6 percent and protein nitrogen 80.1 percent of the total nitrogen in hydrolysed wheat flour. Substracting ammonia nitrogen and protein nitrogen from total nitrogen would give the amount of other nonprotein nitrogen of free amino acids and low molecular weight peptide which were obviously present in an insignificant quantity in the hydrolysed product. The presence of 21.6$ of ammonia nitrogen was a good indication of almost complete deamidation of wheat flour protein during acid hydrolysis. Failure to detect any other nonprotein nitrogen implied that during acid hydrolysis, high molecular weight wheat flour proteins were degraded to lower molecular weight compounds, but degradation did not proceed to the extent of producing large quantities of free amino acids and low mole-cular weight peptides. The peaks at the nonprotein nitrogen position as detected by the chloranil reagent and by measuring absorbance at 280 nm were probably due to ammonium chloride and to browning compounds, respectively. G. Gel f i l t r a t i o n analysis of carbohydrate Fig. 3 and Fig. 4 showed that high molecular weight starch was broken down to lower molecular weight carbohydrates during acid hydrolysis. The low molecular weight carbohydrate peak in Fig. 4 was pooled for further investigation with gel f i l t r a t i o n i n a smaller pore size gel. Fig.. 13 i s the elution profile of the carbohydrate peak pooled from Fig. 4 and eluted in a Sephadex G-100 column. Only one huge peak emerged ° - 5 l 100 11,700 < 5,000 200 Elution volume (ml.) 500 F i g . 13 Gel f i l t r a t i o n elution curve on Sephadex G-100 column f o r the major carbohydrate peak of hydrolysed wheat f l o u r c o l l e c t e d from Sepharose 4B column. Eluant: phosphate buffer. ON 57 towards the end of the elution profile corresponding to mole-cular weight less than 5,000. The carbohydrates thus existed mainly as oligosaccharides after acid hydrolysis. The d i s t r i -bution of the oligosaccharides was further studied by gel f i l t r a t i o n i n the polyacrylamide gel, Biogel P-2 minus 400 mesh, at an elevated temperature of 65* C, Using an automated detection system, this technique was reported to be capable of fractionating oligomers containing two to twenty one glucose units as well as separating a mixture of isomeric compounds with similar molecular weights (85). Fig. 14 was the elution profile of carbohydrates of the hydrolysed wheat flour eluted from an Biogel P-2 column. The column was capable of resolv-ing glucose and i t s polymers up to 8 glucose units. The resolution of higher oligomers of glucose would probably improve with a continuous automated system of carbohydrate detection and with better methods for sample application. Fig. 14 shows that most of the carbohydrates after acid hydro-l y s i s were low molecular weight saccharides with glucose pre-sent in the highest concentration of 18.7 percent. This was followed by the disaccharide maltose which constituted 11.3 percent of the total carbohydrates. The concentration of the individual oligomers decreased as the glucose units increased. This observation was in close agreement with a chromatographic study of the acid hydrolysed products of starch showing a log-arithmatic decrease in the concentration of the degraded pro-ducts with increasing degree of polymerization (86). A 120 G11 A N D H I G H E R 160 2 0 0 Elution volume (ml.) 2 4 0 2 8 0 Fig.' 14 E l u t i o n curve of carbohydrates of hydrolysed wheat f l o u r eluted from Biogel P-2 column with d i s t i l l e d water. 00 59 commercial "brand of corn syrup containing glucose and a con-tinuous series of i t s polymers was used to c a l i b r a t e the Biogel P-2 column. F i g . 15 i s the c a l i b r a t i o n curve showing the r e l a t i o n s h i p between the logarithm of the molecular weight and the r a t i o of elu t i o n volume to void volume. The curve was l i n e a r f o r oligosaccharides with 3 to 8 glucose units . Oligo-saccharides with 9 and 10 glucose units also f e l l on the l i n e a r portion of the curve (85) although they were not c l e a r l y resolved into separate single peaks i n the chromatogram. F i g . 14 indicates that over two-thirds of the carbo-hydrates a f t e r a c i d hydrolysis were oligosaccharides with 7 or less glucose u n i t s . Carbohydrates with 8 or more glucose units constituted 32.9 percent of the t o t a l carbohydrate of hydrolysed wheat f l o u r . This f r a c t i o n eluted as a single peak from a Sephadex G-50 column,.as shown i n F i g . 16, showing very few saccharides had molecular weights greater than 5»000 (75). Gel f i l t r a t i o n chromatography of the hydrolysed wheat f l o u r c l e a r l y indicated that during hydrolysis, wheat starch was extensively degraded to glucose, maltose and o l i g o -saccharides with very few saccharides possessing more than 30 glucose units i n the molecules. This explained the S l i g h t l y sweet taste and the low v i s c o s i t y of an aqueous solution of the hydrolysed product. 2.2 2 .0 H 1 . 8 H V 1 . 6 4 1.4-•J32 X G 3 V G 4 \ G 5 ^ 6 \ G 7 \ G 8 \ G 9 \ G 1 0 2 3 4 5 6 7 8 9 10 Molecular weight (xi<r s) F i g . 15 Calib r a t i o n curve f o r Biogel P-2 column using corn syrup, 20 30 ON o 0.9-1 0.7H 0.5H o cn CU C_3 c= ca CD 0 .3 -1 0 .1 -1 140 180 220 2 6 0 Elution volume (ml.) 3 0 0 3 4 0 F i g . 16 E l u t i o n curve of higher saccharides (G8 and above) of hydrolysed wheat f l o u r eluted from Sephadex G-50 (fine) column with Phosphate buffer. ON 62 H. SDS-PAGE analysis Gel f i l t r a t i o n chromatography with AUC gave a general picture of the changes in molecular weight of wheat flour pro-teins during acid hydrolysis. More specific information on the degradation of polypeptides during acid hydrolysis could be obtained through a study with sodium dodecyl sulfate-polyacry-lamide gel electrophoresis (SDS-PAGE). This technique has been used to demonstrate the compositions of glutenin and gliadin polypeptides in terms of molecular weights (40,47,48). Fig. 17 i s a calibration curve forjthe standard proteins in the SDS-PAGE analysis. The result of SDS-PAGE analysis of the untreat-ed and the acid hydrolysed wheat flour is shown in Fig. 18. The 24,000 daltons protein peak pooled from the AUC eluted Sephadex G-150 column was further separated to two diffuse bands during electrophoresis, one with molecular weight around 3 0 , 0 0 0 and another fast moving band with molecular weight less than 1 1 , 7 0 0 . In addition to the above two bands, a third diffuse band of 53,500 daltons was detected in the whole hydro-lysed wheat flour. Native wheat flour proteins showed a series of typical bands ranging from molecular weights of 17.500 to 1 0 9 , 0 0 0 . The SDS-PAGE provided good evidence of polypeptide degradation during acid hydrolysis. The presence of two diffuse bands in the electrophoresis of the 24,000 daltons peak indicates the possibility of some smaller polypeptides joined by intermolecular disulfide bonds in this peak. 63 1.0 • ^ c y t o c h r o m e C lysozyme 0.8 0-6 -fl j8 - l a c t o g l o b u l i n ' « t r yps in inhibitor ova lbumin 0.4 0 2 - i .bovine serum albumin (monomer) I i t i l l 5 6 7 8 9 1 0 M o l e c u l a r w e i g h t ( x i c r 4 ) bovine serum • a l b u m i n (dimerj bovine ,serum albumin (trimer) 20 F i g . 17 C a l i b r a t i o n curve f o r proteins i n SDS-pplyacry lamide gel electrophoresis. 6k mol. wt. mol. wt. 26,000 30,000 33,500 33:888 57, 200 15,600 19,000 2 3,000 mm 28 , 700 32,400 0— 36 , 400 45,400 mm 50 , 600 tm* 60 , 200 - 89 f 0 2 \106 109 188 600 000 500 000 B C F i g . 18 SDS-Polyacrylamide Gel Electrophoretic Pattern of (A) major peak of acid-hydrolysed wheat f l o u r protein (from AUC-eluted Sephadex G-150 column), (B) whole acid-hydrolysed wheat f l o u r protein, (G) whole wheat f l o u r protein, (D) an enlarged picture of (C). 65 F i g . 19 i s a comparision of the electrophoretic pattern of wheat f l o u r proteins with or without reduction p r i o r to electrophoresis. Without p r i o r reduction, the 24,000 daltons peak from Sephadex G-150 column showed great decrease i n i n t e n s i t y i n the f a s t moving band with simultaneous i n -crease i n i n t e n s i t y i n the 30,000 daltons band. This provides further evidence of intermolecular d i s u l f i d e bonds i n the 24,000 daltons peak. Whole hydrolysed wheat f l o u r protein without reduction exhibited two additi o n a l diffuse bands bet-ween the f a s t moving band and the 30,000 daltons band. These two new bands were apparently made up of molecules with i n t e r -molecular d i s u l f i d e bonds which upon reduction, s p l i t t e d into smaller polypeptides migrating i n the f a s t moving band. The presence of SS- groups i n acid hydrolysed wheat protein was also demonstrated i n another study of s o l u b i l i z e d gluten hydrolysed under d i f f e r e n t hydrochloric acid concentrations (70). No s i g n i f i c a n t decrease i n SS- groups and no increase i n SH- groups were detected i n the s o l u b i l i z e d gluten although the p o s s i b i l i t y of i n t e r - and intra-molecular d i s u l f i d e i n t e r -change could not be excluded. Different electrophoretic patterns were exhibited by native wheat f l o u r proteins with or without reduction. An appreciable amount of proteins remained at the s t a r t i n g point of the gel when the proteins were not reduced. This was probably glutenin molecules which were too large to enter the gel matrices (34). 66 mol. wt. 1 1 , 7 0 0 1 8 , 4 0 0 4 5 , 0 0 0 6 7 , 0 0 0 1 3 4 , 0 0 0 A B C D E F 19 SDS-Polyacrylamide Gel Electrophoretic Pattern of (A) major peak of acid-hydrolysed wheat f l o u r protein (from AUC-eluted Sephadex G-150 column) with reduction p r i o r to electrophoresis, (B) same protein peak as (A) but without reduction, (C) whole acid-hydrolysed wheat f l o u r protein with reduction, (D) whole acid-hydrolysed wheat f l o u r protein without reduction, (E) whole wheat f l o u r protein with reduction, (F) whole wheat f l o u r protein without reduction. 67 Gel f i l t r a t i o n and SDS-PAGE analysis of hydrolysed wheat flour protein clearly showed s p l i t t i n g of peptide bonds during acid hydrolysis. High molecular glutenin and gliadin were degraded mainly to lower molecular weight compounds of about 24,000 daltons according to gel f i l t r a t i o n . Some of these low molecular weight compounds possessed intermolecular disulfide bonds which might be derived from the original bonds t in native glutenin or from disulfide interchange reactions. In SDS-PAGE, those compounds with intermolecular disulfide bonds, when reduced by /8-mercaptoethanol, were s p l i t into smaller fragments and migrated in the fast moving band. I. Amino acids analysis The amino acids composition of acid hydrolysed wheat flour proteins and the untreated proteins was shown in Table 5» No significant loss of essential amino acids was observed. Tryptophan could not be detected in both the hydrolysed and the untreated wheat flour samples. This could be due to the high carbohydrate content which would decrease the recovery of tryptophan during p-toluenesulfonic acid hydrolysis (78). According to the study by Wu (70), only a slight decrease in glycine and tryptophan was observed even under the most severe condition of gluten hydrolysis with 0.5N HC1. Amino acids analysis did not reveal anyrdecrease i n essential amino acids of the wheat flour solubilized by acid hydrolysis. 68 Table 5 Comparison of amino acid content of untreated wheat f l o u r and hydrolysed wheat f l o u r . Amino acids Amino acid -content (g/100 g protein) Untreated Hydrolysed wheat f l o u r wheat f l o u r Aspartic acid 4.28 4.38 •Threonine 1.95 1.88 Serine 4.61 4.62 Glutamic acid 32.50 31.40 Proline 15.65 13.67 Glycine 3.73 3.88 Alanine 3.13 2.45 •Valine 3.60 4.22 •Methionine 1.06 1.12 •Isoleucine 2.67 3.31 •Leucine 5.15 6.92 Tyrosine 3.12 3.19 •Phenylalanine 5.29 5.48 •Lysine 2.37 2.37 ._Histidine 2.26 2.20 •Tryptophan 0.00 0.00 Arginine 3.62 3.89 Cysteine • E s s e n t i a l amino acids. 69 J . Foamability and foam s t a b i l i t y Table 6 shows the coraparision of foamability and foam s t a b i l i t y of the hydrolysed wheat f l o u r protein, casein and soy protein. Using 0.5 percent protein solutions, the hydrolysed wheat' f l o u r was shown to be a better foaming agent than casein although the foam s t a b i l i t y was much lower. Soy protein exhibited r e l a t i v e l y poor foamability but with much better s t a b i l i t y . Increasing protein concentration to 1$ did not improve foamability to appreciable degree but s l i g h t l y increased the foam s t a b i l i t y of the hydrolysed wheat f l o u r . Good foaming property was e s s e n t i a l f o r hydrolysed wheat f l o u r i n making ice-cream and milk shake l i k e products. K. Flavour improvement An undesirable wheaty o f f - f l a v o u r appeared during a c i d hydrolysis of wheat f l o u r . The same of f - f l a v o u r was observed when v i t a l gluten was hydrolysed with d i l u t e hydro-c h l o r i c acid (70). Since milk i s a bland product, the wheaty of f - f l a v o u r has to be removed before the hydrolysed wheat f l o u r can be used as dairy substitute bases. The most e f f e c t i v e method i n reducing the of f - f l a v o u r was found to be passing an aqueous solution of hydrolysed wheat f l o u r through a heated column of granulated carbon. By t h i s treatment, the o f f -flavour was greatly reduced and there was a simultaeous appearance of a pleasant malty flavour. The malty flavour was 70 Table 6 Foamability and foam s t a b i l i t y of hydrolysed wheat flour, casein and soy protein isolate solutions. Sample Foamability* Foam st a b i l i t y ($ volume (seconds) increase) Hydrolysed wheat flour solution (0.5$ protein) 401.4 ± 16.8 160.0 ± 5.0 Hydrolysed wheat flour solution (1.0$ protein) 403.4+5.8 195.0 + 5.0 Casein solution (0.5$ protein) 322.8 ± 12.6 451.7 + 67.9 Soy protein isolate solution (0.5$ protein) 176.0 ± 17.6 1463.3 ±142.2 * Average of 7 determinations and standard deviation. ** Foam s t a b i l i t y was taken as the time elapsed when the drain volume increased from 10 ml to 30 ml. The value was an average of 3 determinations and standard devia-tion. 71 probably masked by the wheaty o f f - f l a v o u r before treatment. In addition to flavour improvement the hot carbon treatment also improved the colour and the storage s t a b i l i t y of the dry hydrolysed product. Removal of the wheaty of f - f l a v o u r was more e f f i c i e n t at higher temperature. A column operating at 90*0 was found to remove the o f f - f l a v o u r better than a column operating at 60*C. The spent carbon can be regenerated by heating to 600'C i n a furnace. One of the major factors which hindered the success-f u l removal of the wheaty of f - f l a v o u r was the lack of knowledge of the nature and o r i g i n of the flavour. Solvent washing of wheat f l o u r was attempted to remove flavour precursors before acid hydrolysis. Washing wheat f l o u r with water, with 95$ ethanol or with an! aqueous so l u t i o n sodium hexametaphosphate only s l i g h t l y reduced the o f f - f l a v o u r i n the hydrolysed product. Washing wheat f l o u r with water saturated butanol was more e f f e c t i v e i n reducing the o f f - f l a v o u r but did not eliminate i t completely. Water saturated butanol removed both free and bound l i p i d s from wheat f l o u r (61,62). It was possible that l i p i d material was the precursor to the wheaty o f f - f l a v o u r . Our observation that the o f f - f l a v o u r s t i l l persisted i n the hydrolysed wheat f l o u r a f t e r a single water saturated butanol wash was probably due to incomplete removal of l i p i d materials. A number of ion exchange r e s i n s , anionic, c a t i o n i c or mixed bed were tested f o r removing o f f - f l a v o u r from an 72 aqueous solution of hydrolysed wheat f l o u r , but none were found to be s a t i s f a c t o r y . Since the wheaty o f f - f l a v o u r might a r i s e through an oxidation reaction during acid hydrolysis, incorporation of a reducing agent i n the hydrolysing solu t i o n might stop flavour development. However, addition of cysteine hydrochloride or sodium s u l f i t e did not appear to reduce the wheaty o f f - f l a v o u r i n the hydrolysed product. From our study, i t appeared that the best method to minimize the wheaty o f f - f l a v o u r was an extraction... of l i p i d material from wheat f l o u r exhaustively with a suitable l i p i d solvent l i k e water saturated butanol. Any r e s i d u a l o f f - f l a v o u r developed during a c i d hydrolysis could be removed by hot carbon treatment. An_autoclaved solution of hydrolysed wheat f l o u r required n e u t r a l i z a t i o n with a l k a l i . Sodium hydroxide was used i n t h i s study. The r e s u l t i n g sodium chloride gave the solution a s a l t y t a s t e . A mixture of potassium hydroxide and sodium hydroxide could be used to reduce s a l t i n e s s . Calcium hydroxide was undesirable because of the b i t t e r taste of calcium chloride. L. Imitation milk product An imi t a t i o n milk product was prepared from hydro-lysed wheat f l o u r deordourized with a single washing of water saturated butanol and with hot activated carbon treatment. 73 A ten percent solu t i o n was homogenized with 4 percent unsalted butter i n a 2 stage homogenizer. The homogenized product had the same appearance as commercial homogenized milk and tasted s l i g h t l y sweet and s a l t y with a very mild wheaty flavour. The emulsion was stable at 4*C f o r 2 weeks with no sign of s p o i l -age. The carbohydrate to protein r a t i o was about 7«1 i n t h i s product. In order to decrease the r a t i o to a l e v e l s i m i l a r to milk, either one of the two approaches could be used. The f i r s t one involved f o r t i f i c a t i o n with other soluble proteins l i k e f i s h , soybean, cottonseed, rapeseed, peanut or s o l u b i l i z e d feather proteins. The high l y s i n e content of some of these proteins e.g. soybean protein would increase the n u t r i t i v e value of the imitation milk product. The second approach u t i l i z e d a dough washing technique (71) which washed off the excess starch. The carbohydrate to protein r a t i o was success-f u l l y reduced to a l e v e l s i m i l a r to that i n milk a f t e r a ser i e s of washings of the dough (87). Besides butter, other animal f a t s and vegetable o i l s l i k e coconut o i l , soybean o i l , cottonseed o i l and corn o i l could be used as a source of l i p i d i n preparation of the i m i t -ation dairy product. 74 CONCLUSION Autoclaving a 10$ wheat f l o u r suspension i n 0.1N HC1 at 121 *C f o r 15 minutes provided a rapid, effecient and econo-mical method of s o l u b i l i z i n g wheat f l o u r . The s o l u b i l i z e d product exhibited a low v i s c o s i t y , excellent s o l u b i l i t y and appearance and could be used as dairy substitute bases. The low v i s c o s i t y and the improved s o l u b i l i t y of the hydrolysed product are due to the extensive degradation of wheat gluten and starch, e s p e c i a l l y due to almost complete deamidation of gluten. From gel f i l t r a t i o n chromatography i n AUC and SDS-polyacrylamide gel electrophoresis, random breakage of both g l i a d i n and glutenin polypeptides was evident. However, the degradation did not proceed to the point of producing large amount of free amino acids and low molecular weight peptides. Apparently, some intermolecular d i s u l f i d e bonds were s t i l l present i n the hydrolysed polypeptides. Gel f i l t r a t i o n chromatography i n neutral phosphate buffe r was not suitable f o r studying the extent of wheat protein degradation i n hydrolysis because of the tendency of the hydro-lysed protein to aggregate i n aqueous s o l u t i o n . Wheat starch was degraded to low molecular weight saccharides with increasing concentration as the number of glucose units decreased. The hydrolysed wheat f l o u r had an Dextrose Equivalence (D.E.) value of 36 and can be used as a 75 substitute of a s i m i l a r D.E. corn syrup i n food processing. The n u t r i t i o n a l value of the hydrolysed product i s expected to be s i m i l a r to that of wheat f l o u r since there was no s i g n i f i c a n t destruction of e s s e n t i a l amino acids. Both the n u t r i t i o n a l value and the protein to carbohydrate r a t i o can be improved by the addition of a protein high i n l y s i n e content. The foamability of the hydrolysed wheat protein was as good as that of casein. An activated carbon treatment greatly reduced the wheaty o f f - f l a v o u r of the hydrolysate. Complete elimination probably requires washing of f <$£ a l l the l i p i d materials from wheat f l o u r with a suitable solvent p r i o r to hydrolysis. 76 LITERATURE CITED 1. "Food Protein From Grains and Oilseeds - A Development Study Projected to 1980". Published by the Office of the Minister Responsible f o r the Canadian Wheat Board. House of Common, Ottawa, Sept. 1972. 2. Hedrick, T. I. 1969. Dairy Industries 3J H 127. 3. Wilkens, W. F., Mattick, L. 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