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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.), U n i v e r s i t y of Manitoba,  1971  M.P.H., U n i v e r s i t y o f C a l i f o r n i a , Los Angeles,  A t h e s i s 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 o f Master of S c i e n c e i n the  Department of  FOOD SCIENCE F a c u l t y of A g r i c u l t u r a l S c i e n c e s  We  accept t h i s t h e s i s as conforming t o the  r e q u i r e d standard '  THE UNIVERSITY OF BRITISH COLUMBIA June (c)  1976  Chi-Pun Fung, 1976  1972  In  presenting  this  thesis  an advanced degree at the I  Library shall  f u r t h e r agree  for  scholarly  by h i s of  this  written  make  it  Columbia,  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  It  for financial  i s understood gain s h a l l  permission.  SOENCB  University of B r i t i s h  |5'  for  the requirements  this  JOi-/ ,  Columbia  that copying or  not  for  that  study. thesis  purposes may be granted by the Head of my Department  20 75 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  freely available  that permission  Department of The  fulfilment of  the U n i v e r s i t y of B r i t i s h  representatives. thesis  in p a r t i a l  or  publication  be allowed without my  ii  ABSTRACT  A main problem i n preparing dairy substitute bases from wheat f l o u r i s the i n s o l u b i l i t y of wheat gluten and starch. In t h i s study, wheat f l o u r was s o l u b i l i z e d by acid hydrolysis. More than 90 percent of the dry weight of wheat f l o u r s o l i d s were s o l u b i l i z e d 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. hydrolysate  The dried  was s i m i l a r i n colour to the o r i g i n a l f l o u r and  had excellent s o l u b i l i t y .  The apparent v i s c o s i t y at 4*C of a  5 percent wheat f l o u r hydrolysate  was 4.9 centipoises as  compared to 5*7 centipoises f o r pasteurized skimmilk. analysis of the dried hydrolysate  Proximate  showed the t o t a l carbohydrate  and protein contents s i m i l a r to those of the o r i g i n a l f l o u r . 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 c e t y l t r i m e t h y l ammonium bromide) revealed an extensive  degradation of most of  the glutenin and the high molecular weight g l i a d i n into a protein f r a c t i o n of 24,000 daltons during hydrolysis.  The  c h l o r a n i l t e s t did not indicate a s i g n i f i c a n t increase i n amino acids or i n small molecular weight  peptides.  According to a Kjeldahl analysis subsequent to d i a l y s i s , ammonia cations accounted f o r 22$ of the t o t a l nitrogen, i n d i c a t i n g almost complete deamidation during acid hydrolysis. hydrolysate.  Other nonprotein nitrogen was not detacted  i n the  'iii  SDS-polyacrylamide disappearance  g e l e l e c t r o p h o r e s i s showed  o f the t y p i c a l wheat p r o t e i n "bands and appear-  ance o f 3 d i f f u s e d bands o f m o l e c u l a r weights 11,700  and  upon h y d r o l y s i s .  30,000  and  A h i g h temperature  30,000  The 2 4 , 0 0 0 d a l t o n s f r a c t i o n  from Sephadex G - 1 5 0 column appeared m o l e c u l a r weights  53,500,  as 2 d i f f u s e d "bands o f  11,700.  g e l f i l t r a t i o n chromatography on  B i o g e l P-2 e x h i b i t e d t h e f o l l o w i n g s a c c h a r i d e composition: 18.7  percent g l u c o s e , 1 1 . 3 percent maltose,  3 7 percent  oligo-  s a c c h a r i d e s ( 3 - 7 glucose u n i t s ) and 3 3 percent h i g h e r s a c c h a r i d e (more than 7 g l u c o s e u n i t s ) .  A g e l f i l t r a t i o n chromatography  on Sephadex G - £ 0 i n d i c a t e d o n l y a t r a c e amount o f p o l y s a c c h a r i d e s w i t h more than 2 0 glucose u n i t s . No s i g n i f i c a n t d e s t r u c t i o n o f e s s e n t i a l amino a c i d s d u r i n g h y d r o l y s i s was observed. U s i n g 0 . 5 percent p r o t e i n s o l u t i o n s , the f o a m a b i l i t y of t h e h y d r o l y s e d wheat f l o u r was found t o be s l i g h t l y b e t t e r than t h a t o f c a s e i n , although the foam s t a b i l i t y was i n f e r i o r . A s t r o n g wheaty o f f - f l a v o u r appeared lysis.  after  hydro-  The most e f f e c t i v e method f o r d e c r e a s i n g t h i s o f f -  f l a v o u r was a treatment w i t h g r a n u l a r a c t i v a t e d carbon a t 90*C. A m i l k - l i k e beverage made by adding b u t t e r was a c c e p t a b l e i n t a s t e , appearance and s t a b i l i t y .  The p r o t e i n t o carbohydrate  r a t i o o f the product was lower than t h a t o f cow's m i l k .  Add-  i t i o n o f other p r o t e i n s o r d e c r e a s i n g wheat s t a r c h by a s e l e c t i v e washing o f wheat f l o u r was suggested  t o improve the r a t i o .  iv  TABLE OF CONTENTS PAGE INTRODUCTION  1  LITERATURE REVIEW  4  A.  Chemistry of wheat protein and starch 1. Soluble proteins 2. Gluten proteins 3. Carbohydrates 4. L i p i d s  4 4 5 9 12  B.  Hydrolysis of wheat f l o u r  12  MATERIALS AND METHODS  16  A.  Materials  16  B.  Preparation of a c i d - s o l u b i l i z e d wheat f l o u r  17  C.  V i s c o s i t y measurement  17  D.  Methods of proximate analysis 1. Carbohydrates  17 17  2. Protein 3. Moisture 4. Ash  18 19 19  E.  C h l o r a n i l 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. B i o g e l P-2 column  24  7. Sephadex G-50  26  column  H.  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s  27  I.  A n a l y s i s o f ammonia, p r o t e i n - n i t r o g e n and nonprotein nitrogen  28  J.  Amino a c i d a n a l y s i s  29  K.  Foamability  29  L.  F l a v o u r improvement o f the s o l u b i l i z e d wheat flour  and foam s t a b i l i t y  . ^  1. S o l v e n t  :  30  washing  30  2. Heated a c t i v a t e d carbon treatment  RESULTS AND DISCUSSION of a c i d - h y d r o l y s e d  3Q  31  A.  Preparation  wheat f l o u r  31  B.  Proximate a n a l y s i s  32  C.  Apparent v i s c o s i t y  34  D.  G e l f i l t r a t i o n a n a l y s i s of the h y d r o l y s a t e  34  E.  G e l , f i l t r a t i o n a n a l y s i s of p r o t e i n  36  F.  A n a l y s i s o f ammonia, n o n p r o t e i n nitrogen  52  and p r o t e i n  G.  G e l f i l t r a t i o n a n a l y s i s of carbohydrate  55  H.  SDS-PAGE a n a l y s i s  62  I.  Amino a c i d a n a l y s i s  67  J.  F o a m a b i l i t y and foam s t a b i l i t y  69  K.  F l a v o u r improvement  69  L.  I m i t a t i o n m i l k product  72  VI  PAGE CONCLUSION  74  LITERATURE CITED  76  vii  LIST OF FIGURES FIGURE  PAGE  1.  E l u t i o n curve of proteins of hydrolysed wheat f l o u r eluted from Sepharose 4B column with phosphate buffer.  35  2.  E l u t i o n curve of proteins of wheat f l o u r hydrolysed with higher acid concentration (0.15 N HC1).eluted from Sepharose 4B column with phosphate buffer.  37  3.  E l u t i o n curve of unhydrolysed wheat starch eluted from Sepharose 4B column with phosphate buffer.  38  4.  E l u t i o n curve of carbohydrates of hydrolysed wheat f l o u r eluted from Sepharose 4B column with phosphate buffer.  39  5.  C a l i b r a t i o n curve of Sephadex G-150 (superfine) column eluted with phosphate buffer.  40  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 b u f f e r .  43  7.  C a l i b r a t i o n curve of Sephadex G-150 eluted with AUC.  44  8.  E l u t i o n curve of wheat f l o u r proteins eluted from Sephadex G-150 column with AUC.  47  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.  48  C n l o r a n i l t e s t of hydrolysed wheat f l o u r proteins eluted from Sephadex G-150 column i n AUC.  49  10.  column  •viii  PAGE  FIGURE 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.  51  12.  Ninhydrin test and Choranil t e s t of hydrolysed wheat f l o u r proteins eluted from Sephadex G-200 column i n phosphate buffer.  53  13.  Gel f i l t r a t i o n e l u t i o n 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.  56  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. > "  58  15.  C a l i b r a t i o n curve f o r Biogel P-2 column using corn syrup.  60  i  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.  61  17.  C a l i b r a t i o n curve f o r proteins i n SDS-polyacrylamide g e l electrophoresis.  63  18.  SDS-Polyacrylamide Gel Electrophoretic Pattern of the major peak of acid-hydrolysed wheat f l o u r protein, whole acid-hydrolysed wheat f l o u r protein and whole wheat f l o u r protein.  64  19.  SDS-Polyacrylamide Gel Electrophoretic Pattern of the major peak of acid-hydrolysed wheat f l o u r protein, whole acid-hydrolysed wheat f l o u r protein and whole wheat f l o u r protein, with or without reduction p r i o r to e l e c t r o phoresis.  66  ix  LIST OF TABLES TABLE  PAGE  1.  Proximate analysis of acid hydrolysed wheat flour.  33  2.  Proteins used i n c a l i b r a t i o n of Sephadex G-150 superfine column eluted with 0.01M phosphate buffer, pH 7.  42  3.  Proteins used i n c a l i b r a t i o n of Sephadex G-150 column eluted with AUC.  45  4.  Total-nitrogen, protein-nitrogen and ammonianitrogen content of the freeze-dried hydrolysed wheat f l o u r .  5^  5.  Comparison of amino acid content of untreated wheat f l o u r and hydrolysed wheat f l o u r .  68  6.  Foamability and foam s t a b i l i t y of hydrolysed wheat f l o u r , casein and soy protein i s o l a t e solutions.  70  X  ACKNOWLEDGEMENT  I would l i k e t o express my deepest g r a t i t u d e t o Dr. S. Nakai  o f the Department o f Food Science f o r h i s  i n v a l u a b l e a d v i c e , c o n s t r u c t i v e c r i t i s m and appropiate guidance  d u r i n g the course of t h i s  study.  I would a l s o l i k e t o thank the f a c u l t y members and graduate  students i n the Department o f Food S c i e n c e  1  f o r t h e i r suggestions and a s s i s t a n c e .  ;  1  INTRODUCTION  World average per capita protein i s expected to exceed average human n u t r i t i o n a l requirement i n 1980. Projections indicate there w i l l be a net positive world supply-demand balance of about 5»7 m i l l i o n metric tons of protein i n I98O.  Plant seed protein w i l l be a positive  supply-demand balance of about 8.2 m i l l i o n metric tons and animal proteins a negative balance of about 2 . 5 metric tons.  million  Wheat, coarse grains and oilseeds w i l l a l l  be i n general under supply (1). The short supply of animal proteins has pointed towards the d i r e c t i o n of more r a t i o n a l use of vegetable proteins as supplement or p a r t i a l replacements of animal proteins i n food.  Since the tastes of people f o r t h e i r  p r o t e i n - r i c h food of animal o r i g i n i s not going to change r a d i c a l l y , the preference w i l l s t i l l be f o r meats, dairy products, eggs, etc.  Therefore, i f food demands are to be  met by food producers, t r a d i t i o n a l products must be made available e s s e n t i a l l y i n t h e i r commonly accepted form at a high l e v e l of n u t r i t i o n a l q u a l i t y and organoleptic appeal. Vegetable proteins, l i k e 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 l i k e 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 o r i g i n ( 2 ) .  Recent r e a l i z a t i o n of surplus  plant protein and milk protein d e f i c i t has resulted i n a worldwide research trend  directed towards creation of dairy  product analogues based f u l l y or p a r t i a l l y on vegetable protein raw materials.  Soy ( 3 , 4 , 5 , 6 , 7 * 8 , 9 , 1 0 , 1 1 , 1 2 ) , peanut  (13»14) and coconut ( 1 5 » 1 6 , 1 7 ) proteins are the centre  of  i n v e s t i g a t i o n f o r the development of a m i l k - l i k e beverage. Among these three, soy protein has been most thoroughly studied.  Indeed the f i r s t successful commercial venture i n  producing a vegetable-based,  m i l k - l i k e beverage i s a sweetened  soy milk under the name •Vitasoy* i n 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 m i l l i o n metric tons of plant protein surplus ( 1 ) .  Over the l a s t 14  years the domestic consumption of wheat was only 27% of the t o t a l production.  The surplus wheat was exported and amounted  to between 20 - 2 5 $ of the t o t a l world trade of wheat (19,20, 21,22).  Since t h i s cereal i s rather well accepted by most  people and i s r e a d i l y available: i n t h i s country,  inexpensive  conversion of the bland f l o u r y endosperm to high q u a l i t y  3  m i l k - l i k e beverage o r o t h e r d a i r y s u b s t i t u t e s would c a n t l y broaden the u t i l i z a t i o n home and abroad.  signifi-  of t h i s c e r e a l g r a i n b o t h a t  LITERATURE.  A.  REVIEW  Chemistry o f wheat p r o t e i n and s t a r c h The  wheat g r a i n i s d i v i d e d r o u g h l y i n t o 3 p a r t s i n  the  f o l l o w i n g proportions  and  (c) husk 1 3 $ .  (a) germ 2?o, (b) endosperm 85#  D u r i n g m i l l i n g , the endosperm i s separated  from the outer husks and germs t o g e t h e r w i t h the r e d u c t i o n of the endosperm i n t o f l o u r which i s made up o f agglomerates o f the broken endosperm, f r e e s t a r c h g r a n u l e s ,  broken s t a r c h and  a broken p r o t e i n a c e o u s m a t r i x . There a r e 4 main c l a s s e s o f p r o t e i n i n wheat f l o u r s albumin, g l o b u l i n , g l i a d i n and g l u t e n i n .  The water s o l u b l e  albumin and s a l t s o l u b l e g l o b u l i n c o n s t i t u t e about 15 - 20$ of the t o t a l f l o u r p r o t e i n whereas the remaining 80 - 85$ i s the g l u t e n p r o t e i n c o n s i s t i n g o f g l i a d i n and g l u t e n i n . G l i a d i n i s s o l u b l e i n 70$ e t h a n o l whereas g l u t e n i n i s n o t . These 2 g l u t e n p r o t e i n s a r e present i n r o u g h l y the same proportion. 1.  Soluble  Proteins  Soluble  proteins are f l o u r proteins  dilute salt solution.  soluble i n  They a r e composed o f what i s known  c l a s s i c a l l y as albumins and g l o b u l i n s t o g e t h e r w i t h minor quantities of glycoproteins,  nucleoproteins  and many o f the  l i p i d - p r o t e i n complexes found i n wheat f l o u r . proteins  S a l t soluble  c o n t a i n about h a l f o f a l l the f l o u r SH and 20$ o f  the SS groups ( 2 3 ) .  The s a l t s o l u b l e p r o t e i n s  i n wheat f l o u r  5  are very heterogeneous.  In a gel electrophoretic analysis,  three d i f f e r e n t protein groups, with over 20  constituents  were found i n saline extract of wheat f l o u r with d i s t i n c t differences i n d i s t r i b u t i o n between Triticum vulgare and  T.  durum (24). 2.  Gluten Proteins The gluten proteins are prepared t r a d i t i o n a l l y as  a wet e l a s t i c mass by kneading a dough i n water to remove starch.  Gluten can be separated  into g l i a d i n and glutenin  f r a c t i o n s by the s o l u b i l i t y of g l i a d i n i n 70% ethyl a l c o h o l . The unusally high glutamic  acid content and high proline  content are d i s t i n c t i v e 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 i s p r o l i n e . R e l a t i v e l y large number of non-polar side chains contribute p o s s i b i l i t i e s f o r 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 l y s i n e , h i s t i d i n e and arginine r e s u l t s i n a low ionic character f o r the gluten proteins.  Certain  e s s e n t i a l amino acids such as l y s i n e , methionine and tryptophan are present i n r e l a t i v e l y small amount r e s u l t i n g i n low n u t r i t i o n a l q u a l i t y of the proteins.  The gluten proteins  have strong aggregation tendencies r e s u l t i n g from the hydrogenbonding p o t e n t i a l of the unusually large number of glutamine side chains, the p o t e n t i a l f o r apolar bonding of the many nonpolar side chains and t h e i r low i o n i c character.  Conse-  6  q u e n t l y they are u s u a l l y i n s o l u b l e near t h e i r i s o e l e c t r i c points  i n most aqueous s o l v e n t s .  the g l u t e n p r o t e i n s  The i s o e l e c t r i c p o i n t o f  g e n e r a l l y f a l l i n the pH range 6 - 9  (27,28). G l i a d i n and g l u t e n i n d i f f e r i n t h e i r p h y s i c a l p r o p e r t i e s , most n o t a b l y i n t h e i r v i s c o e l a s t i c i t y .  Gliadin  i s cohesive w i t h low  e l a s t i c i t y , whereas g l u t e n i n i s both  cohesive and  (29).  elastic  G l i a d i n i s composed of  proteins  of r e l a t i v e l y low m o l e c u l a r weight i n coraparision w i t h h i g h m o l e c u l a r weight p r o t e i n s  of g l u t e n i n f r a c t i o n .  the These  f r a c t i o n s a l s o d i f f e r s l i g h t l y i n amino a c i d compositions g l i a d i n tends to have l a r g e r amount of p r o l i n e , glutamic a c i d p l u s glutamine, c y s t i n e , i s o l e u c i n e , p h e n y l a l a n i n e and nitrogen  than g l u t e n i n whereas g l u t e n i n has  g l y c i n e , l y s i n e and  tryptophan  f o r various g l i a d i n  ranges from about 30,000 t o 80,000.  molecules are made up of s i n g l e p o l y p e p t i d e f o l d e d conformation by i n t r a m o l e c u l a r The  g l u t e n i n i s often considered  polypeptide  l a r g e r amount o f  (30).  M o l e c u l a r weight r e p o r t e d preparations  The  chains h e l d i n a  t o be made up of two and  or more  intramolecular  m o l e c u l a r weights of g l u t e n i n components  extend over a wide range and those of the g l i a d i n .  Gliadin  d i s u l f i d e bonds (31).  chains j o i n e d by i n t e r m o l e c u l a r  d i s u l f i d e bonds.  amide  i n general  are much h i g h e r than  Average m o l e c u l a r weight  ranges from 150,000 to 3 m i l l i o n s  (32).  reported  7  Moving boundary e l e c t r o p h o r e s i s aluminum l a c t a t e b u f f e r a t pH 3.1 r e v e a l e d y8 , T  o f wheat g l u t e n i n 3 major peaks, ot,  and a f o u r t h minor peak, Od (33). E l e c t r o p h o r e s i s i n  s t a r c h g e l showed t h a t the OL-component o f g l u t e n was a c t u a l l y made up o f the o t - g l i a d i n s and t h e g l u t e n i n which was t o o l a r g e t o migrate i n t o t h e s t a r c h g e l  (34)• From e i g h t t o t h i r t y  g l i a d i n components were d e t e c t e d phoresis  (34,35).  using starch gel e l e c t r o -  The g l i a d i n components were e l u t e d as a  s i n g l e peak i n g e l f i l t r a t i o n .  E v i d e n t l y the components which  were shown t o have d i f f e r e n t m o b i l i t i e s upon g e l  electropho-  r e s i s had a p p r o x i m a t e l y the same s i z e (36). Attempts t o determine m o l e c u l a r weights o f g l i a d i n components on columns c a l i b r a t e d with proteins  o f known m o l e c u l a r weight y i e l d e d a  range from 25,000 t o 50,000 (37,38,39). The  m o l e c u l a r weight o f the d i f f e r e n t g l i a d i n  components has been e x t e n s i v e l y  s t u d i e d by v a r i o u s  techniques.  M o l e c u l a r weight o f t h e o t - g l i a d i n f r a c t i o n was found t o be 49,000 by g e l f i l t r a t i o n (38).  and 55,000 from l i g h t s c a t t e r i n g  SDS-PAGE a n a l y s i s o f t h e ot-gliadin f r a c t i o n r e v e a l e d  6 bands r a n g i n g  i n m o l e c u l a r weight from about 30,000 t o  75,000 w i t h 2 major bands a t 32,000 and 36,000 (40),  Molecular  weight o f t h e ^ - g l i a d i n f r a c t i o n was found t o be 31,000 by ultracentrifugation  (41).  SDS-PAGE a n a l y s i s o f t h e j8-gliadin  f r a c t i o n showed 3 major s p e c i e s  and s e v e r a l minor ones.  The  major s p e c i e s  l i e d i n t h e m o l e c u l a r weight range o f 36,000 and  40,000 (40).  Minimum m o l e c u l a r weights o f T , - , 7k-,  and .  8  % -gliadins were estimated to be 16,00, 17»000 and 18,000 respectively from amino acid analysis (42).  Using u l t r a -  centrifugation, d i f f e r e n t molecular weights have been reported f o r r g l i a d i n i n d i f f e r e n t solvents. Values of 26,000 (43), 31,000 (44) and 37.000 (41) were reported f o r r g l i a d i n i n  4M  guanidine hydrochloride, i n 6M guanidine hydrochloride and i n aluminum lactate buffer r e s p e c t i v e l y .  Using sedimentation i n  a v a r i e t y of solvents, Sexson and Wu assigned molecular weight of 30,300 and 34,700 to 7[- and % - g l i a d i n s respectively (45). SDS-PAGE analysis showed that the main constituents of the T - g l i a d i n f r a c t i o n were within a molecular weight range of 38,000 to 44,000 (40).  Booth and Ewart (46) separated three  00 -protein components from Wichita f l o u r and one from Cappelle f l o u r by ion exchange chromatography on carboxylmethylcellulose. These proteins had very high content of proline and lanine but no cystine or methionine.  phenyla-  Molecular weight of the  2 Wichita proteins was found to be 73»000 and 7^.000 by u l t r a cent r i f ugation.  These values were consistent with the subunit  molecular weight of 69,300 and 78,100 f o r 0)-gliadin as determined by SDS-PAGE analysis (47).  The g l i a d i n peak separated  by g e l f i l t r a t i o n gave r i s e to 3 bands i n SDS  polyacrylamide  g e l electrophoresis corresponding to molecular weights 46,000, 38,000 and 33,000 (48).  SDS-polyacrylamide  g e l electrophoresis  of whole g l i a d i n prepared by alcohol extraction revealed that most g l i a d i n proteins have molecular weight near 36,500 (47). The whole g l i a d i n f r a c t i o n also contained 11,400 molecular  9  weight polypeptides, 44,200 and  a major.polypeptide of molecular weight  W-gliadins.  Glutenin constituted approximately 30 - 40$ of the protein i n wheat f l o u r and about h a l f of that i n gluten. Glutenin can be extracted from wheat f l o u r with d i l u t e acetic acid a f t e r previous extraction with saline and 70% alcohol. Some glutenin remained as g e l protein and could be extracted with solution of mercuric chloride, d i s s o c i a t i n g agent (urea or guanidine hydrochloride), detergents, agents (49).  a l k a l i or reducing  Although glutenin migrated as a single component  i n moving boundary electrophoresis with a mobility equivalent to the fastest moving g l i a d i n component (33), i"t was eluted f i r s t when a solution of gluten protein was applied to the gel column ( 3 6 ) .  SDS-polyacrylamide g e l electrophoresis of  reduced glutenin prepared by extracting gluten i n 70% ethanol which was 0.01N i n acetic acid and p r e c i p i t a t i n g at pH 6 . 5 , showed that glutenin was composed of subunits  of at least 15  d i s t i n c t molecular weights ranging from 11,600 to 133,000 (47). In another study, SDS-polyacrylamide g e l electrophoresis of reduced glutenin prepared by g e l f i l t r a t i o n on Sephadex G-100 column showed at l e a s t 8 bands (48). 3.  Carbohydrates The carbohydrates of wheat f l o u r are made up of  starch, pentosan, hemicellulose, c e l l u l o s e and sugars. Analysis of a low extraction f l o u r with 73•&% carbohydrate  10  showed t h a t there were 70.4$ s t a r c h , 2$ pentosan, 0.9$ and  sugars  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 s a c c h a r i d e s , glucose, f r u c t o s e , sucrose, maltose, r a f f i n o s e and a s e r i e s o f o l i g o s a c c h a r i d e s composed of D-fructose and D-glucose r e f e r r e d t o l e v o s i n e or g l u c o fructans  (50,51,52,53). Pentosan and h e m i c e l l u l o s e belong t o a group of  non-starchy  polysaccharide.  Pentosan i s u s u a l l y r e f e r r e d t o  as the water s o l u b l e and h e m i c e l l u l o s e as the water i n s o l u b l e pentose-containing  polysaccharide.  Upon h y d r o l y s i s , the hemi-  c e l l u l o s e s and pentosans y i e l d d e r i v a t i v e s of pentoses and hexoses.  The monomeric u n i t s most f r e q u e n t l y found i n the 2  p o l y s a c c h a r i d e s are the pentose sugars  D-xylose and L - a r a b i n o s e .  The major p o r t i o n o f the s o l u b l e pentosans i s a s t r a i g h t  chain  of anhydro-D-xylopyranosyl r e s i d u e s l i n k e d beta-1, 4 t o which are attached anhydro L - a r a b i n o f u r a n o s y l r e s i d u e s a t the 2 or 3 p o s i t i o n s o f i n d i v i d u a l anhydro-xylose u n i t s (54).  The b u i l d -  i n g b l o c k s o f i n s o l u b l e pentosan or h e m i c e l l u l o s e are s i m i l a r t o those of s o l u b l e pentosan (55)«  The w a t e r - s o l u b l e  and  water i n s o l u b l e . p e n t o s a n s were s t r u c t u r a l l y s i m i l a r and the main d i f f e r e n c e was t h e g r e a t e r degree o f b r a n c h i n g i n the latter  (56).  The unique b r a n c h i n g  s t r u c t u r e of water s o l u b l e  pentosan polymers i s r e s p o n s i b l e f o r t h e i r a b i l i t y t o form s o l u t i o n s o f high v i s c o s i t y and t h e i r a b i l i t y t o imbibe c o n s i d e r a b l e q u a n t i t i e s of water.  11  Starch i s the major component of wheat endosperm and hence, of the f l o u r prepared from i t .  Starch i s made up of 2  types of molecules, amylose, a l i n e a r or straight chain polysaccharides 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 i n t r o duced by ot-1, 6 linkages ( 5 7 ) .  There i s a branch, on the  average f o r 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 i n 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 d i r e c t l y or through water hydrate bridges to form r a d i a l l y oriented, micelles or c r y s t a l l i n e area of various degrees of order.  An intercon-  nected three dimensional m i c e l l a r l a t t i c e i s formed by the p a r t i c i p i t a t i o n of segments of i n d i v i d u a l molecules i n several m i c e l l a r areas.  As a r e s u l t , wheat starch i s insoluble i n  cold water although the starch molecule i s highly hydroxylated and therefore very hygroscopic.  Starch granules however  12  e x h i b i t a l i m i t e d c a p a c i t y f o r absorbing  water and  swell  reversibly. 4.  Lipids Commercial s t r a i g h t grade f l o u r c o n t a i n s l e s s than  2$ t o t a l l i p i d s .  Only a p a r t of the l i p i d s , the f r e e l i p i d s  can be e x t r a c t e d from wheat w i t h the u s u a l nonpolar f a t s o l v e n t s such as petroleum e t h e r .  P o l a r s o l v e n t l i k e water-  s a t u r a t e d n - b u t a n o l can e x t r a c t the more d i f f i c u l t l y bound l i p i d s as w e l l . water-saturated  About 1.5$  butanol  about t w o - t h i r d s  was  the t o t a l l i p i d was  (6l,62).  f r e e and  extracted  f l o u r has been removed by Of t h i s 1.5$  total  o n e - t h i r d bound.  lipid,  About h a l f of  p o l a r and almost a l l the bound l i p i d s  i n t o t h i s category.  T h i n l a y e r chromatography r e s o l v e d  t o t a l l i p i d f r a c t i o n i n t o 23 l i p i d c l a s s e s  (63).  fell  the  Wheat f l o u r  l i p i d c o n s i s t e d of a spectrum of compounds r a n g i n g from v e r y n o n p o l a r substances, such as s t e r o l s and t r i g l y c e r i d e s , n e u t r a l p o l a r l i p i d s , such as g a l a c t o g l y c e r i d e s and phospholipids  B.  to  charged  (64).  H y d r o l y s i s o f wheat f l o u r Wheat f l o u r i s u n s u i t a b l e f o r d i r e c t use as a d a i r y  s u b s t i t u t e 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 p r o p e r t i e s and the i s o e l e c t r i c p o i n t of 6.6 gluten proteins  (65)•  of  the  In a d d i t i o n , wheat s t a r c h e x i s t s as  13  g r a n u l e s i n wheat f l o u r and i s i n s o l u b l e i n c o l d water. s t a r c h s o l u b i l i z e d by h e a t i n g hibits  -  The  or chemical treatment s t i l l  h i g h v i s c o s i t y a t low s o l i d  ex-  content.  Wheat f l o u r can be s o l u b i l i z e d by enzyme or a c i d hydrolysis.  I n the enzymatic p r o c e s s , a p r o t e o l y t i c enzyme  i s r e q u i r e d t o break down the g l u t e n and a carbohydrase t o break down the wheat s t a r c h .  Wheat g l u t e n 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 o f 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 increase  undesirable  i n s m a l l fragments and f r e e amino groups (65).  l a t t e r potentiates  The  toward f l a v o u r changes, such as from  glutamate, and t o storage i n s t a b i l i t y by way o f the M a i l l a r d browning r e a c t i o n . and  P e p s i n treatment produced l a r g e r fragments  thus r e l a t i v e l y s m a l l amount of f r e e amino groups (66).  A scheme i n p r e p a r i n g  a m i l k - l i k e product from enzymatic  h y d r o l y s i s o f 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 w i t h 10 ppm p e p s i n f o r 2 t o 4 hours a t 30*C. c o n t a i n e d most of the p r o t e i n .  The s t a r c h c o n t a i n i n g  p i t a t e was washed and c e n t r i f u g e d . fuged s t a r c h was t r e a t e d w i t h  The supernatant preci- .  About 30% of the c e n t r i -  ot.-amylase, heated a t 70 *C f o r  30 minutes and f i n a l l y heated t o 90 *C f o r another 30 minutes t o destroy  the enzyme.  The n e u t r a l i z e d  pepsin-hydrochloric  a c i d e x t r a c t o f wheat f l o u r was mixed w i t h the OL-amylase t r e a t e d s t a r c h f r a c t i o n and a s t a b i l i z e r , p o l y s a c c h a r i d e B-14-59 was added a t a l e v e l o f 0.7 t o 1%.  gum  The enzyme p e p s i n  14  produces an excellent type and degree of hydrolysis but suffers from the disadvantages of r e s t r i c t e d supply and the low condition required f o r e f f e c t i v e action. cessing i s undesirable  because i t may  Low  pH  pH during pro-  lead to a b i t t e r flavour (67).  and a somewhat astringent mouthful i n the f i n a l product  A neutral protease prepared from B a c i l l u s s u b t i l i s has been used f o r the desirable type of l i m i t e d hydrolysis of f l o u r protein without recourse to low pH conditions. f l o u r protein was was  as low as 10$  Up to 85$  of  rendered d i s p e r s i b l e and dialyzable nitrogen (67).  S o l u b i l i z a t i o n of wheat f l o u r f o r m i l k - l i k e products using acid hydrolysis has not been reported.  The amide groups  on wheat protein are more r e a d i l y susceptible to acid hydrol y s i s than the peptide linkages.  Mild hydrolyzing  conditions  can break o f f ammonia from the amide to a considerable without appreciable hydrolysis of the peptide chain.  degree Such mild  and l i m i t e d hydrolysis of the amide group would increase  the  s o l u b i l i t y and decrease r e l a t i v e i n t e r a c t i n g tendencies of the protein (68). out at 100'C  Deamidation of a 1$ g l i a d i n solution was i n 0.008N to 0.04N hydrochloric a c i d 4 6 8 ) .  hydrolysis of the peptide bonds was 50$  When the degree of  greater than 10$,  became r e a d i l y soluble i n water at pH ?. g l i a d i n was  N6  observed even when over  of'*the amide groups were hydrolysed.  hydrolysis of the amide was  carried  also c a r r i e d by heating a 5.5$  g l i a d i n protein  Deamidation of g l i a d i n solution  i n 0.07N hydrochloric acid f o r 3 hours at 96*-98 G ,  (69).  15  M i l d a c i d h y d r o l y s i s has a l s o "been used t o s o l u b i l i z e wheat gluten  (70).  G l u t e n was s o l u b i l i z e d when the amide content  was reduced by approximately 1 0 $ . a t 85 - 92% y i e l d .  S o l u b l e g l u t e n was r e c o v e r e d  A c e t i c a c i d was found t o be m i l d e r than  h y d r o c h l o r i c a c i d f o r the g l u t e n m o d i f i c a t i o n .  Increased f r e e  amino groups were d e t e c t e d i n the s o l u b i l i z e d g l u t e n as t h e c o n c e n t r a t i o n o f a c i d i n c r e a s e d showing an i n c r e a s e d breakage of the p o l y p e p t i d e c h a i n . The a c i d h y d r o l y s i s o f s t a r c h i s w e l l e s t a b l i s h e d i n the glucose syrup and sugar i n d u s t r y .  When s t a r c h i s hydro-  l y s e d w i t h a c i d as t h e c a t a l y s t , a random cleavage o f t h e -C-O-C l i n k a g e occurs w i t h the p r o d u c t i o n o f glucose and many o f i t s polymers.  Upon c o n t i n u i n g the h y d r o l y s i s there i s an  i n c r e a s e i n t h e number o f the lower molecular weight s u g a r s . The  extent o f c o n v e r s i o n depends on a c i d c o n c e n t r a t i o n , time,  temperature  and p r e s s u r e d u r i n g the r e a c t i o n .  Acid conversion  has a p r a c t i c a l t o p l i m i t o f 55 Dextrose E q u i v a l e n c e w i t h 32$ dextrose because above t h i s v a l u e t h e r e i s much c o l o u r development and appearance o f a b i t t e r t a s t e element ( 7 1 ) .  Starch  h y d r o l y s i s i s complicated by secondary r e v e r s i o n r e a c t i o n . When breakdown has p r o g r e s s e d t o a c e r t a i n l e v e l , some o f the sugars formed a r e themselves  converted t o unwanted  which cause c o l o u r and b i t t e r t a s t e .  substances  Use of enzymes i n the  h y d r o l y s i s e l i m i n a t e the p r o d u c t i o n o f r e v e r s i o n and thus h i g h D. E . syrup can be produced.  16  MATERIALS. AND  A.  METHODS  Materials Wheat f l o u r was  Purpose E n r i c h e d F l o u r . from MCB.  C a s e i n was  a commercial  Wheat s t a r c h was  a purified  product  a s o l u b i l i z e d and f r e e z e - d r i e d sample  prepared i n the l a b o r a t o r y . of General M i l l I n c .  brand of Robin Hood A l l -  Soy p r o t e i n i s o l a t e was  L i l y White c o m  syrup was  a  a product  commercial  product from B e s t Foods. The sources were:  standard p r o t e i n s used i n t h i s study and ovalbumin  (5x  c r y s t a l l i z e d ) was f»; •  their  a product of  Calbiochem} ovomucoid and lysozyme were from Worthington chemical C o r p o r a t i o n ; conalbumin  and  yS-lactoglobulin  were from N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n .  Bio-  (bovine)  Chymotryp-  s i n o g e n A (bovine pancreas, 6x c r y s t a l l i z e d ) , cytochrome C (horse h e a r t ) and myoglobin Mann B i o c h e m i c a l . serum albumin trypsin  (sperm whale) were from  r - G l o b u l i n (human, Cohn f r a c t i o n I I ) ,  (bovine, f r a c t i o n V ) , t r y p s i n  inhibitor  pancreas, type 1-A,  Schwartz-  (bovine, type X I ) ,  (soybean, type 1-S), r i b o n u c l e a s e A 5x c r y s t a l l i z e d ) ,  oi-chymotrypsin  (bovine (bovine  pancreas, type I I , 3x c r y s t a l l i z e d ) , c a t a l a s e (bovine l i v e r ) and t h y r o g l o b u l i n (bovine, type 1) Chemical  were purchased from Sigma  Co. The Blue Dextran 2000, the Sepharose 4B g e l s and  Sephadex G - s e r i e s g e l s , G-50  f i n e , G-100, G-150, G-150  f i n e and G-200 were the products of Pharmacia F i n e  the  super-  Chemicals.  17  Biogel P-2 minus 400 mesh, was from Bio-Rad Laboratories.  B.  Preparation of a c i d - s o l u b i l i z e d wheat f l o u r Ten grams of wheat f l o u r was suspended i n 100 ml of  0.1N HCI. i n a 1000 ml erlenmeyer f l a s k .  The suspension was  heated to 121*C f o r 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 l i q u i d cool at the end of autoclaving).  The autoclaved  sample was cooled r a p i d l y , adjusted to pH 7 with concentrated NaOH s o l u t i o n and centrifuged at 8000 x g f o r 15 minutes. supernatant was freeze-dried.  The  The extent of s o l u b i l i z a t i o n  was determined from the weight of the freeze dried residue.  C.  V i s c o s i t y measurement The v i s c o s i t y of a 5$ solution of the hydrolysed  wheat f l o u r and a commercial skimmilk sample was measured with a Haake Rotovisko Viscometer.  The v i s c o s i t y was measured at 5  d i f f e r e n t shear rates ranging from 152 to 1370 s e c " using an 1  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 f o r carbohydrate determination. To a test tube containing  18  2 ml o f carbohydrate, 0.1  ml o f 80$ phenol was added f o l l o w e d  by a r a p i d a d d i t i o n o f 5 ml o f c o n c e n t r a t e d s u l f u r i c a c i d  from  a 50 ml b u r e t t e , w i t h t h e stream o f a c i d d i r e c t e d a g a i n s t t h e l i q u i d surface.  The t e s t tube was allowed t o s t a n d f o r 10  minutes b e f o r e t h e content was t h o r o u g h l y mixed. down, t h e absorbance  o f the r e a c t i o n mixture was measured a t  4-90 nm a g a i n s t a blank  (without carbohydrate) i n a 1 cm l i g h t  path c u v e t t e w i t h a Beckman DB spectrophotometer. of carbohydrate  Upon c o o l i n g  The amount  (glucose o r glucose polymers) was determined  by r e f e r r i n g t o a glucose s t a n d a r d curve and w i t h based on t h e degree  adjustment  o f polymerization o f the glucose  A l l samples were prepared and determined  polymers.  i n triplicate.  To prepare:a wheat f l o u r s o l u t i o n f o r carbohydrate d e t e r m i n a t i o n , about  0.4 gram o f wheat f l o u r was e x a c t l y  weighed out and d i s s o l v e d i n a minimum amount o f IN NaOH.  The  a l k a l i n e s o l u t i o n was n e u t r a l i z e d w i t h c o n c e n t r a t e d 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 t o 2 l i t r e s i n a v o l u m e t r i c f l a s k w i t h d i s t i l l e d water. (2)  Protein N i t r o g e n content of the u n t r e a t e d wheat f l o u r and  the h y d r o l y s e d wheat f l o u r was determined by a r a p i d K j e l d a h l procedure  (73)•  micro-  The p r o t e i n content was c a l c u l a t e d  by m u l t i p l y i n g the amount o f n i t r o g e n by a f a c t o r o f 5«7.  19  (3)  Moisture Moisture content of the u n t r e a t e d wheat f l o u r  the h y d r o l y s e d wheat f l o u r was sample a t 90'G  determined  and  by h e a t i n g a 10 gram  i n a i r oven u n t i l constant weight was  obtained.  (4) Ash Ash content was i n a type 1300  Chloranil The  by a s h i n g 1 gram sample  Thermolyne E l e c t r i c Furnace  or u n t i l constant weight  E.  determined  a t 600*C f o r 6 hours  reached.  test choloranil  test  (74) was  used t o determine  micro-  gram amount of amino a c i d s and s m a l l p e p t i d e s e l u t e d from g e l filtration  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  ( e t h a n o l s a t u r a t e d w i t h c h l o r a n i l ) , 2 ml of borate b u f f e r (0.05M sodium b o r a t e s o l u t i o n , added. 1*C  The  solution  f o r 1 hour.  was  pH 9)  and 5 ml of d i s t i l l e d water were  t h o r o u g h l y mixed and i n c u b a t e d at  Absorbance was  measured a t 350  nm  in a 1  65 cm  l i g h t path c u v e t t e a g a i n s t blank prepared i n the same way without  F.  sample.  Ninhvdrin test The n i n h y d r i n t e s t was  used t o d e t e c t ammonia, amino  a c i d s and s m a l l p e p t i d e s which emerged as the n o n p r o t e i n n i t r o g e n peak i n g e l f i l t r a t i o n . pared by d i s s o l v i n g  N i n h y d r i n reagent was  1 gram of n i n h y d r i n and 0.15  gram of  pre-  20  hydrindatin i n 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 i n a b o i l i n g water bath f o r 15 minutes.  The mixture was diluted with 3 ml of 5$ ethanol,  mixed and absorbance measured at 570 nm i n a 1 cm l i g h t path cuvette with a Beckman DB spectrophotometer  against blank  prepared i n 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 a p p l i c a t i o n . A 4 ml aliquot was applied and the column was eluted with phosphate buffer at a flow rate of 30 ml per hour. ions of 4 ml were c o l l e c t e d .  Fract-  The elution p r o f i l e of protein  was obtained by measuring absorbance of each f r a c t i o n at 280  nm.  The e l u t i o n p r o f i l e of carbohydrate was obtained by determining the carbohydrate content of each f r a c t i o n by the phenol-sulf u r i c acid method.  #  Eluants used i n g e l 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 d i s s o c i a t i n g medium of 0.1 M acetic acid, 3 M urea and 0.01 M cetyltrimethylammoniura bromide (AUC). The f r a c tions were collected i n a Gilson Medical Electronics f r a c t i o n c o l l e c t o r . Absorbance was measured-in a 1 cm l i g h t path cuvette with a Beckman DB spectrophotometer.  21  A s o l u b l e wheat s t a r c h sample was s o l v i n g 20 mg  of wheat s t a r c h i n 8 ml of IN NaOH.  a l k a l i n e s o l u t i o n was a c i d and was  dis-  The  n e u t r a l i z e d with concentrated s u l f u r i c  made up to 10 ml w i t h d i s t i l l e d water.  A s t a n d a r d i z a t i o n r u n was of  prepared by  c a r r i e d out u s i n g a sample  10 mg Blue Dextran 2000, 2 mg tryptophan and 5 mg t h y r o -  g l o b u l i n d i s s o l v i n g i n 4 ml of phosphate b u f f e r .  V o i d 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 (2)  Sephadex G-150  respectively.  s u p e r f i n e column (2.5  x 45.0  cm)  F i f t e e n grams of dry Sephadex-G-150 s u p e r f i n e suspended i n 1 l i t r e  of d i s t i l l e d water was  a b o i l i n g water bath f o r 5 hours. was  allowed t o s w e l l i n  The s w o l l e n g e l suspension  c o o l e d , deaerated and packed i n a 2.5  x 45.0  Column u s i n g an e x t e n s i o n tube and f o l l o w i n g the recommended by the manufacturer. 15 cm of water was was  cm Pharmacia procedure  An o p e r a t i n g p r e s s u r e of  employed d u r i n g p a c k i n g .  The packed column  e q u i l i b r a t e d f o r 2 days w i t h phosphate b u f f e r i n an upward  flow d i r e c t i o n .  The  o p e r a t i n g p r e s s u r e was  adjusted to give a  f l o w r a t e of, about 10 ml per hour d u r i n g e q u i l i b r a t i o n and i n subsequent r u n s . The  column was  operated i n an upward flow d i r e c t i o n .  The  i n l e t t u b i n g was  j o i n e d t o a 3-way v a l v e which was  ted  t o a one l i t r e m a r i o t t e f l a s k f i l l e d w i t h phosphate b u f f e r  and t o a 5 ml s y r i n g e t u b e . column v i a the s y r i n g e .  Sample of 3 ml was  The e l u a n t was  connec-  a p p l i e d t o the  collected i n 2  ml  22  fractions immediately a f t e r the sample drained clear of the syringe.  The column was eluted with another 3 ml of phosphate  buffer from the syringe before started e l u t i n g from the mariotte f l a s k .  A t o t a l of 130 fractions were c o l l e c t e d .  The elution p r o f i l e of protein was absorbance of each f r a c t i o n at 280  obtained by measuring the 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)  F i f t e e n grams of dry Sephadex G-150 1 l i t r e of AUC  was suspended i n  and was allowed to swell at room  f o r 3 days with occasional s t i r r i n g .  The swollen gel was  deaerated and packed as i n the previous column. column was equilibrated f o r 2 days with AUC direction.  temperature  The packed  i n an upward flow  A flow rate of about 20 ml per hour was  during e q u i l i b r a t i o n and i n subsequent runs.  maintained  The column set  up, sample application, f r a c t i o n c o l l e c t i o n and protein detect i o n were the same as i n the previous column except that AUC was used both as eluant and as sample solvent. An AUC wheat f l o u r extract was prepared by blending 4 grams of wheat f l o u r with 69 ml of AUC f o r 2 minutes i n a S o r v a l l omnimixer operated at the top speed.  After standing  at 25*C f o r 2 hours, the blended mixture was centrifuged at 35,000 x g f o r 30 minutes.  A 3 ml aliquot of the deaerated  supernatant was applied to the column.  1  23  An AUC extract of the freeze-dried hydrolysed wheat f l o u r was prepared s i m i l a r l y .  Again a 3 ml aliquot of the  deaerated supernatant was applied to the column.  Protein  eluted from t h i s run was monitored both by absorbance at 280 nra and by the c h l o r a n i l t e s t . The column was c a l i b r a t e d with standard proteins. Void Volume was estimated from the e l u t i o n volume of Blue Dextran 2000. AUC.  The*Blue Dextran 2000 dissolved very slowly i n  Thus i n preparing t h i s 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 c a l i b r a t e d . * * A sample containing 0.3 gram of the freeze-dried hydrolysed wheat f l o u r i n 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 t o t a l of 120  four-ml f r a c t i o n s were c o l l e c t e d . The absorbance of each f r a c t i o n was measured at 280 nm.  One m i l l i l i t r e from each f r a c t i o n was used f o r the  ninhydrin t e s t .  Another m i l l i l i t r e from each of the fractions  within the absorbance peak was used f o r the c h l o r a n i l t e s t . (5) Sephadex G-100  column (2.5 x 100.0  cm)  The upward flow column was packed and c a l i b r a t e d . * * ** The column was prepared and calibrated by Mr. C. Y. Ma, Dept. of Food Science, U. B. C.  24  The major carbohydrate peak of h y d r o l y s e d wheat f l o u r e l u t e d from the Sepharose 4B column was A sample c o n t a i n i n g 5 mg i n 5 ml o f phosphate column was  pooled and  freeze-dried.  of the f r e e z e - d r i e d product d i s s o l v e d  b u f f e r was  a p p l i e d t o the column.  The  e l u t e d w i t h phosphate b u f f e r from a r e s e r v o i r .  t o t a l o f one hundred  5-ml  f r a c t i o n s were c o l l e c t e d .  l i t r e s from each f r a c t i o n was  Two  A milli-  t r a n s f e r r e d t o a t e s t tube f o r  carbohydrate d e t e r m i n a t i o n by p h e n o l - s u l f u r i c a c i d method. (6) B i o g e l P-2  Column  About 200  grams o f d r y g e l was  shaken through 2  s i e v e s , 200 mesh and 325 mesh, such t h a t 160  grams o f dry g e l  o f p a r t i c l e s i z e between 43 - 74/*. were o b t a i n e d .  The g e l was  a l l o w e d t o s w e l l over n i g h t i n excess d i s t i l l e d water. h y d r a t i o n , the g e l was  After  f r a c t i o n a t e d by r e p e a t e d s e t t l i n g  and  d e c a n t i n g the f i n e s u n t i l a sharp zone o f s e t t l i n g g e l p a r t i c l e s was  attained.  The g e l s l u r r y was  deaerated b e f o r e p a c k i n g .  The chromatographic column was made up of 2 jacketed pyrex g l a s s tubes (1.5 x 100.0  cm) p u l l e d out a t the bottom  and plugged on the top by rubber s t o p p e r s .  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 o t h e r was from the r e s e r v o i r .  the i n l e t f o r eluant  The lower end of the f i r s t tube  was  j o i n e d t o the top of the second, tube by narrow p l a s t i c tubing: so as t o g i v e an e f f e c t i v e column l e n g t h of 200  cm.  chromatographic tubes were j a c k e t e d by 2 l a r g e r tubes 95.0  cm) f i t t e d w i t h d r i l l e d rubber stoppers a t both  The 2 (4.4 x. ends.  25  The 2 tubes were packed i n d i v i d u a l l y b e f o r e connecting.  Each tube was f i r s t  f i l l e d t o h a l f i t s length with  deaerated d i s t i l l e d water.  Deaerated g e l suspension was then  poured i n t o the tube... When a g e l l a y e r o f 2 - 4 cm was formed at  the bottom of t h e tube, a slow stream o f water was allowed  to  flow-out.  the  More g e l suspension was poured i n t o the tube as  water l e v e l f e l l u n t i l t h e tube was packed t o about 1 cm  from the t o p .  The 2.packed  tubes were connected.  T i g h t and  c l o s e p a c k i n g was a c h i e v e d by pumping a s o l u t i o n o f 0.1$ NaCI w i t h a M i l t o n Roy Minipump a t a flow r a t e of 40 ml per hour. A f t e r compacting, f u r t h e r a d d i t i o n o f t h e g e l suspension was n e c e s s a r y t o b r i n g t h e l e v e l o f the packed g e l t o about 1.5 cm from the t o p .  The packed and connected column was e l u t e d w i t h  b o i l e d d i s t i l l e d water from a r e s e r v o i r maintained a t 80*C i n a constant temperature water bath w i t h a r e g u l a t e d flow r a t e of 28 ml p e r hour by a M i l t o n Roy Minipump.  After eluting for  5 hours t o e l i m i n a t e d i s s o l v e d oxygen, t h e pumping and the flow were stopped.  The column was heated by a constant temp-  e r a t u r e 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 j a c k e t s and maintained a temperature o f 65 *C.  After heating  up t o t h e r e q u i r e d temperature, the pump was t u r n e d on a g a i n to  s t a r t the flow.  The column was e l u t e d f o r 10 hours f o r  equilibration. In a t y p i c a l run, t h e column was f i r s t heated up t o 65*C. ing  No eluant was pumped through the g e l bed d u r i n g h e a t -  up.  The column was e l u t e d w i t h 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 c a l i b r a t i o n , 0 . 3 ml of 5 0 $ com solution was injected into the column.  syrup  The eluant was moni-  tored f o r carbohydrates using an E-C column monitor which measures the r e f r a c t i v e index. For analysis of carbohydrate composition of the hydrolysed wheat f l o u r , 0 . 2 ml of a 2 0 $ hydrolysed wheat f l o u r solution was injected into the column.  The eluant was  collec-  ted i n 2 ml fractions and 1 5 0 f r a c t i o n s were c o l l e c t e d .  The  e l u t i o n pattern of the carbohydrates was determined by taking 0 . 4 ml from each f r a c t i o n , d i l u t i n g to 2 ml with d i s t i l l e d water and performing the phenol-sulfuric acid t e s t .  The  amount of carbohydrate i n each peak was obtained by pooling the f r a c t i o n s of i n d i v i d u a l peak and determine the carbohydrate by the phenol-sulfuric acid method?. ( 7 ) Sephadex G-50  column (2 x 9 5  cm)  The column operated i n a downward flow d i r e c t i o n was packed and calibrated ( 7 5 ) P-2  The f r a c t i o n s (from Biogel  column) of the hydrolysed wheat f l o u r 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 r e s e r v o i r . tions were c o l l e c t e d .  A t o t a l of two hundred; 2-ml f r a c -  The carbohydrate of each f r a c t i o n was  27  determined by the p h e n o l - s u l f u r i c a c i d method.  H.  SDS-polyacrvlamide g e l  electrophoresis  Both the wheat f l o u r and h y d r o l y s e d  wheat f l o u r were  d e f a t t e d by s e v e r a l e x t r a c t i o n s w i t h dry n - b u t a n o l .  AUC  e x t r a c t s of d e f a t t e d samples were prepared as d e s c r i b e d viously.  The  two  AUC  The The  hydrolysed w i t h AUC  f o r 3 days w i t h 2 changes  d i a l y s e d samples were f r e e z e - d r i e d . 24,000 m o l e c u l a r weight p r o t e i n peak of  wheat f l o u r e l u t e d from the Sephadex G-150  was  prepared by p o o l i n g the peak f r a c t i o n s .  pooled peaks from s e v e r a l runs were d i a l y s e d a g a i n s t volumes of d i s t i l l e d water a t 4*C o f water.  The  d i a l y s e d sample was  b o i l i n g ethanol before  by Weber and  Osborn (77).  et a l (76)  A s l i g h t l y modified Instead  procedure was  1%  modified  p-mercaptoethanol)  cytochrome C electropho-  adopted f o r  of i n c u b a t i n g at 37*C f o r  2 hours, the p r o t e i n sample i n 0.01M Ifo SDS,  carried  Bovine serum albumin, ovalbumin,  £-lactoglobulin, lysozyme and  the p r o t e i n s .  500  use.  were used as m o l e c u l a r weight standards i n each  reducing  The  r e c r y s t a l l i z e d from  t o the method of S h a r p i r o  trypsin inhibitor,  column  freeze-dried.  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s was out a c c o r d i n g  the  f o r 3 days w i t h 2 changes  Sodium dodecyl s u l f a t e was  r e t i c run.  500  e x t r a c t s were d i a l y s e d a g a i n s t  volumes of d i s t i l l e d water a t V C o f water.  pre-  was  phosphate b u f f e r (pH incubated  at 95"-100*C  7»  28  for  5 minutes.  /S-Mercaptoethanol  was  omitted  from Samples  not r e q u i r i n g r e d u c t i o n .  Amount of p r o t e i n used i n each g e l  ranged from 15 t o 40 jag.  The  the s e p a r a t i n g g e l was at  7mA  per g e l f o r 3-4  7.5$.  The  Bromophenol blue was  used as  an  distance After elec-  i n a Pharmacia g e l d e s t a i n e r ,  photographs of the g e l s were taken.  The  m o b i l i t y of a p r o t e i n  c a l c u l a t e d as d i s t a n c e of p r o t e i n m i g r a t i o n ( i n photograph)  Mobility =  I.  i n a Pharmacia g e l  s t a i n i n g were measured.  t r o p h o r e t i c a l l y destained  in  c a r r i e d out  l e n g t h of the g e l and the  moved by the dye b e f o r e  band was  of acrylamide  E l e c t r o p h o r e s i s was  hours a t 15'C  e l e c t r o p h o r e s i s apparatus. i n t e r n a l standard.  concentration  :  length a f t e r destaining ( i n photograph)  X  A n a l y s i s of ammonia, p r o t e i n - n i t r o g e n and  —  length before staining d i s t a n c e of dye migration  nonprotein  nitrogen F i v e grams of the h y d r o l y s e d s o l v e d i n 20 ml of d i s t i l l e d water. i n d i a l y s i s c a s i n g a g a i n s t 100 4"C  wheat f l o u r were d i s -  The  s o l u t i o n was  volumes of d i s t i l l e d water a t  f o r 3 days w i t h 2 changes of d i s t i l l e d water.  d i a l y s i s , the d i a l y s e d sample was with d i s t i l l e d The f l o u r was  dialysed  After  c a r e f u l l y made up to 50  ml  water. t o t a l n i t r o g e n content  of the h y d r o l y s e d  wheat  determined by a r a p i d M i c r o - K j e l d a h l procedure  u s i n g 1 ml sample of 10fo h y d r o l y s e d  wheat f l o u r s o l u t i o n .  (73)  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 f l o u r s o l u t i o n with the same procedure but omitting the digestion step. nonprotein nitrogen was  J.  The  determined by subtraction.  Amino acid analysis Freeze-dried hydrolysed wheat f l o u r and wheat f l o u r  were hydrolysed with p-toluenesulfonic acid and 3 - (2 - amino ethyl) indole at 110*C  f o r 24 hours (78).  Amino acids were  determined on a single column system (Durrum Chem. Corp., Palo A l t o , C a l i f . ) 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 f o r 5 minutes i n a Sunbeam Mixmaster at a speed s e t t i n g of 10. 500  The foam was  quickly transferred to a  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 f o r  a c e r t a i n volume of drainage to be c o l l e c t e d .  30 L.  Flavour improvement of the s o l u b i l i z e d wheat f l o u r  (1) Solvent washing  ''  One hundred grams of wheat f l o u r were suspended i n 1000 ml of a solvent.  The suspension was beaten i n a kitchen  a i d mixer f o r 30 minutes at 58 rpm.  The beaten suspension was  centrifuged at 16,000 xg f o r 20 minutes.  The supernatant was  discarded and the residue was washed i n 1000 ml of water before resuspended i n 1000 ml 0.1 N HC1 and autoclaved at 121*C f o r 15 minutes.  The solvents used were water, water  saturated butanol, ethanol (95$) phosphate solution at pH  and 0.05  M sodium hexameta-  3.  (2) Heated activated carbon treatment Granular activated carbon from Whitco Chemical Co. 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 jacketed column (2 x 40 cm) to a l e v e l of 2/3  of the column length.  The column was heated up to and  maintained at 90 *C with a constant temperature c i r c u l a t o r . A 10$ solution of the hydrolysed wheat f l o u r was b o i l e d and passed through the heated column at a rate of about 30 ml per  minute.  31  RESULTS AND DISCUSSION  (A)  P r e p a r a t i o n o f a c i d - h y d r o l y s e d 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 a c i d h y d r o l y s i s i n  the present  i n v e s t i g a t i o n . Two groups o f a c i d s c o u l d be used,  o r g a n i c a c i d s and m i n e r a l a c i d s .  A c e t i c a c i d was found t o be  s i g n i f i c a n t l y m i l d e r than h y d r o c h l o r i c a c i d i n the s o l u b i l i z a t i o n o f wheat g l u t e n (70). r e q u i r e d f o r phosphoric  A much h i g h e r c o n c e n t r a t i o n was  a c i d than h y d r o c h l o r i c a c i d t o s o l u -  b i l i z e wheat f l o u r t o a s i m i l a r extent  (79).  Consequently,  h y d r o c h l o r i c a c i d 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 a c i d was n o t c o n s i d e r e d because o f i t s p o s s i b l e damage on the n u t r i t i o n a l q u a l i t y o f p r o t e i n s . found t o impart The  S u l f u r i c a c i d was  a b i t t e r t a s t e t o the h y d r o l y s a t e .  degree o f 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 a c i d c o n c e n t r a t i o n , the f l o u r c o n c e n t r a t i o n and the extent o f heat t r e a t m e n t .  Increased  s o l u b i l i z a t i o n was  observed w i t h i n c r e a s i n g a c i d c o n c e n t r a t i o n , i n c r e a s i n g heat treatment and d e c r e a s i n g f l o u r c o n c e n t r a t i o n . s o l u b i l i z e d product f l o u r suspension minutes.  An acceptable  was obtained by a u t o c l a v i n g a 10 percent  i n 0.1N h y d r o c h l o r i c a c i d a t 121*C f o r 15  Over 90 percent  under t h i s c o n d i t i o n .  o f f l o u r s o l i d s was s o l u b i l i z e d  Higher a c i d c o n c e n t r a t i o n and heat  treatment i n c r e a s e d the y i e l d but r e s u l t e d i n an u n d e s i r a b l e brown-coloured product.  Higher a c i d c o n c e n t r a t i o n a l s o  32  i n c r e a s e d the s a l t i n e s s of the t a s t e of the h y d r o l y s e d a f t e r n e u t r a l i z a t i o n w i t h sodium h y d r o x i d e .  product  Using f l o u r con-  c e n t r a t i o n s g r e a t e r than 10 percent i n c r e a s e d the browning and decreased  the y i e l d , however, u s i n g c o n c e n t r a t i o n s l e s s  than  10 percent would be uneconomical. The  f r e e z e - d r i e d powder of the s o l u b i l i z e d wheat  f l o u r had c o l o u r s i m i l a r t o t h a t of the u n t r e a t e d f l o u r . was  r e a d i l y s o l u b l e i n c o l d water.  The aqueous s o l u t i o n  l i g h t l y straw c o l o u r e d and possessed salty taste. i n the  B.  It was  a s l i g h t l y sweet and  A s t r o n g wheaty o f f - f l a v o u r was  also detected  solution.  Proximate a n a l y s i s Table 1 shows the r e s u l t of proximate a n a l y s i s of  the h y d r o l y s e d wheat f l o u r as compared to the o r i g i n a l wheat flour.  Hydrolysed wheat f l o u r I and  I I were two  separate  p r e p a r a t i o n s h y d r o l y s e d under the same c o n d i t i o n s . of the h y d r o l y s e d products I and I I were 95.3$ and respectively.  The  yields  93.3$,  The s l i g h t l y h i g h e r y i e l d i n product I  was  probably,  due t o a l o n g e r t e m p e r a t u r e - r i s i n g time d u r i n g auto-  claving.  The  carbohydrate  and p r o t e i n content of the t r e a t e d  and u n t r e a t e d samples were q u i t e s i m i l a r . was  Apparently  there  s i m i l a r degree of s o l u b i l i z a t i o n of wheat p r o t e i n and  wheat s t a r c h d u r i n g a c i d h y d r o l y s i s .  The  ash content i n the h y d r o l y s e d samples was  significantly due  t o the  higher  presence  33  Table 1  Proximate a n a l y s i s o f a c i d h y d r o l y s e d  wheat f l o u r .  Proximate composition •  Sample Protein*  Carbohydrate**  Moisture*  Ash*  11.3$  73.7$  11.1$  0.3$  10.6$  74.0$  6.7$  6.0$  10.7$  76.9$  5.4$  6.1$  Untreated Wheat Flour  Hydrolysed Wheat F l o u r (I)  Hydrolysed Wheat F l o u r (II)  *  Average o f d u p l i c a t e a n a l y s i s .  **  Average o f t r i p l i c a t e a n a l y s i s . The values from the g l u c o s e standard curve had been a d j u s t e d by a c o r r e c t i o n f a c t o r o f 0.9 f o r the wheat s t a r c h and a c o r r e c t i o n f a c t o r of 666/720 f o r the s a c c h a r i d e s o f the h y d r o l y s e d wheat f l o u r (assuming an average degree o f p o l y m e r i z a t i o n o f 4 f o r the s a c c h a r i d e s ) .  34  of sodium c h l o r i d e r e s u l t e d from n e u t r a l i z a t i o n w i t h 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° s o l u t i o n of  the h y d r o l y s e d wheat f l o u r , as c a l c u l a t e d by a v e r a g i n g the apparent v i s c o s i t i e s at d i f f e r e n t shear r a t e s , was poises  at 4'C.  4.9  A commercial skimmilk sample measured  c a l c u l a t e d s i m i l a r l y gave a v a l u e of 5.7  centiand  centipoises.  A p p a r e n t l y , d u r i n g a c i d h y d r o l y s i s , s t a r c h g r a n u l e s , molecules of amylose and  amylopectin and wheat g l u t e n were  extensively  degraded or m o d i f i e d , 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 a n a l y s i s of the  hvdrolvsate  G e l f i l t r a t i o n chromatography was  used t o study  extent of degradation of the wheat p r o t e i n s and s t a r c h , caused by a c i d h y d r o l y s i s . used t o f r a c t i o n a t e  (36,80) and  the wheat  T h i s technique has  been  t o determine the molecular  weight d i s t r i b u t i o n of the wheat p r o t e i n s  (39).  F i g . 1 shows  the e l u t i o n p r o f i l e of h y d r o l y s e d wheat f l o u r p r o t e i n w i t h phosphate b u f f e r from a Sepharose 4B absorbance peaks were observed.  column.  A major peak was  eluted  Three eluted  the v o i d volume corresponding to m o l e c u l a r weights of 3 m i l l i o n s or over.  One  peak was  the  observed at the  elution  at  2.0  Fig. 1  E l u t i o n curve of p r o t e i m of h y d r o l y s e d wheat f l o u r e l u t e d from Sepharose 4B column w i t h phosphate b u f f e r .  36  volume of tryptophan corresponding to a molecular weight of 3,00,000 or under.  The t h i r d broad peak was eluted between  the above two peaks.  F i g . 2 shows the elution p r o f i l e of  wheat f l o u r proteins hydrolysed at a higher acid concentration.  A considerable increase i n the size was observed f o r  the peak at the trytophan p o s i t i o n suggesting either more extensive degradation of proteins or the appearance of more browned or conjugated small compounds. Fig.  3 shows the elution p r o f i l e of untreated wheat  starch eluted from Sepharose 4-B column.  Most of the carbohy-  drate appeared at the void volume i n d i c a t i n g molecular weight ••I  of 3 m i l l i o n s or over.  F i g . 4- shows the elution p r o f i l e of  carbohydrates of hydrolysed wheat f l o u r eluted from the same column.  The major carbohydrate peak s h i f t e d from the void  volume to the e l u t i o n volume of tryptophan.  This c l e a r l y  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 p l o t t i n g the logarithm of the molecular weights of the standard proteins against the r a t i o s of t h e i r elution volumes to the void volume of the column (81). Fig.  5 i s the c a l i b r a t i o n curve of a Sephadex G-150 superfine  o  03 CO  00 CN  c= ca  CO  Elution volume ( m l . ) Fig. 2  E l u t i o n curve of p r o t e i n s of wheat f l o u r h y d r o l y s e d w i t h h i g h e r a c i d concentrat i o n (0.15 N HCI) e l u t e d from Sepharose 4B column w i t h phosphate b u f f e r .  -N3  F i g . :3  E l u t i o n curve o f unhydrolysed wheat s t a r c h e l u t e d from Sepharose 4-B column w i t h phosphate "buffer. CO  2.0-1  1.54  cn  c=  1.0-1  CO  -C2  0.54 6 7 ),000  160  E l u t i o n volume Fig.  /  """"  '- ^ m  320  '  1  :—i 480  (ml.)  E l u t i o n curve of carbohydrates of h y d r o l y s e d wheat f l o u r e l u t e d Sepharose 4-B column w i t h phosphate b u f f e r .  from  40  3.0-|  26H B> c y t o c h r o m e C 8  ribonuclease A  2.2-1 •chymotrypsin  \  /3-lactog l o b u l i n  1.8 ovomucoid i  ^ovalbumin  conalbumin.  1.4-  J i o v i n e serum a l b u m i n (monomer)  (dimer) bovine serum albumin »catalase  I 3  .8 .9 1  Molecular  Fig. 5  I  4  weight  I 5  I  I I I I 6 7 8 9 1 0  20  30  (xicr )  C a l i b r a t i o n curve o f Sephadex G-150 e l u t e d w i t h phosphate b u f f e r .  4  ( s u p e r f i n e ) column  41  column operated i n phosphate b u f f e r . used were l i s t e d i n Table 2.  The standard  proteins  The curve i s a s t r a i g h t l i n e  between m o l e c u l a r 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-  l y s e d wheat f l o u r p r o t e i n s from the Sephadex G-150 column.  superfine  The major peak a g a i n appeared a t the v o i d volume  s u g g e s t i n g a m o l e c u l a r weight of 400,000 o r h i g h e r .  The.  other absorbance peak had m o l e c u l a r weight l e s s than 10,000 and  c o u l d be n o n p r o t e i n n i t r o g e n  compounds due t o p r o t e i n  degradation during a c i d h y d r o l y s i s . Gel f i l t r a t i o n patterns proteins  of the h y d r o l y s e d wheat  i n phosphate b u f f e r might not g i v e a t r u e  picture  o f the e x t e n t of p r o t e i n d e g r a d a t i o n d u r i n g a c i d h y d r o l y s i s . Hydrolysed wheat p r o t e i n s might aggregate i n a n e u t r a l phosphate b u f f e r s o l u t i o n .  The huge absorbance peak a t the v o i d  volume from both Sepharose 4B and Sephadex G-150 gel  superfine  f i l t r a t i o n s t u d i e s c o u l d be due t o aggregates of hydro-  l y s e d wheat p r o t e i n s .  In a d d i t i o n , as g l u t e n i s i n s o l u b l e i n  phosphate b u f f e r , a d i r e c t comparision of g e l f i l t r a t i o n patterns  between the u n t r e a t e d  p r o t e i n s were i m p o s s i b l e .  and the h y d r o l y s e d wheat f l o u r  A h i g h l y d i s s o c i a t i n g solvent  t a i n i n g 0.1M a c e t i c a c i d , 3M u r e a and 0.01M  cetyltrimethy-  lammonium bromide (AUC) was used f o r t h i s purpose. solvent  con-  This  c o u l d d i s s o l v e about 95f° of f l o u r p r o t e i n which was  d i s s o c i a t e d t o 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 p r o t e i n s  (Table  3), e l u t e d from a  42  Table 2  Proteins used i n c a l i b r a t i o n of Sephadex G-150 superfine column eluted with 0.01M phosphate buffer, pH 7.  Proteins  Ve/Vo  Catalase  Mol. wt.  (Reference)  1.11  244,000  ( 89  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  1.83  36,800  ( 77  1.66  27,000  ( 81  2.04  22,500  ( 81  Ribonuclease A  2.38  13.600  ( 81  Cytochrome C  2.43  11,700  ( 77  Bovine serum albumin  /3-Lactoglobulin Ovomucoid od -Chymo t r y p s i n  Ve:  E l u t i o n volume  Vo:  Void volume  (Dimer)  Fig. 6  E l u t i o n curve of p r o t e i n s of h y d r o l y s e d wheat f l o u r e l u t e d from Sephadex G-150 ( s u p e r f i n e ) column w i t h phosphate b u f f e r .  44  3.0-  • ribonuclease A  2.6-t  ^myoglobi n •  2.2chymotrypsinogen  A  t rypsi n  ••ovomucoid  1.8-1  ovalbumi n  1.4-  • b o v i n e s e r u m a l b u m i n (monomer) •.conalbumin  T T T  .8 9 1  T  2  b o v i n e s e r u m a l b u m i n ( d i m e r1 ?1* * — • " " m a n 7 globulin  3  4  Molecular weight Fig. 7  5  |  6  [111  7 8  (xicr  C a l i b r a t i o n curve of Sephadex G-150 AUC.  4  910  |  20  ) column e l u t e d  with  ?  30  45  Table 3  Proteins used i n c a l i b r a t i o n of Sephadex G-150 column eluted with AUC.  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  13 ,-600  ( 81 )  Tryptophan  3.38  2  240  N-Ethylraaleimide  3.34  4  125  Proteins  Vet Vos  E l u t i o n volume Void volume  *Mol. wt. provided by supplier.  l  )  46  Sephadex G-150  column with AUC.  S i g n i f i c a n t deviation from  the c a l i b r a t i o n curve was observed f o r ovalbumin.  Fig. 8  shows an elution p r o f i l e of untreated wheat f l o u r protein from the AUC eluted Sephadex G-15Q  column.  Unlike an elution  p r o f i l e of wheat f l o u r proteins on an AUC eluted Sephadex G-200 column which separated into d i s t i n c t glutenin and g l i a d i n peaks (39)» no clear separation of glutenin and g l i a d i n peaks was observed.  Possible explanations f o r t h i s difference are  i n poorer resolution of Sephadex G-150  g e l at a higher mole-  cular weight range and i n v a r i e t y difference.  Elution profiles  of wheat proteins s i m i l a r to F i g . 8 were also reported i n another study using a Sephadex G-150  g e l column (82).  Both  the albumin and the nonprotein nitrogen peaks i n F i g . 8 were quite small and most of the g l i a d i n s were found between molecular weights of 40,000 and 100,000.  F i g . 9 i s an elution  curve of hydrolysed wheat f l o u r proteins from the AUC Sephadex G-150  column.  eluted  Comparision between F i g . 8 and F i g . 9  shows that almost a l l the glutenins and higher molecular weight g l i a d i n s 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 y i e l d i n g a large amount of,nonprotein nitrogen during acid hydrolysis.  F i g . 10  shows an elution p r o f i l e of the hydrolysed wheat f l o u r proteins eluted from the same Sephadex G-150  column but detected by the  0.5 10,000  80  120  160  200  240  E l u t i o n volume ( m l . ) Fig. 8  E l u t i o n curve o f wheat f l o u r p r o t e i n s e l u t e d from Sephadex G - 1 5 0 column with AUC.  100,000  80  25,000  120  Elution Fig. 9  10,000  160  200  240  volume (ml.)  E l u t i o n curve o f p r o t e i n s of h y d r o l y s e d wheat f l o u r e l u t e d from Sephadex G-150 column w i t h AUC.  100,000  80  25,000  120  Elution F i g . 10  C h l o r a n i l t e s t of hydrolysed G-150 column i n AUG.  10,000  160  200  240  volume (ml.) wheat f l o u r p r o t e i n s e l u t e d from Sephadex  50 chloranil test.  The two elution p r o f i l e s are quite s i m i l a r  except f o r the r e l a t i v e l y smaller nonprotein nitrogen peak detacted by the c h l o r a n i l t e s t .  In the c h l o r a n i l reaction,  the amino groups i n amino acid and protein form n-ir charge transfer complex with c h l o r a n i l , r e s u l t i n g i n a red s h i f t of the c h l o r a n i l band on complexing (83)•  The t e s t i s capable  of measuring microgram amounts of amino acids with no i n t e r ference from urea and claimed to be more sensitive than the ninhydrin method (7b).  Proteins e.g. tyrosinase, gave a  spectral peak s i m i l a r 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 c h l o r a n i l test was a more s p e c i f i c test f o r 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 f l o u r with a neutral phosphate buffer from a Sephadex G-200 column.  The e l u t i o n p r o f i l e 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 c h l o r a n i l t e s t  Fig.  11  E l u t i o n curve of p r o t e i n s of h y d r o l y s e d wheat f l o u r e l u t e d Sephadex G-200 column w i t h phosphate b u f f e r .  from  52  demonstrated only milder reactions with the nonprotein compounds, aihuge peak was 12).  nitrogen  detacted "by the ninhydrin test ( F i g .  During mild acid hydrolysis of gluten, ammonia was  released by s e l e c t i v e hydrolysis of the amide groups of g l u t amines and asparagines  (68).  The ninhydrin reagent, unlike  the c h l o r a n i l reagent i s s e n s i t i v e 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 i n 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 f l o u r protein did not provide conclusive quantitative results f o r the proportion of ammonia-nitrogen, other nonprotein nitrogen and protein nitrogen,  Nonprotein nitrogen  i n wheat f l o u r may be separated from protein nitrogen by heat coagulation, TCA p r e c i p i t a t i o n and d i a l y s i s . d i a l y s i s was  Of these,  found to be the most reproducible method on the  basis of molecular  s i z e (84).  A combination of d i a l y s i s and  a rapid micro-Kjeldahl procedure was. used to study the quant i t a t i v e d i s t r i b u t i o n of nitrogen i n the hydrolysed wheat flour.  The r e s u l t i s shown i n Table 4.  Ammonia constituted  160  Elution F i g . 12  320  volume  480  (ml.)  Ninhydrin t e s t ( ) and C h o r a n i l t e s t (. ••• ) of h y d r o l y s e d wheat f l o u r p r o t e i n s e l u t e d from Sephadex G-200 column i n phosphate b u f f e r . A-0  Table 4  Total-nitrogen, protein-nitrogen and ammonianitrogen content of the freeze-dried hydrolysed wheat f l o u r .  Nitrogen content ($ by weight of  1  freeze-dried hydrolysate)  % T o t a l nitrogen  Total-nitrogen  1.827 ± 0.041- '  Protein-nitrogen  1.464 ± 0.041  80.1  Ammonia-nitrogen  0.394 ± 0.010  21.6  •a-  100.0  Average of 4 determinations and standard deviation.  55  21.6 percent and protein nitrogen 80.1 percent of the t o t a l nitrogen i n hydrolysed wheat f l o u r .  Substracting ammonia  nitrogen and protein nitrogen from t o t a l nitrogen would give the amount of other nonprotein nitrogen of free amino acids and low molecular weight peptide which were obviously present i n an i n s i g n i f i c a n t quantity i n the hydrolysed product. presence of 21.6$  The  of ammonia nitrogen was a good i n d i c a t i o n of  almost complete deamidation of wheat f l o u r protein during acid hydrolysis.  F a i l u r e to detect any other nonprotein nitrogen  implied that during acid hydrolysis, high molecular weight wheat f l o u r 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 molecular weight peptides.  The peaks at the nonprotein nitrogen  p o s i t i o n as detected by the c h l o r a n i l reagent and by measuring absorbance at 280 nm were probably due to ammonium chloride and to browning compounds, r e s p e c t i v e l y .  G.  Gel f i l t r a t i o n analysis of carbohydrate Fig.  3 and F i g . 4 showed that high molecular weight  starch was broken down to lower molecular weight during acid hydrolysis.  The low molecular weight  carbohydrates carbohydrate  peak i n F i g . 4 was pooled f o r further investigation with gel f i l t r a t i o n i n a smaller pore size g e l . Fig.. 13 i s the e l u t i o n p r o f i l e of the carbohydrate peak pooled from F i g . 4 and eluted i n a Sephadex G-100  column.  Only one huge peak emerged  11,700  °- l  < 5,000  5  100  Elution F i g . 13  500  200  volume  (ml.)  G e l f i l t r a t i o n e l u t i o n curve on Sephadex G-100 column f o r the major carbohydrate peak o f h y d r o l y s e d wheat f l o u r c o l l e c t e d from Sepharose 4B column. E l u a n t : phosphate b u f f e r . ON  57  towards the end of the e l u t i o n p r o f i l e corresponding to molecular weight less than 5,000.  The carbohydrates thus existed  mainly as oligosaccharides a f t e r acid hydrolysis.  The d i s t r i -  bution of the oligosaccharides was further studied by g e l f i l t r a t i o n i n the polyacrylamide g e l , Biogel P-2 minus 400 mesh, at an elevated temperature  of 65* C,  Using an automated  detection system, t h i s technique was reported to be capable of f r a c t i o n a t i n g oligomers containing two to twenty one glucose units as w e l l as separating a mixture of isomeric compounds with s i m i l a r molecular weights  (85).  F i g . 14 was the elution  p r o f i l e of carbohydrates of the hydrolysed wheat f l o u r eluted from an Biogel P-2 column.  The column was capable of resolv-  ing glucose and i t s polymers up to 8 glucose u n i t s .  The  r e s o l u t i o n of higher oligomers of glucose would probably improve with a continuous automated system of carbohydrate detection and with better methods f o r sample a p p l i c a t i o n . Fig.  14 shows that most of the carbohydrates a f t e r acid hydro-  l y s i s were low molecular weight saccharides with glucose present i n the highest concentration of 18.7 percent.  This was  followed by the disaccharide maltose which constituted percent of the t o t a l carbohydrates.  11.3  The concentration of the  i n d i v i d u a l oligomers decreased as the glucose units increased. This observation was i n close agreement with a  chromatographic  study of the acid hydrolysed products of starch showing a l o g arithmatic decrease i n the concentration of the degraded products with increasing degree of polymerization (86).  A  G11  120  AND HIGHER  160  200  240  280  Elution volume (ml.) Fig.' 14  E l u t i o n curve of carbohydrates of h y d r o l y s e d wheat f l o u r e l u t e d from B i o g e l P-2 column with d i s t i l l e d water.  00  59  commercial  "brand of c o r n syrup c o n t a i n i n g glucose and a con-  t i n u o u s s e r i e s o f i t s polymers B i o g e l P-2  column.  was  used t o c a l i b r a t e the  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 l o g a r i t h m of the molecular and the r a t i o of e l u t i o n volume t o v o i d volume.  weight  The curve  l i n e a r f o r o l i g o s a c c h a r i d e s with 3 to 8 glucose u n i t s .  was  Oligo-  s a c c h a r i d e s w i t h 9 and 10 glucose u n i t s a l s o f e l l on the l i n e a r p o r t i o n of the curve  (85) although they were not  clearly  r e s o l v e d i n t o separate s i n g l e peaks i n the chromatogram. F i g . 14 i n d i c a t e s t h a t over t w o - t h i r d s of the carbohydrates a f t e r a c i d h y d r o l y s i s were o l i g o s a c c h a r i d e s w i t h 7 o r l e s s glucose u n i t s . u n i t s c o n s t i t u t e d 32.9  Carbohydrates  w i t h 8 or more g l u c o s e  percent of the t o t a l carbohydrate  h y d r o l y s e d wheat f l o u r . peak from a Sephadex G-50  of  T h i s f r a c t i o n e l u t e d as a s i n g l e column,.as shown i n F i g . 16,  showing  v e r y few s a c c h a r i d e s had m o l e c u l a r weights g r e a t e r than 5»000  (75). G e l f i l t r a t i o n chromatography of the h y d r o l y s e d wheat f l o u r c l e a r l y i n d i c a t e d t h a t d u r i n g h y d r o l y s i s , wheat s t a r c h was  e x t e n s i v e l y degraded  t o g l u c o s e , maltose and  oligo-  s a c c h a r i d e s w i t h v e r y few s a c c h a r i d e s p o s s e s s i n g more than g l u c o s e u n i t s i n the m o l e c u l e s .  T h i s e x p l a i n e d the  Slightly  sweet t a s t e and the low v i s c o s i t y of an aqueous s o l u t i o n of the h y d r o l y s e d p r o d u c t .  30  2.2  •J32 2.0 H  XG3 1.8H  VG4  \G5  V 1.64  ^ 6  \ G 7 \G8 \G9 \G10  1.4-  2  3  4  5  6  Molecular weight F i g . 15  7  8  9  10  (xi<r )  20  30  s  C a l i b r a t i o n curve f o r B i o g e l P-2 column u s i n g c o r n syrup,  ON  o  0.9-1  0.7H  o cn  0.5H  CU C_3  c=  ca CD  0.3-1  0.1-1  140  180  220  260  300  340  Elution volume (ml.) Fig.  16  E l u t i o n curve of h i g h e r s a c c h a r i d e s (G8 and above) of h y d r o l y s e d wheat f l o u r e l u t e d from Sephadex G-50 ( f i n e ) column w i t h 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 i n molecular weight of wheat f l o u r proteins during acid hydrolysis.  More s p e c i f i c information on  the degradation of polypeptides during acid hydrolysis could be obtained through a study with sodium dodecyl sulfate-polyacrylamide g e l electrophoresis (SDS-PAGE).  This technique has been  used to demonstrate the compositions of glutenin and g l i a d i n polypeptides i n terms of molecular weights (40,47,48).  F i g . 17  i s a c a l i b r a t i o n curve forjthe standard proteins i n the SDSPAGE a n a l y s i s .  The r e s u l t of SDS-PAGE analysis of the untreat-  ed and the acid hydrolysed wheat f l o u r i s shown i n F i g . 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 f a s t moving band with molecular weight less  than 1 1 , 7 0 0 .  In addition to the above two bands, a t h i r d  d i f f u s e band of 5 3 , 5 0 0 daltons was detected i n the whole hydrolysed wheat f l o u r .  Native wheat f l o u r proteins showed a series  of t y p i c a l bands ranging from molecular weights of 1 7 . 5 0 0 to 109,000.  The SDS-PAGE provided good evidence of polypeptide  degradation during acid h y d r o l y s i s .  The presence of two  d i f f u s e bands i n the electrophoresis of the 24,000 daltons peak indicates the p o s s i b i l i t y of some smaller polypeptides joined by intermolecular d i s u l f i d e bonds i n t h i s peak.  63  1.0  •^cytochrome C lysozyme j8-lactoglobulin ' « t r y p s i n inhibitor  0.8  0-6 -fl ovalbumin  0.4  .bovine serum albumin (monomer)  02-i bovine serum • a l b u m i n (dimerj bovine ,serum albumin (trimer) I  5  Molecular F i g . 17  weight  i  6  t  i  l  l  7 8 910  20  (xicr ) 4  C a l i b r a t i o n curve f o r p r o t e i n s i n SDS-pplyacry lamide g e l e l e c t r o p h o r e s i s .  6k  mol. wt. 15,600 19,000 2 3,000  mol. wt. 26,000 30,000 33,500  33:888  mm  28 , 7 0 0 32,400  0—  36 , 400 45,400  mm tm*  57, 200  50 , 600  F i g . 18  89  -  f  B  188  60 , 2 0 0 0  2  \106 109  600 000 500 000  C  SDS-Polyacrylamide G e l E l e c t r o p h o r e t i c P a t t e r n o f (A) major peak o f a c i d - h y d r o l y s e d wheat f l o u r p r o t e i n (from AUC-eluted Sephadex G-150 column), (B) whole a c i d - h y d r o l y s e d wheat f l o u r p r o t e i n , (G) whole wheat f l o u r p r o t e i n , (D) an e n l a r g e d p i c t u r e o f ( C ) .  65  F i g . 19 i s a comparision  o f the e l e c t r o p h o r e t i c  p a t t e r n o f wheat f l o u r p r o t e i n s w i t h or without r e d u c t i o n prior to electrophoresis.  Without p r i o r r e d u c t i o n , the 24,000  d a l t o n s peak from Sephadex G-150 column showed great decrease in intensity  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  f u r t h e r evidence o f i n t e r m o l e c u l a r d i s u l f i d e bonds i n the 24,000 d a l t o n s peak.  Whole h y d r o l y s e d wheat f l o u r p r o t e i n  without r e d u c t i o n e x h i b i t e d two a d d i t i o n a l  d i f f u s e bands b e t -  ween the f a s t moving band and t h e 30,000 d a l t o n s band.  These  two  inter-  new bands were a p p a r e n t l y made up of molecules w i t h  molecular  d i s u l f i d e bonds which upon r e d u c t i o n , s p l i t t e d  s m a l l e r p o l y p e p t i d e s m i g r a t i n g i n t h e f a s t moving band. presence o f SS-  (70).  of s o l u b i l i z e d  under d i f f e r e n t h y d r o c h l o r i c a c i d  No s i g n i f i c a n t decrease i n SS-  i n SH-  The  groups i n a c i d h y d r o l y s e d wheat p r o t e i n was  a l s o demonstrated i n another study hydrolysed  into  gluten  concentrations  groups and no i n c r e a s e  groups were d e t e c t e d i n the s o l u b i l i z e d g l u t e n  although  the p o s s i b i l i t y o f i n t e r - and i n t r a - m o l e c u l a r d i s u l f i d e change c o u l d n o t be excluded.  Different  inter-  electrophoretic  p a t t e r n s were e x h i b i t e d by n a t i v e wheat f l o u r p r o t e i n s w i t h o r without r e d u c t i o n .  An a p p r e c i a b l e amount o f p r o t e i n s remained  at t h e s t a r t i n g p o i n t o f t h e g e l when the p r o t e i n s were n o t reduced.  T h i s was probably  g l u t e n i n molecules which were t o o  l a r g e t o enter the g e l m a t r i c e s (34).  66  mol. wt. 1 1 , 700 18,400  45,000  6 7,000  134,000  A 19  B  C  D  E  F  SDS-Polyacrylamide G e l E l e c t r o p h o r e t i c P a t t e r n o f (A) major peak o f a c i d - h y d r o l y s e d wheat f l o u r p r o t e i n (from AUC-eluted Sephadex G-150 column) with reduction prior to electrophoresis, (B) same p r o t e i n peak as (A) b u t without r e d u c t i o n , (C) whole a c i d - h y d r o l y s e d wheat f l o u r p r o t e i n with reduction, (D) whole a c i d - h y d r o l y s e d wheat f l o u r p r o t e i n without reduction, (E) whole wheat f l o u r p r o t e i n w i t h r e d u c t i o n , (F) whole wheat f l o u r p r o t e i n without r e d u c t i o n .  67 Gel  f i l t r a t i o n and SDS-PAGE analysis of hydrolysed  wheat f l o u r protein c l e a r l y showed s p l i t t i n g of peptide bonds during acid hydrolysis.  High molecular glutenin and g l i a d i n  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 d i s u l f i d e bonds which might be derived from the o r i g i n a l bonds t i n native glutenin or from d i s u l f i d e interchange reactions. In SDS-PAGE, those compounds with intermolecular d i s u l f i d e bonds, when reduced by  /8-mercaptoethanol, were s p l i t into  smaller fragments and migrated i n the f a s t moving band.  I.  Amino acids analysis The amino acids composition of acid hydrolysed wheat  f l o u r proteins and the untreated proteins was shown i n Table 5» No s i g n i f i c a n t loss of  e s s e n t i a l amino acids was  observed.  Tryptophan could not be detected i n both the hydrolysed and the untreated wheat f l o u r 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 s l i g h t decrease i n  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 e s s e n t i a l amino acids of the wheat f l o u r s o l u b i l i z e d by acid hydrolysis.  68  Table 5  Comparison of amino a c i d content  o f u n t r e a t e d wheat  f l o u r and h y d r o l y s e d wheat f l o u r . Amino a c i d s  Amino a c i d -content  (g/100 g p r o t e i n )  Untreated wheat f l o u r  Hydrolysed wheat f l o u r  4.28  4.38  1.95 4.61  1.88 4.62  32.50  31.40  Proline  15.65  Glycine  3.73 3.13 3.60 1.06  13.67 3.88  Aspartic acid •Threonine Serine Glutamic  acid  Alanine •Valine •Methionine •Isoleucine •Leucine Tyrosine •Phenylalanine •Lysine ._Histidine •Tryptophan Arginine Cysteine  • E s s e n t i a l amino a c i d s .  2.67 5.15 3.12  2.45 4.22 1.12 3.31 6.92 3.19 5.48  5.29 2.37 2.26 0.00  2.37 2.20 0.00  3.62  3.89  69  J.  F o a m a b i l i t y and foam s t a b i l i t y Table 6 shows the coraparision o f f o a m a b i l i t y and  foam s t a b i l i t y o f the h y d r o l y s e d wheat f l o u r p r o t e i n , c a s e i n and  soy p r o t e i n .  Using 0.5 percent p r o t e i n s o l u t i o n s , the  h y d r o l y s e d wheat' f l o u r was shown t o be a b e t t e r foaming agent than c a s e i n although the foam s t a b i l i t y was much lower.  Soy  p r o t e i n e x h i b i t e d r e l a t i v e l y poor f o a m a b i l i t y but w i t h much better s t a b i l i t y .  I n c r e a s i n g p r o t e i n c o n c e n t r a t i o n t o 1$ d i d  not improve f o a m a b i l i t y t o a p p r e c i a b l e degree b u t s l i g h t l y i n c r e a s e d the foam s t a b i l i t y o f the h y d r o l y s e d wheat f l o u r . Good foaming p r o p e r t y was e s s e n t i a l f o r h y d r o l y s e d wheat f l o u r i n making ice-cream and m i l k shake l i k e  K.  products.  F l a v o u r improvement An u n d e s i r a b l e wheaty o f f - f l a v o u r appeared d u r i n g  a c i d h y d r o l y s i s o f wheat f l o u r .  The same o f f - f l a v o u r was  observed when v i t a l g l u t e n was h y d r o l y s e d w i t h d i l u t e hydrochloric acid  (70). Since m i l k i s a b l a n d product, the wheaty  o f f - f l a v o u r has t o be removed b e f o r e the h y d r o l y s e d wheat f l o u r can be used as d a i r y s u b s t i t u t e bases.  The most e f f e c t i v e  method i n r e d u c i n g the o f f - f l a v o u r was found t o be p a s s i n g an aqueous s o l u t i o n o f h y d r o l y s e d wheat f l o u r through a heated column o f g r a n u l a t e d carbon. f l a v o u r was g r e a t l y reduced  By t h i s treatment,  the o f f -  and t h e r e was a simultaeous  appearance o f a p l e a s a n t malty f l a v o u r .  The malty f l a v o u r was  70  Table 6  Foamability and foam s t a b i l i t y of hydrolysed wheat f l o u r , casein and soy protein i s o l a t e s o l u t i o n s .  Sample  Foamability* ($ volume increase)  Foam s t a b i l i t y (seconds)  Hydrolysed wheat f l o u r solution (0.5$ protein)  401.4 ± 16.8  160.0  ±  5.0  403.4+5.8  195.0  +  5.0  322.8 ± 12.6  451.7  +  67.9  Hydrolysed wheat f l o u r solution (1.0$ protein) Casein solution (0.5$ protein) Soy protein i s o l a t e solution (0.5$ protein) 176.0 ± 17.6 1463.3 ± 1 4 2 . 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 deviation.  71  probably masked by the wheaty o f f - f l a v o u r before  treatment.  In a d d i t i o n to f l a v o u r improvement the hot carbon  treatment  a l s o improved the c o l o u r and the storage s t a b i l i t y of the dry h y d r o l y s e d product.  Removal of the wheaty o f f - f l a v o u r  more e f f i c i e n t at h i g h e r temperature. 90*0 was  was  A column o p e r a t i n g at  found t o remove the o f f - f l a v o u r b e t t e r than a column  o p e r a t i n g at 60*C.  The spent carbon can be regenerated  by  h e a t i n g t o 600'C i n a f u r n a c e . One  of the major f a c t o r s which hindered the s u c c e s s -  f u l removal of the wheaty o f f - f l a v o u r was  the l a c k of knowledge  of the nature and o r i g i n of the f l a v o u r .  Solvent washing of  wheat f l o u r was  attempted  acid hydrolysis.  t o remove f l a v o u r p r e c u r s o r s b e f o r e  Washing wheat f l o u r w i t h water, with  95$  e t h a n o l or w i t h an aqueous s o l u t i o n sodium hexametaphosphate !  o n l y s l i g h t l y reduced the o f f - f l a v o u r i n the h y d r o l y s e d Washing wheat f l o u r w i t h water s a t u r a t e d b u t a n o l was  product.  more  e f f e c t i v e i n r e d u c i n g the o f f - f l a v o u r but d i d not e l i m i n a t e i t completely.  Water s a t u r a t e d b u t a n o l removed both f r e e  bound l i p i d s from wheat f l o u r l i p i d m a t e r i a l was  (61,62).  I t was  and  possible that  the p r e c u r s o r t o the wheaty o f f - f l a v o u r .  Our o b s e r v a t i o n t h a t the o f f - f l a v o u r s t i l l p e r s i s t e d i n the h y d r o l y s e d wheat f l o u r a f t e r a s i n g l e water s a t u r a t e d b u t a n o l wash was  probably due t o incomplete removal of l i p i d m a t e r i a l s . A number of i o n exchange r e s i n s , a n i o n i c , c a t i o n i c  or mixed bed were t e s t e d f o r removing o f f - f l a v o u r from  an  72  aqueous s o l u t i o n o f h y d r o l y s e d wheat f l o u r , but none were found t o be  satisfactory.  S i n c e the wheaty o f f - f l a v o u r might a r i s e through an o x i d a t i o n r e a c t i o n d u r i n g a c i d h y d r o l y s i s , i n c o r p o r a t i o n of a r e d u c i n g agent i n the h y d r o l y s i n g s o l u t i o n might development.  stop f l a v o u r  However, a d d i t i o n of c y s t e i n e h y d r o c h l o r i d e or  sodium s u l f i t e d i d not appear t o reduce the wheaty o f f - f l a v o u r i n the h y d r o l y s e d p r o d u c t . From our study, i t appeared t h a t the b e s t method t o minimize the wheaty o f f - f l a v o u r was  an extraction... of l i p i d  m a t e r i a l from wheat f l o u r e x h a u s t i v e l y w i t h a s u i t a b l e s o l v e n t l i k e water s a t u r a t e d b u t a n o l .  Any r e s i d u a l  lipid  off-flavour  developed d u r i n g a c i d h y d r o l y s i s c o u l d be removed by hot  carbon  treatment. An_autoclaved s o l u t i o n of h y d r o l y s e d 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 c h l o r i d e gave the s o l u t i o n  a salty taste.  A mixture o f potassium hydroxide and sodium  hydroxide c o u l d be used t o reduce s a l t i n e s s . was  u n d e s i r a b l e because  L.  I m i t a t i o n milk product  Calcium hydroxide  of the b i t t e r t a s t e of c a l c i u m c h l o r i d e .  An i m i t a t i o n m i l k product was  prepared from  hydro-  l y s e d wheat f l o u r d e o r d o u r i z e d w i t h a s i n g l e washing of water s a t u r a t e d b u t a n o l and w i t h hot a c t i v a t e d carbon treatment.  73 A t e n percent s o l u t i o n was  homogenized w i t h 4 percent u n s a l t e d  b u t t e r i n a 2 stage homogenizer.  The homogenized product  the same appearance as commercial  homogenized m i l k and  had  tasted  s l i g h t l y sweet and s a l t y w i t h a v e r y m i l d wheaty f l a v o u r . emulsion was age.  s t a b l e a t 4*C  f o r 2 weeks w i t h no s i g n of s p o i l -  The carbohydrate t o p r o t e i n r a t i o was  product.  about 7«1  in this  In order t o decrease the r a t i o t o a l e v e l s i m i l a r t o  m i l k , e i t h e r one of the two approaches first  The  c o u l d be used.  The  one i n v o l v e d f o r t i f i c a t i o n w i t h o t h e r s o l u b l e p r o t e i n s  l i k e f i s h , soybean, c o t t o n s e e d , rapeseed, peanut or s o l u b i l i z e d feather proteins.  The h i g h l y s i n e content of some of these  p r o t e i n s e.g. soybean p r o t e i n would i n c r e a s e the n u t r i t i v e v a l u e of the i m i t a t i o n m i l k p r o d u c t . u t i l i z e d a dough washing technique excess s t a r c h .  The  second  approach  (71) which washed o f f the  The carbohydrate to p r o t e i n r a t i o was  success-  f u l l y reduced t o a l e v e l s i m i l a r t o t h a t i n m i l k a f t e r a s e r i e s o f washings of the dough  (87).  B e s i d e s b u t t e r , other animal f a t s and vegetable l i k e coconut  oil,  soybean o i l ,  oils  cottonseed o i l and corn o i l  c o u l d be used as a source of l i p i d i n p r e p a r a t i o n of the i m i t a t i o n d a i r y product.  74  CONCLUSION  A u t o c l a v i n g a 10$ wheat f l o u r suspension at  i n 0.1N HC1  121 *C f o r 15 minutes p r o v i d e d a r a p i d , e f f e c i e n t and econo-  m i c a l method of s o l u b i l i z i n g wheat f l o u r . product  The s o l u b i l i z e d  e x h i b i t e d a low v i s c o s i t y , e x c e l l e n t s o l u b i l i t y and  appearance and c o u l d be used as d a i r y s u b s t i t u t e bases. The low v i s c o s i t y and t h e improved s o l u b i l i t y of the h y d r o l y s e d product  are due t o the e x t e n s i v e degradation of  wheat g l u t e n and s t a r c h , e s p e c i a l l y due t o almost complete deamidation of g l u t e n . From g e l f i l t r a t i o n polyacrylamide  chromatography i n AUC and SDS-  g e l e l e c t r o p h o r e s i s , random breakage of both  g l i a d i n and g l u t e n i n p o l y p e p t i d e s was e v i d e n t . degradation  d i d not proceed  However, the  t o the p o i n t o f producing  large  amount o f f r e e amino a c i d s and low molecular weight p e p t i d e s . Apparently,  some i n t e r m o l e c u l a r d i s u l f i d e bonds were  p r e s e n t i n the h y d r o l y s e d Gel f i l t r a t i o n  still  polypeptides. chromatography i n n e u t r a l phosphate  b u f f e r was not s u i t a b l e f o r s t u d y i n g the extent of wheat p r o t e i n degradation  i n h y d r o l y s i s because of the tendency of the hydro-  l y s e d p r o t e i n t o aggregate i n aqueous s o l u t i o n . Wheat s t a r c h was degraded t o low molecular  weight  s a c c h a r i d e s w i t h i n c r e a s i n g c o n c e n t r a t i o n as the number of glucose u n i t s decreased. Dextrose E q u i v a l e n c e  The h y d r o l y s e d wheat f l o u r had an  (D.E.) v a l u e of 36 and can be used as a  75  s u b s t i t u t e o f a s i m i l a r D.E. The  c o r n syrup i n food  processing.  n u t r i t i o n a l value of the h y d r o l y s e d product i s  expected t o be s i m i l a r to t h a t of wheat f l o u r s i n c e there no  s i g n i f i c a n t d e s t r u c t i o n of e s s e n t i a l amino a c i d s .  n u t r i t i o n a l v a l u e and  the be  i n l y s i n e content.  f o a m a b i l i t y of the h y d r o l y s e d wheat p r o t e i n  as good as t h a t of An  Both  the p r o t e i n t o carbohydrate r a t i o can  improved by the a d d i t i o n of a p r o t e i n h i g h The  was  was  casein.  a c t i v a t e d carbon treatment g r e a t l y reduced  wheaty o f f - f l a v o u r of the h y d r o l y s a t e .  the  Complete e l i m i n a t i o n  p r o b a b l y r e q u i r e s washing o f f <$£ a l l the l i p i d m a t e r i a l s wheat f l o u r w i t h a s u i t a b l e s o l v e n t p r i o r t o h y d r o l y s i s .  from  76  LITERATURE CITED  1.  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