A study of the starch and protein in barley
by Clarence A Ryan
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry at Montana State College
Montana State University
© Copyright by Clarence A Ryan (1956)
Abstract:
An investigation was made to determine the per cent amylose in the starch fraction of ninety-seven
barley samples. Two barley flours, prepared from Compana and Vantage varieties, were selected for a
study on the preparation of starch. The starch was separated from the protein fraction by Dimler'S
alkali process to see if possible commercial application could be made. The protein fractions were
analyzed chromatographically for their amino acid content to see if there was a correlation between
amino acid content and malting characteristics. A STUDY OF THE STARCH AM) PROTEIN IN BARLEY
I
by
CLARENCE A. RYAN JR .
A THESIS
S u b m itte d t o t h e G r a d u a te F a c u l t y
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in
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p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n ts
f o r th e d e g re e o f
M a s te r o f S c ie n c e i n C h e m is try at
M o n tan a S t a t e C o lle g e
A p p ro v e d :
H e a d /' M a jo r 1D e p a rtm e n t
E kam iningC C om m ittee
< D e a n , G ra d u a te " D i v i s i o n
Bozem an, M ontana
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.
:
_ 2 _
TABLE OF CONTENTS
C-o-xs. 2S e c tio n
P age Mo•
I.
A b s t r a c t ........................................................................................................................
3
II.
I n t r o d u c t i o n ...............................................................................................................
k
III.
E x p e r im e n ta l P a r t 1 ..............................................................................................
8
M e th o d s ....................................................................................................................
8
R e s u l t s .......................................................................................................................11
IV .
E x p e r im e n ta l P a r t I I .......................................................................................
18
M e th o d s .......................................................................................................................18
R e s u l t s .......................................................................................................................19
V.
E x p e r im e n ta l P a r t I I I .............................................................................................. 23
M e th o d s .................................................................................................................... 23
R e s u l t s ...............................
27
V I.
Summary............................................................................................................................. 30
V II.
L i t e r a t u r e C i t e d ........................................................................................................31
V III.
A c k n o w le d g e m e n ts..................................................................................................... 33
118853
“ 3 "
ABSTRACT
An i n v e s t i g a t i o n w as made t o d e te r m i n e t h e p e r c e n t a m y lo se i n t h e
s t a r c h f r a c t i o n o f n i n e t y - s e v e n b a r l e y s a m p le s* Two b a r l e y f l o u r s , p r e ­
p a r e d fro m Compana a n d V a n ta g e v a r i e t i e s , w e re s e l e c t e d f o r a s tu d y on
th e p r e p a r a tio n o f s ta rc h *
The s t a r c h w a s - s e p a r a t e d fro m t h e p r o t e i n
f r a c t i o n b y D i m le r 1s a l k a l i p r o c e s s t o s e e i f p o s s i b l e c o m m e rc ia l a p p l i ­
c a t i o n c o u ld b e m ade. The p r o t e i n f r a c t i o n s w e re a n a ly z e d c h ro m a to g ra p h ­
i c a l l y f o r t h e i r am ino a c i d c o n t e n t to s e e i f t h e r e was a c o r r e l a t i o n
b e tw e e n am ino a c i d c o n t e n t a n d m a l t i n g c h a r a c t e r i s t i c s *
-UII.
INTRODUCTION
T h e re h a s b e e n ' c o n s i d e r a b l e i n t e r e s t r e c e n t l y ( I , 2 ) i n f i n d i n g c e r e ­
a l s w i t h a h i g h a m y lo s e c o n t e n t .
The -work o f W olf e t . a l ,
( 3 ) h a s shown
t h a t p u r e a m y lo se c a n "be u s e d t o m ake s e l f s u p p o r t i n g f i l m s w i t h m o st o f t h e
d e s ira b le q u a li ti e s o f o th e r p l a s t i c f ilm s .
T hey h a v e a l s o s h o rn t h a t good
f i l m c h a r a c t e r i s t i c s a r e r e t a i n e d w i t h a d d i t i o n s o f s m a ll am o u n ts o f
a m y lo p e c tin .
S t a r c h i s com posed o f tw o f r a c t i o n s ,
am ylosb a n d a m y lo p e c tin .
A m ylose
i s t h e l i n e a r f r a c t i o n , a n d u s u a l l y c o n s t i t u t e s a b o u t tw e n ty p e r c e n t o f
t h e s t a r c h o f m o s t common c e r e a l s .
The r e m a in in g s t a r c h i s t h e b r a n c h e d
■
f r a c t i o n , c a ll e d a m y lo p e c tin .
C o n s i d e r a b l e p r o g r e s s h a s b e e n made i n t h e s e l e c t i v e c r o s s b r e e d i n g o f
com v a rie tie s
(U)
w i t h some r e s u l t i n g c r o s s - b r e e d s sh o w in g a m arked
i n c r e a s e i n a m y lo s e p e r c e n t a g e s
w h ic h i s
T h e refo re, i f
a c e r e a l c o u ld b e f o u n d ,
above a v e r a g e i n a m y lo s e , c r o s s - b r e e d i n g c o u ld b e a tte m p te d t o
i n c r e a s e t h e p e r c e n t a g e o f a m y lo s e .
I f t h i s c o u ld be a c c o m p lis h e d , t h e
r e s u l t i n g s t a r c h c o u ld b e u s e d t o r e p l a c e a c o n s i d e r a b l e am ount o f a lp h a
c e l l u l o s e i n th e p ro d u c tio n o f f il m s .
T h is w o u ld b e d e s i r a b l e w i t h t h e
p r e s e n t s h o r t a g e o f wood p u l p an d .e x c e s s o f c e r e a l g r a i n s »
The p r e s e n t
p r i c e o f a l p h a c e l l u l o s e w o u ld make s t a r c h a c o m p e t i t i v e p r o d u c t .
I n s e a r c h i n g f o r a c e r e a l o f t h i s t y p e , b a r l e y se em e d a n o b v io u s
c h o ic e i n t h e N o r th w e s t.
T h is i s due m a in ly t o t h e f a c t t h a t b a r l e y i s t h e
o n l y c e r e a l t h a t c a n b e grow n o n m uch o f t h e l a n d ta k e n o u t o f w h e a t p r o ­
d u c tio n .
As a r e s u l t l a r g e s u r p l u s e s o f b a r l e y h a v e b e e n b u i ^ t up an d
t h i s c o n d i t i o n w i l l p r o b a b l y becom e much w o r s e .
T h e re fo re , a se a rc h has
b e e n u n d e rta k e n to f i n d v a r i e t i e s o f b a r l e y s u i t a b l e f o r p o s s ib l e c ro s s ­
b re e d in g .
O n ly f o u r b a r l e y v a r i e t i e s h a v e b e e n r e p o r t e d f o r t h e i r p e r c e n t a g e o f
a m y lo s e p r e v i o u s t o t h i s p a p e r ( 5 ) .
Of t h e s e f o u r , o n l y o n e , T r e b i , h a s
b een in c lu d e d in t h i s su rv e y .
B a r le y s t a r c h h a s s e v e r a l o t h e r p o t e n t i a l i t i e s .
O rd in a rily over
n i n e t y - f i v e p e r p e n t . o f t h i s c o u n t r y ’ s s t a r c h i s p r o d u c e d fro m c o rn a n d
m ilo m a iz e .
A s m a l l am ount i s p r o d u c e d fro m -w heat, b u t t h i s i s d i f f i c u l t
t o o b t a i n an d t h e r e f o r e e x p e n s i v e .
I t p r o b a b l y -would n o t be p ro d u c e d i f
i t w e re n o t f o r t h e f a c t t h a t t h e w h e a t p r o t e i n i s u s e d f o r t h e m a n u f a c tu r e
o f so d iu m g l u t a m a t e .
A s m a ll am ount o f p o t a t o s t a r c h i s a l s o p r o d u c e d fro m
c u l l p o ta to e s *
I n t h e c o r n b e l t , w h e re m o st s t a r c h p l a n t s a r e l o c a t e d , b a r l e y h a s n o t
b e e n s e r i o u s l y . c o n s i d e r e d a s a s o u rc e f o r p t a r c h p r o d u c t i o n *
p la c e ,
In th e f i r s t
b a rle y and c o rn a re a b o u t eq u al on a c o s t p e r pound, and s ta r c h p e r
p o u n d ..b a s is .
T h is m akes c o r n t h e b e t t e r c h o ic e b e c a u s e o f a v a i l a b i l i t y
and e ase o f p ro c e s s in g .
An e v e r i n c r e a s i n g dem and f o r s t a r c h
w e s t , t h e lo w c o s t o f b a r l e y p e r b u s h e l ,
and s t a r c h p r o d u c t s i n t h e N o rth ­
and i t s a v a i l a b i l i t y , w o u ld t e n d
t o make b a r l e y s t a r c h p r o d u c t i o n m ore f e a s i b l e h e r e t h a n i n t h e c o rn b e l t .
F u r th e r m o r e , good i r r i g a t e d b a r l e y s h o u ld y i e l d a b o u t a s much s t a r c h a s
c o m on a pound p e r pound b a s i s .
S t a r c h p r o d u c t i o n i n t h e N o rth w e s t w o u ld
a l s o c u t s h i p p i n g c o s t s now p a i d b o t h w ays on t h e s e p r o d u c t s .
A commer­
c i a l o u t l e t f o r b a r l e y w o u ld b e h i g h l y d e s i r a b l e i n t h i s a r e a .
B e c a u se o f t h e h i g h c o s t o f . a c t u a l l y b u i l d i n g a p l a n t f o r t h e p r o -
- 6 d u c t i o n o f b a r l e y s t a r c h , i t w o u ld b e n e c e s s a r y t o d e v e lo p a p r o c e s s w h ic h
c o u ld b e u t i l i z e d b y u s i n g e q u ip m e n t a l r e a d y i n t h e a r e a .
A p r o c e s s f o r t h e p r o d u c t i o n o f s t a r c h fro m w h e a t h a s b e e n d e v e lo p e d
r e c e n t l y b y D im le r , e t a l .
( 6) .
T h i s p r o c e s s c o u ld b e a p p l i e d t o b a r l e y
i n a s u g a r b e e t p l a n t w i t h a s m a l l a d d i t i o n o f e q u ip m e n t.
T h is s i t u a t i o n
w o u ld be i d e a l b e c a u s e t h e s e p l a n t s do n o t o p e r a t e tw e lv e m o n th s a y e a r
an d c o u ld p r o d u c e s t a r c h d u r in g t h e l a y o f f m o n th s .
W ith a few m in o r
a d d i t i o n s t h e y c o u ld make d e x t r o s e s y r u p fro m t h e ra w s t a r c h .
The p r o t e i n fro m t h e b a r l e y c o u ld be s o l d a s f e e d o r f e e d s u p p le m e n t,
t h u s f u r .t h u r i n g t h e e c o n o m ic s o f s u c h a n o p e r a t i o n .
The s e c o n d e x p e r i m e n t a l p a r t o f t h i s t h e s i s c o n c e r n s a p p l i c a t i o n s o f
D i m le r t S p r o c e s s t o t h e p r e p a r a t i o n o f b a r l e y s t a r c h .
B a r le y i s u s e d a lm o s t u n i v e r s a l l y a s m a l t f o r t h e p r o d u c t i o n o f b e e r .
The m a l t i n g p r o c e s s , d u r in g w h ic h t h e b a r l e y g r a i n i s i n d u c e d t o s p r o u t
u n d e r i d e a l t e m p e r a t u r e a n d m o is t u r e c o n d i t i o n s , p r o d u c e s h ig h a l p h a
a m y la s e a c t i v i t y .
T h is a c t i v i t y b r e a k s down t h e
s ta r c h o f t h e b a rle y g r a in
a n d o t h e r c a r b o h y d r a te s u b s t r a t e s p r e s e n t t o f e r m e n t a b le s u g a r s and lo w
m o le c u la r w e ig h t d e x t r i n s .
N ot a l l b a r l e y v a r i e t i e s a r e good m a l t i n g b a r l e y s .
T h is seem s t o b e
b e c a u s e o f t h e i n a b i l i t y o f some b a r l e y g r a i n s t o p r o d u c e en o u g h a lp h a
a m y la s e f o r c o m p le te c o n v e r s i o n .
D u rin g t h e m a l t i n g s t a g e s , no p r o t e i n o r am ino a c i d s u b s t r a t e s a r e
added to th e in c u b a tin g b a r l e y .
T h is w o u ld i n d i c a t e t h a t I . )
a n am ino a c id
p o o l w as p r e s e n t t o s y n t h e s i z e a lp h a a m y la s e , 2 . ) t h a t s y n t h e s i s o c c u r r e d
fro m t r a n s f e r o f am ino a c i d s fro m o t h e r p r o t e i n s , o r 3 . ) t h a t a n o th e r
- 7 enzym e s y s te m may b e t h e p r e c u r s o r o f a l p h a a m y la s e .
I f o n e o f t h e s e p o s s i b i l i t i e s w e re p r e s e n t , i t w o u ld seem p o s s i b l e
t h a t good m a l t i n g q u a l i t i e s w e r e t i e d up i n am ino a c i d c o n t e n t o f th e
b a r l e y . ■I f s o , i t c o u ld p o s s i b l y f u r n i s h a c l u e t o t h e c o m p o s itio n o f
a l p h a a m y la s e , o r a t l e a s t t o t h e am ino a c id s n e c e s s a r y f o r i t s
s y n th e s is .
S in c e some v a r i e t i e s a r e d e f i n i t e l y known t o b e good m a l t i n g v a r i e t i e s
w h i l e ;o t h e r s a r e n o t t h i s p ro b le m i s o f some i n t e r e s t . .
As f a r . a s i s known n o t h i n g h a s b e e n done i n t h i s d i r e c t i o n ,
a lth o u g h
some common am ino a c i d s h a v e b e e n a d d e d t o m a l t w i t h no im p ro v e m e n t shown
i n m a ltin g q u a l i t i e s o f th e b a r l e y .
A p a r t fro m t h e m a l t i n g a s p e c t , i t w o u ld b e d e s i r a b l e t o know t h e am ino
a c i d c o m p o s itio n o f some b a r l e y v a r i e t i e s w h ic h c o u ld bq grow n i n t h e
N o r th w e s t.
T h is i s b e c a u s e o f t h e c o m m e rc ia l p o s s i b i l i t i e s o f b a r l e y p r o ­
t e i n a s a b y - p r o d u c t o f b a r l e y s t a r c h p r o d u c t i o n ..
As a f e e d o r f e e d
s u p p le m e n t t h e am ino a c i d c o n t e n t w o u ld b e v a l u a b l e k n o w le d g e when c o n ­
s i d e r i n g t h e m e to b o lic dem ands o f a n im a ls b e i n g f e d .
The g lu ta m ic a c i d i n b a r l e y c o u l d b e a p o s s i b l e
so u rc e f o r th e p ro d u c ­
t i o n o f so d iu m g l u t a m a t e , w h ic h i s u s e d w i d e l y a s a f l a v o r e x te n d e r i n m e a t.
The am o u n t p r e s e n t i n b a r l e y p r o t e i n w o u ld b e o f c o n s i d e r a b l e im p o r ta n c e .
W ith t h e s e i d e a s i n m in d , a c h r o m a to g r a p h ic d e t e r m i n a t i o n w as made o f
t h e am ino a c i d c o n t e n t o f Compana b a r l e y , w h ich i s c o n s i d e r e d a good m a l t ­
i n g b a r l e y , and V a n ta g e , w h ic h h a s p r o v e n t o p o s s e s s v e r y p o o r m a l ti n g
c h a ra c te ris tic s .
Compana i s n o t t h e b e s t b a r l e y v a r i e t y w h ic h c o u ld h a v e
b e e n c h o s e n , b u t i t w as s e l e c t e d b e c a u s e o f i t s a b i l i t y t o grow w e l l i n
th e G a l la t in V a lle y .
- 8 III.
EXPERIMENTAL PART I
METHODS
D u rin g t h e s t u d y o f am yl o s e i n b a r l e y , t h e f i r s t c o n s i d e r a t i o n s w e re
t h e e f f e c t s o f e n v ir o n m e n t a l f a c t o r s o n th e p e r c e n t a m y lo se i n a s i n g l e
v a r ie ty ®
The e f f e c t s o f t h e f o l l o w i n g f a c t o r s w e re c o n s i d e r e d u s in g t h e
Gompana v a r i e t y i n a l l c a s e s , l ) t h e y e a r s w hen p l a n t e d , 2 ) t h e d i f f e r e n t
d a t e s o f o n e y e a r w hen p l a n t e d , 3 ) e f f e c t s o f f e r t i l i z e r s
o f g ro w th w hen h a r v e s t e d , a n d 3 ) l o c a t i o n s w h e re grown*
a d d e d ; i±) s t a g e
A l l s a m p le s
e x c e p t 5 ) l o c a t i o n s , w e r e grow n i n t h e G a l l a t i n V a l l e y .
T h is k n o w le d g e i s n e c e s s a r y i f a s u r v e y o f d i f f e r e n t v a r i e t i e s i s t o
b e m ade; b e c a u s e i f t h e e n v ir o n m e n t d id iqake a d i f f e r e n c e i n a m y lo se
c o n t e n t , t h e n i t w o u ld n o t o n l y b e a m a t t e r o f c r o s s - b r e e d i n g t o r a i s e t h i s
c o n t e n t , b u t o f h a v in g i d e a l g ro w in g c o n d i t i o n s a s w e l l .
A f u r t h e r s tu d y f o llo w e d i n w h ic h tw e n ty - tw o b a r l e y v a r i e t i e s fro m
e i g h t s t a t e s w e re a n a ly z e d f o r p e r c e n t a m y lo s e .
T w e n ty - e ig h t v a r i e t i e s
fro m tw e lv e f o r e i g n c o u n t r i e s w e re s i m i l a r l y a n a ly z e d .
F i f t e e n b a r l e y s a m p le s o f Compana S hrunlcen E ndosperm a n d f i v e Compana
f r e a k s w e re a l s o a n a l y z e d .
The b a r l e y s a m p le s w e re s e l e c t e d b y M r. R o b e r t E s l i c k o f The M ontana
S t a t e C o lle g e Agronom y D e p a r tm e n t.
A p p r o x im a te ly t e n gram s o f e a c h b a r l e y v a r i e t y w e re p e a r l e d f o r one
m in u te t o rem ove t h e h u l l s .
A p e a r l i n g m a c h in e c o n s i s t s b a s i c a l l y o f a
r o t a t i n g em ory w h e e l, w h ic h s t r i p s th e h u l l s a n d m o s t o f t h e p e r i c a r p fro m
t h e g r a i n , b u t t h i s d o e s n o t a f f e c t t h e r a t i o o f a m y lo se t o a m y lo p e c tin
i n th e b a r le y s a m p le s.
- 9 T h re e t o f i v e gram s o f p e a r l e d b a r l e y w e re g ro u n d i n a W ile y m i l l t o
p a s s a f o r t y m esh s c r e e n , a n d t h e f a t e x t r a c t e d f o r t w e n t y - f o u r h o u r s i n a
S o x h l e t e x t r a c t o r w i t h 8%% m e th a n o l.
The r e s i d u e w as d r i e d a t room
te m p e ra tu re f o r a t l e a s t tw e n ty -fo u r h o u rs b e fo re a n a l y s i s .
A ll p e rc e n ta g e s
a re r e p o r te d on a f a t f r e e b a s i s .
The b a r l e y s a m p le s w e re a n a ly z e d f o r p e r c e n t a g e s o f f i b r e , a s h , p r o - '
t e i n and m o is tu re .
T he p e r c e n t a g e s o f t h e s e f o u r w e re t h e n u s e d a s t o t a l
n o n r - s ta r c h i n e a c h b a r l e y s a m p le .
S i x random sa m p le s o f b a r l e y w ere
c h o s e n a s a n a v e r a g e v a lu e f o r t h e p e r c e n t a s h a n d f i b r e .
M o is tu r e d e t e r ­
m i n a t i o n s w e re m ade a t random on t w e n t y - f o u r s a m p le s and s in c e t h e y a l l
f e l l i n a n a rr o w r a n g e t h e a v e r a g e w a s u s e d t o c a l c u l a t e t h e b a l a n c e .
p r o t e i n d e t e r m i n a t i o n w as made on t h e i n d i v i d u a l b a r l e y s a m p l e s .
e x t r a c t e d g ro u n d b a r l e y s a m p le s w e re u s e d i n e v e r y a n a l y s i s .
A
The
The a v e r a g e
v a l u e f o r f i b r e , a s h a n d m o is t u r e w ere u s e d a s o v e r a l l c o r r e c t i o n s f o r n o n s t a r c h m a t e r i a l , w h i l e i n d i v i d u a l c o r r e c t i o n s w ere made f o r p r o t e i n .
The f i b r e w a s a n a ly z e d ,by t h e s t a n d a r d A .O .A .C . m eth o d ( ? ) .
r e s u l t s v a r i e d b e tw e e n 1 .3 3 $ an d 1 .6 3 $ .
The
The a v e r a g e b e in g l . h 9 $ .
The a s h w as a l s o a n a ly z e d b y t h e s t a n d a r d ,A .O .A .C . ( 8 ) s p e c i f i c a t i o n s .
The a s h c o n t e n t v a r i e d b e tw e e n 1 .9 8 $ a n d 1 .6 9 $ .
F o r th e d e te rm in a tio n o f m o is tu re ,
The a v e r a g e w as 1 .8 8 $ .
a p p r o x im a te ly tw o gram sa m p le s w e re
d r i e d i n a vacuum o v e n f o r t w e n t y - f o u r h o u r s a t IOO0 0 .
w as r e p o r t e d a s p e r c e n t m o i s t u r e .
6 .8 1 $ .
The l o s s i n w e ig h t
The v a l u e s v a r i e d b e tw e e n 6 . 6l $ an d
The a v e r a g e w as 6 .7 1 $ m o i s t u r e .
The t o t a l a v e r a g e o f t h e s e t h r e e a n a l y s e s w as 1 0 .0 8 $ .
T h is f i g u r e w as
u s e d a s a b l a n k e t c o r r e c t i o n on a l l o f t h e s a m p le s a s t h e n o n - p r o t e i n , n o n -
- I O s t a r c h c o m p o n e n t.
I t i s t o b e o b s e r v e d t h a t a n e r r o r a s g r e a t a s -1% on
t h i s t o t a l w o u ld o n l y make a d i f f e r e n c e o f 0 .2 0 t o 0 . 2 ^ o n t h e p e r c e n t
a m y lo se a n d t h i s i s w i t h i n t h e e x p e r m e n t a l e r r o r o f t h e m e th o d .
P r o t e i n d e t e r m i n a t i o n s w e re c a r r i e d o u t b y a m o d if ie d K j e ld a h l ' m eth o d
(9 ).
The p e r c e n t p r o t e i n u s e d i n t h e c a l c u l a t i o n s w as o b t a i n e d b y m u l t i ­
p ly in g th e p e rc e n t n itr o g e n b y th e f a c t o r £ . 7 - ( l 0 ) .
•
The a m y lo se w as d e te r m in e d a c c o r d in g t o t h e m eth o d d e s c r i b e d b y B a t e s ,
B u n d le a n d F r e n c h ( l l )
a s m o d if ie d b y L a n s k i ( 1 2 ) .
To a p p r o x im a te ly OeOli
gram s o f t h e e x t r a c t e d g ro u n d b a r l e y w as a d d e d 10 m l o f b o i l i n g d i s t i l l e d
w a te r t o g e l a t i n i z e th e s t a r c h .
w as a d d e d t o d i s p e r s e t h e
T hen 5. m l o f 1 .0 W p o t a s s i u m h y d r o x id e
s ta r c h i n th e s a m p le s.
The s o l u t i o n was l e f t t o
s t a n d f o r o n e h o u r , ' n e u t r a l i z e d w i t h O .g H h y d r o c h l o r i c a c i d , u s in g m e th y l
o ran g e as th e i n d i c a t o r .
A 10 m l q u a n t i t y o f 0 . 5 W p o t a s s i u m i o d i d e was
p i p e t t e d i n t o . t h e s o l u t i o n and t h e v o lu m e w as b r o u g h t to . 100 m l w ith
d is tille d .w a te r .
T h is s o l u t i o n w as t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h a
s ta n d a r d io d in e i n p o ta s s iu m io d id e , p o ta s s iu m c h lo r id e s o l u t i o n c o n ta in ­
i n g .0 0 0 1 9 7 0 gram s o f i o d i n e p e r m l i n a s o l u t i o n 0 .0 £ K i n b o t h p o ta s s iu m
io d id e a n d p o ta s s iu m c h lo r i d e .
The i n s t r u m e n t u s e d f o r f o l l o w i n g th e
t i t r a t i o n s w as a Beckman M odel H-2 pH m e t e r , s e t t o r e a d i n m i l l i v o l t s , a n d
u s i n g a c a lo m e l r e f e r e n c e e l e c t r o d e a n d a p l a t in u m i n d i c a t o r e l e c t r o d e .
The t i t r a t i o n i s d e p e n d e n t upon t h e a c t i v i t y o f t h e i o d i n e i n s o lu ­
t i o n . . A m ylose fo rm s a co m p le x w i t h i o d i n e i n w h ic h 0 ,2 1 5 gram s o f i o d i n e
com bine w i t h 1 . 0 gram o f a m y lo s e .
p e c t i n an d i o d i n e
(ll).
A co m p le x i s n o t fo rm e d b e tw e e n a m y lo -
A t t h e end p o in t th e r e is a sh a rp in c r e a s e i n t h e
i o d i n e a c t i v i t y w h ic h c a n be f o l l o w e d b y t h e i n c r e a s e i n p o t e n t i a l .
The .
— 11 —
u p p e r and m ore, h o r i z o n t a l p o r t i o n o f t h i s . c u r v e i s e x t r a p o l a t e d t o g iv e t h e
m l o f s t a n d a r d i o d i n e u s e d , s i n c e t h i s p o i n t g i v e s a m ore a c c u r a t e r e s u l t ,
a s shown b y e x p e r i m e n t a t i o n ( F ig u r e l ) „
The i o d i n e s o l u t i o n w a s s t a n d a r d i z e d w i t h a r s e n o u s o x i d e .
The s o l u - .
t i o n u s e d f o r a n a l y s i s o f a m y lo se i s p r e p a r e d b y t e n f o l d d i l u t i o n o f t h e
s ta n d a rd iz e d s to c k s o lu tio n .
A s t a n d a r d c u rV e i s p l o t t e d t y t i t r a t i n g
83 O m g .o f p o t a s s i u m i o d i d e
a n d /373 mg o f p o t a s s i u m c h l o r i d e i n 100 m l o f d i s t i l l e d w a t e r w ith t h e '
s ta n d a rd iz e d io d in e s o lu tio n .
From t h i s s t a n d a r d c u r v e , t h e m il li g r a m s o f
i o d i n e i n s o l u t i o n a r e s u b t r a c t e d fo rm t h e t o t a l i o d i n e u s e d a t th e e n d
p o i n t w hen t i t r a t i n g
a sa m p le o f s t a r c h .
i o d i n e bound i n t h e co m p le x w i t h a m y lo s e .
T h is g i v e s t h e m i l l i g r a m s o f
-Knowing t h a t .2 1 5 gram s o f
i o d i n e i s bo u n d p e r gram o f a m y lo s e , t h e w e ig h t o f a m y lo se w a s c a l c u l a t e d .
T h is w e ig h t o f a m y lo s e , d i v i d e d b y t h e w e i g h t o f s t a r c h i n th e s a m p le ,
t i m e s one h u d r e d , w i l l g i v e t h e p e r c e n t- a m y lo se i n t h e s t a r c h . ,
m e n ta l e r r o r was r a t h e r h i g h p l u s o r m in u s 1.0%,.
t h e i n s t r u m e n t w h ic h c o u ld be r e a d t o
The e x p e r i ­
T h is w as d u e l a r g e l y t o
o n ly tw o s i g n i f i c a n t f i g u r e s .
RESULTS
T h e re w as no s t r i k i n g d i f f e r e n c e f o u n d i n t h e p e r c e n t a g e s o f am y lo se
i n t h e s t a r c h due t o t h e e n v ir o n m e n ta l f a c t o r s w h ic h w e re o b s e r v e d .
Comp a n a , f e r t i l i z e d w i t h I I - I 1.8-O , a p p e a r e d s l i g h t l y h i g h e r i n a m y lo s e .
T h e se s a m p le s w e re r e c h e c k e d a n d t h e r e s u l t s w e re r e p r o d u c a b l e (T a b le I ) .
No b a r l e y v a r i e t i e s w e re fo u n d w i t h a b n o r m a lly h i g h a m y lo s e c o n t e n t .
The h i g h e s t b e in g tw o Compana F r e a k v a r i e t i e s , w h ic h c o n t a i n e d 2$»6% an d
2h*9% a m y lo s e .
The n e x t h i g h e s t v a r i e t y f o u n d was D ekap, fro m T u rk e y ,
12 -
M i l l i l i t e r s o f i o d i n e s o l u t i o n (.0 0 0 1 9 7 0 g /m l)
F ig u re I
A t y p i c a l p o t e n t i o m e t r i c t i t r a t i o n c u rv e o f Compana a m y lo se
u s i n g i o d i n e i n a p o ta s s iu m i o d i d e s o l u t i o n .
- 13 c o n t a i n i n g 2 k *2% a m y lo s e .
The lo w w a s 'P I 220069 fro m P a k i s t a n w ith 1 2 .1 $ .
The a v e ra g e w a s 1 8 .0 ^ a m y lo se ( T a b le s I I ,
I I I . an d I V ) .
I f c r o s s - b r e e d i n g ' i s t o b e a tte m p te d on t h e b a s i s o f t h i s s u r v e y , i t
i s s u g g e s t e d t h a t B ekap ahd t h e tw o Compana F r e a k s be u s e d , a n d p o s s i b l y
B u lk ( 2 3 . 1 $ ) , O t i s ( 23 . 0%) a n d B a r b l e s s ( 2 3 . 0 $ ) v a r i e t i e s .
— llj. —
TABIE I
ENVIROMEKTAL IHFIUM CE ON PERCENT
AMYLOSE IN COMPANA BARLEY STARCH
% P ro te in
Ie
.'
Y e a rs
1950"
1951
1952
1953
195k
1955
2•
F e rtiliz e rs
MHj. NO«j
o-k3-o
3»
H - I tS-O
I l - I tS-O
P l a n t i n g D a te s
May k, 195k . .
May 1 8 , 195k
J u n e 3 , 195k
J u n e 1 7 , 195k
k* L o c a ti o n s
C r e s to n
D r y la n d
Irrig a te d
S id n e y
D ry la n d
Irrig a te d
H u n tle y
H av re
S te v e n s v ille
S p rin g h ill
M is s o u la
R onan
5 . . G row th S ta g e Tillhen H a r v e s te d
L a te M i l k •
S o f t Dough
H ard Dough
When R ip e
A v e ra g e
% S ta rc h
• % A m ylose
i n th e s ta r c h
fra c tio n
12.8%
Ik . 7*
I k o 6%
16.9%
9.2%
9.7%
75.1%
75.3%
75.k%
73.1%
80.8%
80.3%
21.2%
20.k%
21 . 1 %
21.0%
21.0%
20.6%
12.6%
12.k% •
9.3%
8.1%
6.7%
6,7%
77.k%
77,5%
60.7%
81.9%
83.3%
83.3%
20.8%
19.5%
■ 19.1%
21.8%
22.2%
2 2 .k%
6 . 6%
6 . 6%
7.%%
7.2%
8 3 .k%
83.k%
82.8%
82.8%
20.2% .
21.1%
20.k%
22.0%
7o2%
82.8%
82.8%
21.1%
21 . 1%
11.7%
79.9%
71.2%
77.8%.
80.6%
8 2 .k%
78.9%
79.8%
78.3%
2i.k%
22.6%
21.6%
20.1%
21.8%
20.1%
20.2%
21.5%
12 . 1 %
9.k%
9.k%
8.5%.
77.8%
80.6%
80.6%
81.5%
20.7%
21 . 6%
22.0%
21.5%
7.2% •
1 0 . 1%
8 . 8%
1 2 . 1%
9.k%
7.6%
11.1%
10.1%
20 * 6%
■
- I* TABLE I I
PERCENT AMILOSE IN BARLEY STARCHES
OF U .S . ORIGIN
V a rie ty
S ta te
C o lo ra d o
L ic o
C o lo ra d o
O tis
C a lifo rn ia
B u lk
C a lifo rn ia
A t l a s I46 .
C a lifo rn ia
Hooded A t l a s
U ta h
V e lv o n II
U ta h
B o n n e v ille W
a sh in g to n
P r o s s e r #6
N.
D a k o ta
K in d r e d T i t a n
N e b ra s k a
C u s te r
Wyoming
H ila n d
M ontana
S p a rta n
Corapana
M o n ta n a '
G lo s s y M u ta n t
Compana
G lo s s y S e e d li n g
M ontana
S t r i p e d M u ta n t
M ontana
Corapana M a rg e n o t
M ontana
B a r b le s s
B a r b le s s
G lo s s y M u ta n t - M ontana
' M ontana
M ars
M ars
M ontana
G lo s s y M u ta n t
G la c ie r T ita n
M ontana
(U7-7ilO£-V)
M ontana
G la c ie r T ita n
(5 0 -5 6 3 9 -1 2 )
%. P r o t e i n
9.6$
10.7%
8.7%
8 . 1%
9.6%
8 . 6%
8 . 0%
8.6%
8.6%
1 3 . 0%
9.2%
13.3%
% S ta rc h
% A m ylose
i n th e s ta r c h
fra c tio n
80 . 1t%
. 81.3%
81.3%
81.9%
80 . 1*%
81 . 1*% '
82.0%
81 . 1 %
81.1*%
22.9%
16.2%
21 . 1*%
1 8 . 0%
18.9%
19.5%
23.0%
11*. 3%
23.1t%
77.0%
ll*.6%
80.8%
76.7%
16.5%
■13.2%
76.8%
19.5%
8.6%
■ 81,1*%
10.7%
79.3%
73.2%
19.5%
19.1%
23.0%
16,8%
9.2%
10.7% .
. 80.8%
79.3%
20.1*%
22.3%
ll*.3%
8.1%
81.9%
8.6%
8l.l*%
16.7%
15.5%
79.3%
21.0%.
10.7% ■
.
-
-
- 16 TABLE I I I
PERCENT AMIEOSE IN BARLEY STARCHES
OF FOREIGN ORIGIN .
V a rie ty
C o u n try
H a r la n
T ita n
H usky
S a n a tta
M o ro v ia n
P . I . 221377
C anada
C anada
C anada
C anada
■M o ro v ia
Y u g o s la v ia
Y u g o s la v ia
Y u g o s la v ia
Y u g o s la v ia
Y u g o s la v ia
A f g h a n is ta n '
A f g h a n is t a n
A f g h a n is ta n '
A f g h a n is t a n
A f g h a n is t a n
A f g h a n is t a n
P a k is ta n
P a k is ta n
Sw eden
S o . A f ric a
P e ru
Germany '
F in la n d
In d ia
In d ia
T u rk e y
T u rk e y
T u rk e y
P . I . 22130k P . I . 221309
P .I . 221322
. I . 221327
P
P
P
P
P
P
P
P
P
P
P
P
.I .
.I .
.I .
.I .
.I .
.I .
.I .
.I .
.I .
.I .
.I .
211398
212813
21281).7
212830
219860
221U22
220069
717332
220833
219737
213708
P .I . 221072
Tammi
P . I . 211+326
P . I . 216033
Dekap
T re b i
I n t . C . I . 7837
$ P ro te in .
8.8%
9 ,#
■ 8.7#
9.6%
9.5%
12.2%
13.7%
12.3%
12.1%
81.2% '
. 80.3%
81.3%
80.1+%
' 1 3 .3 #
11.9%
■ 13.3%
13.0%
11.3%
Il+ . 8%
11.1%
13.7%
1 3 . 8%
17.3%
12.1+% '
13.5%
13.2%
8.3%
13.7%
U+.9%
8.2%
8 * 2%
8.1+%
% S ta rc h
•
81 . 3%
77.8%
76.3%
77.5%
77.9%
76.7%
78.1%
7k .7%
73.0%
79.7%
73.2%
.79.9%
86.3% .
76.2%
72.7%
77.6%
7k .3%
7 8 .k%
81.3%
76.3%
73.1% •
81.8%
81.8%.
81.6%
% A m ylose .
i n th e s ta r c h
fra c tio n
19.9%
22.3%
22.8%
17.6%
18.9%
19.1%
13.7%
13.9%
16.9%
I k . 3%
13.9%
12.6%
13.1%
13.9%
I k .7%
lk.k%
12 . k%
17.3%
16.6%
17.6%
13.9%
13.0%
16.7%
l k . 8%
19.7%
2 k . 2%
20.2%
21.8%.
■
- 17 . TABLE IV
PERCENT AMYLOSE IN STARCHES OF COMPANA SHRUNKEN ENDOSPERMS
~
:
'
AND COMPANA FREAKS
:
—
S h ru n k e n E ndosperm
A g. Number
7152
71&
71#
7157
7158
7159
7160
. 7162
7163
7161*
7165
7166
' 7167
7168
. 7169
% P ro te in
16.6%
16.2%
16.0%
11.6%
10.1%
11.2%
. 12.1%
■ 11.0%
' 10.2%
11.2%
12.2%
.
10.6%
10.7%
9.2%
■1 1 . 5%
. % S ta rc h '
% A m ylose
i n th e s ta r c h
fra c tio n
73.b%
'
. 73.8%
7l*.0%
78.1*%
79.6%
78.3%
77.9%
79.0%
79.8%
79.8% .
i
' \
77.9%
79.4%
79.3%
80.8%
,
78.5%
12^6%
17.0%
ll*.6%
19.3%
20.1%
17.4%
15.2%
18.1%
18.0%
16.4% '
16.7%
17.6%
15,6%
15.9%
■1 6 . 4 %
F re ak s
A g. Number
7217
7218
7219
7220
7223
■ .11*. 6%
■■ 1 3 ®8%
1 0 . 3%
7.0%
10.7%
75.4%
76.2%
. 79.5%
83.0%
79.3%
.18.3%. .
10.7%
25.6%
■ 18.4% '
24,9% ■
- 18 IV . ■ EXPERIMENTAL PART I I
METHODS '
The p r o c e s s ' d e v e lo p e d b y D im le r ( 6 ) e s s e n t i a l l y i n v o l v e s t h e d i s s o l v ­
i n g o f t h e f l o o r p r o t e i n m t h d i l u t e a l k a l i and c e n t r i f u g i n g , t h u s l e a v i n g
th e s ta rc h - f r a c t i o n a s th e r e s i d u e .
The a l k a l i n e p r o t e i n s o l u t i o n i s t h e n
a c i d i f i e d t o ' p r e c i p i t a t e th e a l k a l i s o lu b le p r o te i n .
The s t a r c h and p r o - '
t e i n can th e n b e p ro c e s s e d as d e s ir e d .
D i m le r 1s p r o c e s s h a s b e e n d e v e lo p e d f o r . t h e p r o d u c t i o n o f w h e a t s t a r c h ,
u s i n g sodium h y d r o x id e a s t h e s o l v e n t , a n d s u l f u r i c a c i d f o r t h e a c i d i f i c a ­
t i o n and p r e c i p i t a t i o n o f th e p r o t e i n .
I n o r d e r f o r t h i s p r o c e s s t o w o rk e f f i c i e n t l y on b a r l e y f l o u r t h e
maximum s o l u b i l i t y a n d th e pH f o r t h e maximum p r e c i p i t a t i o n o f th e b a r l e y
p r o t e i n m u s t b e d e te r m i n e d .
. Gompana b a r l e y f l o u r , p r e p a r e d i n a B u h l e r . m i l l , w as u s e d f o r t h e t e s t ­
in g .
The p r o t e i n i n t h e f l o u r 'w a s fo u n d t o be 9 .1 2 $ b y t h e K j e ld a h l m eth o d .
The a p p a r e n t p r o t e i n s o l u b i l i t y w as b a s e d o n t h i s f i g u r e .
A p p ly in g D i m le r ' s w o rk on w h e a t t o b a r l e y , v a r i o u s n o r m a l i t i e s o f
so d iu m h y d r o x id e w e re u s e d t o f i n d t h e a p p a r e n t maximum s o l u b i l i t y .
T w enty
gram s a m p le s o f b a r l e y f l o u r w e re m ix e d w i t h tw o h u n d r e d m l p o r t i o n s o f
so d iu m h y d r o x id e v a r y i n g fro m 0 .0 1 ^ 'N t o 0 , 03 ^ N u n t i l a hom ogeneous
m ix t u r e r e s u l t e d .
The sa m p le s w e re l e f t t o s t a n d w i t h o c c a s i o n a l s t i r r i n g
f o r t h i r t y m in u te s t o i n s u r e s o l u b i l i t y .
f i v e m in u te s a n d d e c a n t e d .
T hey w e re t h e n c e n t r i f u g e d f o r
The pH o f t h e s o l u t i o n s w e re d e te r m in e d a n d '
t e n ml a l i q u o t s ta k e n f o r K je ld a h l a n a l y s i s .
T h e 'm il li g r a m s o f n i t r o g e n
w e re d i v i d e d b y t w e n ty t o f i n d m i l l i g r a m s o f n i t r p g e n d i s s o l v e d p e r gram
- 1 9 o f flo u r.
The p r o c e d u r e w as c a r r i e d o u t b e tw e e n 20°C a n d 30°C t o p r e v e n t
g e l a t i n i z a t i o n o f th e s t a r c h .
A f t e r t h e pH f o r maximum s o l u b i l i t y w as f o u n d , t h e pH f o r maximum
p r e c i p i t a t i o n o f t h e p r o t e i n w as s o u g h t .
P r o t e i n r e c o v e r y was c a r r i e d o u t o n s i x a l k a l i n e s o l u t i o n s w h ic h w e re
a c i d i f i e d w i t h 0 .3 0 M s u l f u r i c a c i d .
The s o l u t i o n s w e re t h e n c e n t r i f u g e d
f o r f i v e m in u te s and a p r o t e i n a n a l y s i s w as r u n on t h e f i l t r a t e ,
d i f f e r e n c e b e tw e e n t h e p e r c e n t p r o t e i n p e r ml i n th e f i l t r a t e ,
The "
and t h e
p e r c e n t p r o t e i n p e r m l i n t h e o r i g i n a l a l k a l i n e s o l u t i o n s w as t a k e n a s
p e rc e n t p ro te in p r e c ip ita te d .
T h is a s s u m p t io n i s n o t s t r i c t l y v a l i d a s
some p r o t e i n m ay a d h e re to t h e s t a r c h when t h e pH i s l o w e r e d .
However
t o t a l p r o t e i n r e c o v e r i e s on t h i s m a t e r i a l b y a c o -w o rk e r u s i n g t h e sam e .
m e th o d w e re i n good a g re e m e n t w i t h t h e s e v a l u e s .
RESULTS
• I n a p p ly in g D i m le r f S p r o c e s s t o . b a r l e y f l o u r i t w as fo u n d t h a t b a r l e y
p r o t e i n h a s a n a p p a r e n t s o l u b i l i t y o f 100% a t a pH o f 1 1 . 14 an d above '
( T a b le 7 ) . , T h is i n d i c a t e s t h a t b a r l e y s t a r c h o f h ig h p u r i t y c o u ld b e
p r o d u c e d w i t h t h i s p r o c e s s d e p e n d in g u p o n t h e p u r i t y o f t h e f l o u r u s e d .
I h e a t p r o t e i n h a s a n a p p a r e n t s o l u b i l i t y o f 100% a t a pH o f 1 1 . 7 .
I t w as n o t e d d u r in g c e n t r i f u g a t i o n t h a t t h e s t a r c h was q u i t e h e a v y
a n d a p p e a r e d t o s e t t l e m ore r a p i d l y t h a n e i t h e r c o r n o r w h e a t s t a r c h .
I t w as r e a d i l y o b t a i n e d i n 70% y i e l d b u t due t o la c k , o f tim e t h i s f r a c t i o n
w as n o t i n v e s t i g a t e d , .
The p r o t e i n w as 91% p r e c i p i t a t e d a t a pH o f 6 .0 ( T a b le T l ) .
T h is w as
q u i t e h i g h a s c o m p a red t o D im le r f S r e s u l t s o f 77% p r e c i p i t a t i o n o f w h e a t
- '20. p r o t e i n a t a pH o f !?•£.
T h is w o u ld m ean t h a t o v e r 90% o f t h e o r i g i n a l
p r o t e i n i n b a r l e y f l o u r c p u ld b e r e c o v e r e d b y t h i s p r o c e s s .
A lth o u g h much w ork m u st y e t be d o n e t o f u r t h e r e x p l o r e t h e p o s s i b i l i ­
t i e s o f t h i s p ro c e s s w ith b a r le y f lo u r , th e b a s ic s o l u b i l i t y p a tt e r n o f
b a r le y p r o te in g iv e s fa v o ra b le in d ic a tio n s f o r i t s
use.
- 21 TABLE V
THE APPARENT SOLUBILITY OF
BARLEY PROTEEN
N o r m a lity
ES
A p p a re n t S o l u b i l i t y
OeOlBN
9 .8
32.Q #
0.020N
1 0 ,3
73,0#
0.02B N
11.1
8 0 . 0#
0.030N
ll.it
. 1 0 0 . 0#
0.03BN
1 1 .7
100 . 1#
.
22 TABLE YI
PRECIPITATION OF BARLEY PROTEIN.
FROM ALKALINE EXTRACT
ES
It.O
% P ro te in P r e c ip ita te d
72.8#
77.7#
5 .0 .
85.3#
5 .5
90.2#
6.o
91.0#
6 .5
7U.Q#
7 .0
52.6#
- 23 V.
EXPERIMENTAL PAET I I I
METHODS
The am in o a c i d a n a l y s e s o n b a r l e y p r o t e i n s a m p le s w e re c a r r i e d o u t
c h r o m a t o g r a p h i c a l l y a c c o r d i n g t o t h e m eth o d o f M oore a n d S t e i n ( 1 3 ) .
U s in g t h i s m e th o d m o s t o f t h e common am ino a c i d s an d s e v e r a l o f t h e
i n f r e q u e n t l y o c c u r r i n g am ino a c i d s i n p r o t e i n m a t e r i a l c a n b e s e p a r a t e d .
T r y p to p h a n , c y s t e i n e a n d c y s t i n e a r e n o t a b l e e x c e p t i o n s s i n c e t h e y a r e
d e s t r o y e d b y a c i d h y d r o l y s i s i n t h e p r e s e n c e o f c a r b o h y d r a te m a t e r i a l .
The s a m p le s o f b a r l e y p r o t e i n u s e d w e re p r e p a r e d fro m t h e f l o u r o f
Compana a n d V a n ta g e v a r i e t i e s w i t h a m o d i f i c a t i o n o f D i m le r 1s a l k a l i
p ro ce ss.
The p r o t e i n o f t h e tw o w as n o t p u r i f i e d a n d c o n t a i n e d 73«0$ an d
69.3% p r o t e i n r e s p e c t i v e l y b y K j e l d a h l a n a l y s i s .
T h e .r e m a in d e r o f t h e
sa m p le w a s c a r b o h y d r a te m a t e r i a l .
E ach p r o t e i n s a m p le w as d r i e d f o r t w e n t y - f o u r h o u r s i n a vacuum ov en
a t 75°C a n d a p p r o x i m a t e ly 5>0 mg w as h y d r o ly z e d b y r e f l u x i n g w i t h 200 m l
o f 6 N h y d r o c h l o r i c a c i d f o r 21}. h o u r s .
T h is m eth o d (ll} ) f o r h y d r o l y s i s
p e r m i t s t h e p r e s e n c e o f l a r g e a m o u n ts o f c a r b o h y d r a te m a t e r i a l i n t h e
h y d r o l y s a t e w i t h o u t a l t e r i n g t h e v a l u e s o f t h e am ino a c i d s on a n a l y s i s .
The h y d r o l y s a t e was t h e n f i l t e r e d th r o u g h a s i n t e r e d g l a s s f i l t e r
a n d e v a p o r a te d t o d r y n e s s i n a vacuum d e s s i c a t o y .
The r e s i d u e was ta k e n
up w i t h 1 0 m l o f d i s t i l l e d w a t e r a n d a one m l a l i q u o t w as u s e d f o r e a c h
a n a ly s is .
F o r t h e h y d r o l y s i s o f Compana, £0 mg o f c r u d e p r o t e i n w as u s e d ,
o r 3 6 .5 mg o f p u r e p r o t e i n .
F o r th e V a n ta g e a n a l y s i s , 5>2.3 mg o f c ru d e
p r o t e i n w e re h y d r o ly z e d , o r 36.2 mg o f p u r e p r o t e i n .
The c h r o m a to g r a p h ic s e p a r a t i o n s w e re made w i t h e q u ip m e n t a l r e a d y i n
/
- 2 k -
u s e a t t h e E x p e rim e n t S t a t i o n o f M ontana S t a t e C o l l e g e .
The e q u ip m e n t
c o n s i s t e d o f tw o c h r o m a to g r a p h ic c o lu m n s 'p a c k e d w i t h Dowex'5 0 , a s u l f o n a te d p o ly s ty re n e r e s i n .
w as 0 ,9 cm b y 25> cm.
One colum n w as O«,9 cm b y 100 cm, w h i l e t h e o t h e r
The f o r m e r co lu m n w a s j a c k e t e d t o p e r m i t te m p e r a t u r e
c o n tr o l w ith a r e g u la te d w a te r b a th .
The w a t e r w as c i r c u l a t e d th r o u g h t h e
j a c k e t fro m t h e b a t h w i t h a s m a l l pum p.
The s h o r t colum n w as n o t j a c k e t e d ,
a s a l l d e t e r m i n a t i o n s w i t h t h i s colum n w e re c a r r i e d o u t a t 2 £ °C .
A r o t a t i n g w h e e l w h ic h h e l d e i g h t y - e i g h t c o l l e c t i n g t u b e s a ro u n d i t ' s
p e r i m e t e r w as u s e d t o c o l l e c t o n e m l s a m p le s a s , t h e y w e re e l u t e d fro m t h e
c o lu m n .
The w h e e l r o t a t e d t h e d i s t a n c e b e tw e e n t h e c e n t e r s o f a d j a c e n t
c o l l e c t i n g tu b e s e v e ry f i f t e e n m in u te s .
The l o n g colum n w as a d j u s t e d
a b o v e t h e t u b e s a n d b y m eans o f a t a n k o f n i t r o g e n e x e r t i n g p r e s s u r e on
t h e b u f f e r i n t h e co lu m n , a c o n s t a n t f lo w o f o n e m l p e r f i f t e e n m in u te s
w as a c h i e v e d .
The f lo w o f t h e s h o r t c o lu m n w as a d j u s t e d s im p ly , b y r a i s i n g
o r l o w e r i n g t h e b u f f e r r e s e r v o i r w hich, i s f ie ld n e a r t h e l e v e l o f. t h e to p
o f t h e colum n on a n i r o n r i n g . '
The l o n g colum n w as u s e d f o r t h e d e t e r m i n a t i o n o f a l l b u t t h e b a s i c
am ino a c i d s i n t h e h y d r o l y s a t e s .
The s h o r t colum n w as u s e d t o
t h e b a s i c am ino a c i d s , a r g i n i n e , l y s i n e ,
d e te r m in e
an d h i s t i d i n e .
The b u f f e r s e q u e n c e f o r t h e Ip n g co lu m n was a s f o l l o w s ( 1 3 ) j t h e
colum n w as b e g u n w i t h a pH 3*^2 c i t r a t e b u f f e r a t 3 7 ° C u n t i l v a l i n e w as
e l u t e d , t h e n a pH
c i t r a t e b u f f e r a t HO0C was u s e d , b e i n g r a i s e d t o
7H°C b e f o r e t y r o s i n e a n d p h e n y la la n in e - cam e' o u t t o i n s u r e good s e p a r a t i o n
o f t h e tw o .
T h i s s e q u e n c e w o rk e d v e r y w e l l f p r t h e a c i d i c am ino a c i d s o f
b o th b a r l e y p r o te i n d e te r m in a tio n s * ■
- 25! The, s h o r t colum n b u f f e r s e q u e n c e b e g a n w i t h a c i t r a t e b u f f e r o f pH
5>e0 a n d a f t e r 25> m l o f t h i s b u f f q r h a d b e e n p a s s e d t h r o u g h t h e colum n, i t
w as f o ll o w e d b y a pH 6«5 c i t r a t e b u f f e r t o f i n i s h t h e s e p a r a t i o n *
I
One m l p e r 100 m l o f b u f f e r o f a d e t e r g e n t , BRIJ<-35* w as u s e d t o g iv e
b e t t e r s e p a r a t i o n o f t h e am ino a c i d s ( 1 3 ) .
From t h e c o l l e c t i n g t u b e s , 0*7 m l o f s o l u t i o n w as t r a n s f e r r e d t o t e s t
t u b e s f o r d e t e r m i n a t i o n o f t h e am ino a c i d s .
T h is s o l u t i o n w as b r o u g h t t o
pH 3*0 b y a d d i t i o n o f so d iu m h y d r o x id e o r h y d r o c h l o r i c a c i d , a n d one and
.
o n e - h a l f m l o f n i n h y d r i n s o l u t i o n was a d d e d .
The t e s t t u b e s w e re p l a c e d
i n t o a b o i l i n g w a t e r b a t h f o r t h i r t y m in u tp s t o d e v e lo p c o l o r ( i j ? ) .
Two
m o le s o f n i n h y d r i n com bine p e r m ole o f am ino a c id w hen h e l d a t t h e b o i l i n g
t e m p e r a t u r e o f w a t e r f o r t h i r t y m in u te s t o p r o d u c e a c o l o r w h ic h i s a
q u a n t i t a t i v e m e a s u re o f t h e am ino a c id *
The s o l u t i o n w as d i l u t e d t o 10 m l
w i t h 5>0$ e t h y l a l c o h o l , t r a n s f e r r e d t o a c o l o r i m e t e r t u b e , a n d t h e o p t i c a l
d e n s i t y w as r e a d a t 570 mu e x c e p t f o r p r o l i n e w h ic h w as r e a d a t IiliO mu o n
a Beckman M odel B S p e c tr o p h o t o m e te r .
The t r a n s f e r r i n g w as a c c o m p lis h e d w i t h a t u b e r c u l i n s y r i n g e .
A s ta n d ­
a r d c u rv e w as made u s i n g s o l u t i o n s o f l e u c i n e b e tw e e n 0 .0 5 raM olar and 0 .3
m M olar c o n c e n t r a t i o n s a n d d e v e lo p in g c o l o r w i t h n i n h y d r i n a s a b o v e .
The
s t a n d a r d w as r e a d on t h e c o l o r i m e t e r and t h e o p t i c a l d e n s i t y w as p l o t t e d
a g a in s t c o n c e n tra tio n .
From t h i s
c u rv e t h e c o n c e n t r a t i o n s o f a m in o ,a c id s
fro m t h e co lu m n s w e re f o u n d u s i n g t h e i r o p t i c a l d e n s i t i e s .
T h is c o n c e n t r a ­
t i o n was d i v i d e d b y a c o l o r y i e l d f o r e a c h amino a c i d w h ic ji c o r r e c t e d f o r
t h e d i f f e r e n c e i n o p t i c a l d e n s i t y r e a d i n g o f d i f f e r e n t am ino a c i d s o f t h e
sam e c o n c e n t r a t i o n s
- 26 From t h e t o t a l c o n c e n t r a t i o n s o f a l l o f t h e r e a d i n g s o f a p a r t i c u l a r
am ino a c i d a n d t h e v o lu m e i n w h ic h i t w as c o n t a i n e d , t h e p e r c e n t i n t h e
h y d r o l y s a t e w a s f o u n d u s i n g t h e f o l l o w i n g f o r m u la s ( 1 5 ) :
# = c o n e , o f am ino a c i d x v o lu m e x m o le c u la r w e ig h t______
c o l o r y i e l d x w t . o f p r o t e i n u s e d f o r a n a l y s i s i n mg
(l)
Some am ino a c i d s w e re e l u t e d o v e r l a p p i n g one a n o t h e r .
T he f o ll o w i n g
f o r m u la s w e re u s e d i n t h e s e c a s e s ( I ] ? ) :
(2)
A1 = -
,
f l 1'
^ i (IW 2Zv
(3 )
A5 -
2
p
2)
(V p /V l) P qT
F2 (P1 (VgZV1 )Pg)
W here:
V A ^ q u a n t i t i e s i n u m o le s o f am ino a c i d s i n 1 s t a n d 2 n d p e a k s
w hen o p t i c a l d e n s i t y i s p l o t t e d v s . c o n e .
P11 , P'g h e i g h t o f e a c h p e a k i n u m o le s
Vl i V2 e f f l u e n t v o lu m e s a t e a c h p e a k
Fl i Fg c o l o r y i e l d s
T
l e u c i n e e q u i v a l e n t s o f c o m b in e d p e a k s
When A1 an d Ag a r e fo u n d t h e p e r c e n t a g e s can b e c a l c u l a t e d u s in g
fo rm u la , ( l ) .
I t w as n o t e d when a n a l y z i n g t h e b a s i c am ino a c i d s t h a t a n am ino a c i d
a p p e a r e d t h a t i s n o t comm only fo u n d i n p r o t e i n a n a l y s e s .
T h is p a r t i c u l a r
am ino a c i d w as o v e r l a p p i n g t h e t y r o s i n e , p h e n y l a l a n i n e c o m b in a tio n w h ic h
a p p e a r s i n t h e s h o r t c o lu m n .
I n o r d e r .to s e p a ra te t h e
n e c e s s a r y t o r u n t h e l o n g colum n p a s t t h e pH
Ij..2^
am ino a c i d , i t was
b u f f e r a t 7£°C by c o o l i n g
i t t o 25>°C a n d p a s s i n g a pH 6 .8 c i t r a t e b u f f e r t o e l u t e i t
p r e s e n c e w as v e r i f i e d i n b o t h p r o t e i n d e t e r m i n a t i o n s .
(l6 ).
Its
The p r e s e n c e o f t h i s
am ino a c i d i s d i s c u s s e d i n t h e E x p e r im e n ta l P a r t I I I R e s u l t s .
The p e r c e n t a g e s f o r a l l t h e am ino a c i d s f o u n d a r e r e p o r t e d i n T a b le
- 27 V II.
The t o t a l p e r c e n t a g e i s a lm o s t 100$ e v e n th o u g h t r y p t o p h a n , c y s t e i n e ,
c y s t i n e and am m onia w e re n o t p r e s e n t i n t h e c a l c u l a t i o n s .
T h is i s due t o
t h e a d d i t i o n o f w a t e r t o t h e am ino a c i d s a s t h e y a r e b r o k e n fro m th e p r o ­
t e i n on h y d r o l y s i s , t h u s i n c r e a s i n g t h e t o t a l w e i g h t o f h y d r o ly z e d m a t e r i a l
i n th e h y d ro ly s a te .
The e x p e r i m e n t a l e r r o r d u e t o t h e t e c h n i q u e i s a b o u t 1 0 $ .
T h is i s
i n c r e a s e d som ew hat i n u s i n g t h e f a c t o r 5«7 c o n v e r t mg o f n i t r o g e n t o p e r--,
c e n t p r o t e i n d u r in g t h e K j e l d a h l a n a l y s i s .
T h is f i g u r e i s n o t a n a b s o l u t e
v a l u e , b u t h a s b e e n a c c e p te d a s t h e c o n v e r s i o n f a c t o r b y c e r e a l c h e m i s ts .
RESULTS
Of t h e am ino a c i d s a n a ly z e d o n l y o n e , p r o l i n e , w a s f o u n d t o be o f a
n o t i c e a b l y lo w e r p e r c e n t a g e i n V a n ta g e t h a n i n Gompana.
o f p r o l i n e w as
a n d 1 8 .9 $ ' r e s p e c t i v e l y .
w e l l ab o v e t h e e x p e r i m e n t a l e r r o r i n v o l v e d .
The p e r c e n ta g e
T h is d i f f e r e n c e o f 9,)\% i s
A lth o u g h no c o n c lu s i o n s c a n
b e draw n fro m t h i s one c o m p a r is o n , i t w i l l b e o f i n t e r e s t w hen a n a ly z in g
o th e r m a ltin g b a r le y v a r i e t i e s .
T h is l a r g e d i f f e r e n c e i n d i c a t e s t h a t am ino
a c id s i n d i f f e r e n t b a r le y p r o te in s can b e q u ite v a r ie d ,
O th e r am ino a c i d s
w e re o f d i f f e r i n g p e r c e n t a g e s b e tw e e n t h e tw o p r o t e i n s , b u t n o n e so o u t ­
s ta n d in g a s p r o l i n e .
The a d d i t i o n o f am ino a c i d s t o m a l t , a s m e n tio n e d i n
th e I n tr o d u c tio n , d id n o t in c lu d e p r o lin e .
S e v e r a l o f t h e am ino a c i d p e r c e n t a g e s d i d n o t c o r r e l a t e w i t h t h e
a n a ly s e s - a v a i l a b l e i n t h e l i t e r a t u r e
( 1 7 ,1 8 ) w h ic h w e re m i c r o b i o l o g i c a l l y
p e r f o r m e d on unnam ed b a r l e y v a r i e t i e s .
The i m p r e s s i o n g i v e n fro m t h e s e
a r t i c l e s w as t h a t b a r l e y p r o t e i n d o e s n o t v a r y w i t h v a r i e t y .
From th e
p r e s e n t a n a l y s i s t h i s d o e s n o t a p p e a r t o be th e c a s e ( T a b le V I I ) .
- 28 D u rin g t h e a n a l y s i s two am ino a c i d s a p p e a r e d t h a t w e re n o t p o s i t i v e l y
id e n tifie d .
G lu ta m in e (1 9 ) a n d gamma a m in o b u ty r ie a c i d ( 2 ) h a v e l a t e l y
b e e n i d e n t i f i e d i n b a r l e y p r o t e i n and i t i s p r o b a b l y t h e s e a c i d s t h a t a r e
p re s e n t.
F u r t h e r w o r k . i s p la n n e d t o p o s i t i v e l y i d e n t i f y t h e s e a c i d s .
The h i g h p e r c e n t a g e o f g lu ta m ic a c i d ,
i n Compana a n d 31.,3% i n
V a n ta g e , s u g g e s t s t h e p o s s i b l e u s e o f b a r l e y p r o t e i n f o r t h e c o m m e rc ia l
p r o d u c t i o n o f so d iu m g l u t a m a t e .
The c h i e f s o u r c e o f sodium g lu ta m a te a t
t h i s tim e i s w h e a t p r o t e i n , w h ic h c o n t a i n s 3 1 .8 $ g lu ta m ic a c i d ( 2 1 ) .
The am ino a c i d p e r c e n t a g e s a r e p r e s e n t e d i n . T h b le V I I . . T h e se v a l u e s ■
a r e c o m p a red t o a v a i l a b l e p e r c e n t a g e s o f am ino a c i d s i n w h e a t ( 2 2 ,2 3 ) .
'
1.
'i
c o m p le te am ino a c i d a n a l y s i s w as f o u n d . f o r w h e a t p r o t e i n a lt h o u g h t h e
No
p e r c e n t a g e s w e re a v a i l a b l e f p r t h e e s s e n t i a l am ino a c i d s a n d g lu ta m ic a c i d .
T h e se a n a l y s e s w e re made m i c r o b i o l b g i c a l l y ,
From th e s t a n d p o i n t o f t h e b a s i c am ino a c i d s a n d g l u ta m i c a c i d , th e
p r o t e i n s a p p e a r som ew hat a l i k e fro m t h e o v e r a l l r a n g e s o f am ino a c i d s
r e p o r t e d , a lt h o u g h t h e tw o b a r l e y p r o t e i n s r e p o r t e d h e r e a r e h i g h e r i n
g l u ta m i c a c i d and v a l i n e , w h i l e b e in g som ew hat lo w e r i n m e t h i o n i n e .
It
i s p o s s i b l e w hen d e v e lo p in g new v a r i e t i e s f o r f e e d p u r p o s e s t h a t m ore
a t t e n t i o n s h o u ld b e g iv e n t o t h e
to y ie ld p e r a c re .
amino a c i d c o n t e n t o f t h e p r o t e i n t h a n
- 29 TABLE H I
COMPOSITION OF THE PROTEIN IN
■COMPANA AND VANTAGE BARLEY
Amino A c id
Compana
V a n ta g e
' O th e r s
(17,181
2.9%
3.6%
2.2%
37.3%
9.2%
A s p a rtic
. 1.7%
2.0%
S & rin e
T h r e o n in e
1 , .#
G lu ta m ic
364#
18.9%
P ro lin e
G ly c in e
1.2%
A la n in e
2.1%
6,3%
V a lin e
T y r o s in e
3.2%
P h e n y la la n in e
2.1%
H is tid in e
2.1%
L y s in e
3.2%
3.6%
A r g in in e
1.1%
M e th io n in e
L e u c in e
9.1%
Is o le u c in e
3.1%
.21%
Gamma 'a m in b b u ty r ie * ■
G lu ta m in e #
*22%
T o ta ls
99.19%
-“-n o t p o s i t i v e l y I d e n t i f i e d
• 2.1+%
lu 0%
6.3%
2.9%
2.3%
1.1+%
2*6%
3.0%
. 0.2%
6.8%
3.1%
.33%
.30%
-
92.03%
.0 .8 - 1.2%
' 0 .1 - 3.2%
1.9%
22.8-23.9%
I+. 2-12.3%
0 .0 - 1,7%
0.1+— 1 . 2%•
0 .1 - 3.2%
0 .8 - 1.8%
2 . 2- 3.6%
0 .8 - 3.2%
0 .0 - 0.8%
1+.0— 6.0%
. 0.8%
3 .2 — l+«6%
- 3 .2 - U.3%
W heat
(21,22,23)
2 .8 - 3.3% ■
' 31.8%
■ ■ "
— —
3 .6 - l+.2%
3 .7 - 2.7%
1 .0 - 2.2%
2 .7 - 2.9%
0 .2 — l+el+%
1.3%
2 .8 - 8.3%
3.3-'6.9% ——
„
„
- 30 • V I.
1.
SUMMARY"
Compana F r e a k s , n um bers 7219 a n d 7223, D ek ap , B u lk , O t i s , and B a r b le s s
v a r i e t i e s o f b a r l e y a r e s u g g e s t e d f o r u s e i n c r o s s - b r e e d i n g t o a tt e m p t
t o r a i s e t h e p e r c e n t a m y lo se i n t h e i r s t a r c h e s .
2 . ■ P r e l i m i n a r y r e s u l t s i n d i c a t e t h a t b a r l e y may b e u s e d a s a n e c o n o m ic a l­
l y f a v o r a b l e s u b s t r a t e f o r t h e p r o d u c t i o n o f s t a r c h and g lu c o s e s y ru p
i n th e In te rm o u n ta in a r e a ,
3.
Two b a r l e y p r o t e i n s a m p le s fro m Compana an d V a n ta g e v a r i e t i e s w e re
a n a ly z e d c h ro m a to g r a p h i c a l l y and t h e p e r c e n t a g e s o f am ino a c id s i n t h e
tw o p r o t e i n s w e re f o u n d t o d i f f e r .
Ue
The o u t s t a n d i n g v a r i a t i o n i n t h e am ino' a c i d s o f t h e tw o b a r l e y v a r i e ­
t i e s , o n e o f w h ic h i s a good m a l t i n g b a r l e y , . t h e o t h e r p o o r , w as in .
t h e 9ch% d i f f e r e n c e i n p r p l i h e .
Ue
'
B o th v a r i e t i e s w ere h i g h i n g l u ta m i c a c i d w h ic h m ig h t make b a r l e y
p r o t e i n c o m m e r c ia lly f e a s i b l e f o r t h e p r o d u c t i o n o f so d iu m g l u t a m a t e .
V I I . • LITERATURE CITED
( 1 ) H i l b e r t , G. .E. a n d - M acM aste r s , M. M ., J . B i o l . Chem. 1 6 2 , 2 29-38
(1946)
( 2 ) I h i s t i e r , R . L . a n d H i l b e r t , G. E ., I n d . Eng. G hem ., 36 , 796-8
( 3 ) W o lf,
I . A ., D a v is , H. A ., C lu s k e y , JV E.-, Gundrum, L . J . ,
(l9lj.li:)
an d R i s t
C. E . , I n d . E ng. Chem. k 3 , 9 1 5 -1 9 ( l9 .5 l)
(Ij) D e a t h r a g e , W. L . , M a c M a ste rs, M. M.,- V in y a r d , M. L », an d B e a r , R . P . ,
C e r e a l Chem. 3 1 , 5 0 -5 2 (1 9 5 U
( 5 ) D e a th r a g e , W. L . , M a c M a ste rs, M. M ., a n d R i s t , C. E . , A. A. A. C.
T r a n s a c t i o n s , 8_, 31 -lj2 (1 9 5 5 )
( 6 ) D im le r , R . J . , D a v is , H. A ., R i s t , C. E . , a n d H i l b e r t , G. ;E .,: C e r e a l
Chem. 2 1 , 1:30-16 ( i ^ )
'
.
'
(7 ) A. 0 .
A. C ,, p . 1 3 , 2 .2 lj s e v e n t h e d i t i o n ,
(1 9 5 0 ) ■
( 8 ) A. 0 ,
A. C ., p . 3I4.6 , 2 2 .2 9 s e v e n t h e d i t i o n ,
( 9 ) A. 0 .
A . C ., p . • 3U3, 2 2 .9 s e v e n t h e d i t i o n (1 9 5 0 ).
(1 9 5 9 )
( 1 0 ) B a n a s ic k , 0 . J . , H a r r i s , R . H ., a n d S c o t t , G. M ., B re w e rs D i g e s t , 2 9 ,
1 0 9 -1 3 (1 9 5 U
(1 1 ) B a t e s , F . L «, F r e n c h , D ., a n d R u n d le , R . E . , J . Am. Chem. S o c ., 6 5 ,
llj2 (1 9 5 2 )
(1 2 ) L a n s k i, S . , K o o i, M ,, a n d Sc h o c k , T . J . , P r o p e r t i e s o f t h e F r a c t i o n s
a n d L e n i o r S u b f r a c t i o n s fro m V a r io u s S t a r c h e s , i n P r e s s .
( 1 3 ) M oore, S . , and S t e i n , W. H ., J . B i o l . C h e m .,' 1 9 2 , 663 ( l 9 5 l )
'
( l i t ) D u s t i n , J . P . , C z a jk o w s k a ,' C ,, M oore, S . , a n d B igw ood, E . J . , A n a l.
C h im ic a A c ta 9 , 256 (1 9 5 3 )
(1 5 )
M oore, S . , a n d S t e i n , W. H ., J . B i o l . C hem ., 176 , 367-88 (19lj8)
- 32 ( 1 6 ) S t e i n , W. H ., J . B i o l , C hem -.,'2 0 1 , ^ - 5 8
(1 9 5 3 )
( 1 7 ) F o l k e s , B. F . , a n d Temm, E . W ., B iochem . J . 6 2 , I i - I l (1 9 5 6 )
( 1 8 ) B is h o p , L , - R , ,• J . . I n s t . B rew . 3 b f 101 (1 9 2 8 )
( 1 9 ) M acLeod, A. M ., J . I n s t . B rew in g 5 7 , 163-69 ( l 9 5 l )
( 2 0 ) L ju n g d a h l L . j S a n d g re n E.> E u ro p e a n B rew .. C onv. P r o c . C o n g r. 1 7 2 -9
(1 9 5 3 )
(2 1 ) M ille r ,
B. S i , S c a f f e , J . V ., S c h e l l e n b u r g e r , J . A ., an d M i l l e r , G, D . , '
. C e r e a l C hem ., 2 7 , 9 6 -1 0 6 (1 9 5 0 )
■
( 2 2 ) B a r to n - W r ig h t, E . C .,. A n a ly s t T l , 2 6 7 -7 8 ( I 9I46)
(2 3 ) K u rk e n , K. A ., Lyman, C. M ., D i e t r i c h , S . , B r a d f o r d , M», a n d T r a n t , M .,
J» N u t r i t i o n 36 , 3 5 9 -6 8 (1 9 ^ 8 )
^ 33 —
V III.
ACKNOWLEDGEMENTS
My d e e p e s t g r a t i t u d e a n d 't h a n k s a r e e x p r e s s e d t o D r. K e n n e th G o e rin g
f o r h i s p e r s o n a l i n t e r e s t a n d g u id a n c e t h r o u g h o u t t h e r e s e a r c h p ro g ra m , t o
M rs . B e r n ic e D e h a a s o f t h e M o n tan a S t a t e C o lle g e E x p e rim e n t S t a t i o n , t o
M r .’ R o b e r t E s l i c k o f t h e M o n tan a S t a t e C o lle g e Agronom y D e p a r tm e n t, a n d t o
t h e o t h e r m em bers o f t h e C h e m is try D e p a rtm e n t f o r t h e i r w elcom e s u g g e s t i o n s .
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