extraction, composition and some of the physical and
TRANSCRIPT
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EXTRACTION, COMPOSITION
AND SOME OF THE
PHY S ICAL AND CHEMICAL PROPERTIES
OF
COMPONENTS OF D I ETARY FIBRE
by
War r en Oesmond Ho l l owa y
A the s i s pre sented in par ti a l ful f i lment o f the
r equi rements for the degree of
DOCTOR OF PH ILOSOPHY
i n the
Departmen t o f Food Technology
MASSEY UN IVERS ITY
1 98 5
"The value of wha t one knows i s d oubl ed i f one confe s s e s to
not knowi ng wha t one does not know . What one knows i s then r a i sed beyond the suspi c ion to which i t i s expo sed when one
c la im s to know wha t one does n o t know . "
Schopenhauer
ii
iii
ACKNOWLEDGEMENTS
I wi sh to acknowledge the fol l owing people and organ i sations that have
a s s i s ted in the preparation of thi s thes i s . The chi e f s upervi sor
Profe s so r E . L . Ri chard s and the second s upervi sor Or J. Le l i ev r e and
the ac ting supervi sor or M . Ta yl o r , a l l from the Department of Food
Te chnol ogy ; also Appl ied Bi ochemi s try Di vi sion , Departme n t of
Sc i e n ti f i c and Industr ial Re search , Palmer s ton No r th for f i nanc ial and
m a te r i a l s upport tha t enab l ed thi s proj e c t to be und er taken .
Thanks a r e also due to Or J . Li l l , Ho r ti cul tural Re search Sta ti o n ,
Mi n i s tr y o f Ag ricul ture and Fi she r i e s , Levi n , and Appl ied Bi ochemi stry
Divi s i o n for supplying sampl e s .
My thank s a r e a l so due to the s ta f f of ABO f o r the i r encourag eme n t and
guid anc e , par ticul arly to or J . Monro for d i scuss �ons and comme n ts , to
or J. Le e f o r running the Induc tive ly-Coupl ed Argon Pla sma Emi s sion
Spec trome ter for mul ti pl e me tal analys i s ; Mr o . Hopc ro f t and Mr R .
Bennett o f the e lec tron microscopy unit and Mr P . C . van Oi ngenen o f
the wo rkshop .
He lp f ul s ug g e s tions and contributions we re r ece ived from member s o f
the Chem i s tr y , Biochem i s tr y , Bi ophy s i c s Depar tment; Or M . Ha rdman for
acc e s s to a c ompute r i sed nonlinear c urve- f i tting procedure , Mr R .
MacKe nz i e fo r assi s tance wi th measurement o f zinc , iron , sod i um and
pota s s i um and or R. Gr eenway for the use of the osm ometer .
Thanks to Mr G . C . Arnold of the Mathema ti c s Depa r tment for d i scu s s i ons
on the i n te rpr e tation of the s ta ti stical procedures used i n s tudyi ng
the r e l a tionship betwe e n fibre c om po s i tion and m e tal bind ing .
iv
I am a l s o indeb ted to the s ta f f a nd s tud ents of the Fa cul ty of
Technol ogy for advice , comme n ts and referenc e s , par ticularly Dr M .
Ea r l e , Dr G . I . Robertson , Mr R . Greig , Mr M . Re eve s , Mr S e n Wong and
Mr s Bewl e y for her un s ti n ti ng he lp in a s s i s ting to locate chemicals
and equ i pm e n t and Mr Alg er for manufactur ing a cutting tool tha t
enab l ed the UM05 membranes to be cut to s i ze .
Gr a te fu l tha nks to Mr s J . Ti poki for runni ng the g raphi c s prog ram , to
my wi fe fo r typing the drafts , and to Mi s s D . J . Ro sva l l for typing the
final sc ript of thi s the si s .
T A B L E 0 F C 0 N T E N T S
ACKNOWL EDGEMENTS
TABLE OF CONTENTS
LIST OF FIGU RES AND TABL ES
ABSTRACT
INTRODUCTION
P URPOS E OF •rHIS STUDY
P ART I
Isol ation and De te rm i n a tion of the Chem i c al
Compos i tion of some o f the Fi bre Components o f Frui ts , Vege tab l e s , Pastur e Grasses and
Wheat Br an
I n troduc tion
Ma te r i a l s and Me thod s
Re sul ts
D i scus sion
P ART I I
Study o f the vi scosi ty o f Wheat Bran
Hemicel luloses
In troduc tion
Ma te r ia l s and Me thods
Re sul ts
Di scussion
-,
-v
Page
No s
i i i
V
v i i
5
8
10
1 4
1 9
2 5
3 1
3 4
3 6
4 1
PART I I I
Mo rphol ogical Chang e s i n Whea t Bran a s a resul t
o f polymer extraction
In troduc tion
Ma ter ials and Me thod s
Re sul ts
Di scuss ion
PART IV
Me tal Ad sorption to Wa ter Sol uble and
I n so l uble Fibres and from Frui ts , Vege table s , Br an and Grasses
In trod uc tion
Ma te r ials and Me thods
Re s u l ts
Di scussion
P ART V
Zinc Ad so rption to Whe a t Bran , i ts Fibre
Componen ts and to Phyta te
I n tr oduc tion
Ma te r ia l s and Me thods
Re s ul ts
Di scussion
G ENERAL DISCUSS ION
C ONCLUS IONS
REFERENCES
APPENDICES
vi
4 5
49
5 2
6 4
6 7
69
7 4
8 8
9 9
1 04
1 09
1 2 3
1 34
1 4 1
1 42
1 68
vii
L I S T 0 }o, F I G U R E S A N D T A B L E S
Fig u r e 1 :
Figure 2 :
F i g u r e 3 :
Tab l e I :
Tabl e I I :
Tab l e I I I :
Fig u r e 4 :
F i g u r e 5 :
Figure 6 :
Tabl e IV :
Tabl e V :
F i g ur e 7 :
Figur e 8 :
F i g u r e 9
A to D :
Flow diag ram o f the five stages o f the
i nvestig a ti o n s i n thi s thes i s .
Sequence o f extrac tion of fibre c omponen ts .
Separ ati o n o f s ug ars through a colum n packed
wi th 3% ECNSS-M at 190 ° C u s i ng a Hewle tt Packard 5 8 4 0A Ga s Chromatog raph .
Yield in g r am s o f dried polym e r s from the
s tarting m a te r ia l .
Compo s i ti o n o f i solated fibres from frui ts ,
vegetabl e s , g ra s s e s and wheat b r an .
Reg ions o f New zeal and where the foods
a nd g ras se s used for ' fibre ' ex trac ti o n
Pag e
No s
9
20
21
2 2
2 3
were grown . 2 4
Sequence o f s yn the s i s of
N-me thyl-morphol ine-N- oxide .
Kinematic v i scos i ty of wheat b r an
hemi cellulose extracted in alka l i before and afte r d e l i g n i fication .
Ki nemati c v i scosi ty of wheat b r an
hemicellulose a fter solub i l i sation and r ecovery f rom N-m e thyl-morpholine-N-ox id e .
Limi ting v i sc os i ty numbers o f the
hemicellul o s e s extrac ted in alk a l i before and afte r d e l i g ni fication and a f te r recovery
f rom N-methyl-morpholi ne-N-ox id e .
Re lative vi scos i ty o f nond e l ig n i fied
hem icellulose i n alka l i as a fun c tion o f time .
Ca ryops i s o f whe a t ( Tr i ti cum ae s tivum ) and
parts o f i ts pe r i c arp in long i tud inal section .
DMSO c e l l wa l l prepar ation procedur e .
Wheat b r an , b r an ex e thanol , b r an ex oxalate
and bran ex alkali observed at 380 mag n i f i c a ti o n b y l ight microscopy .
3 4
37
38
39
4 0
4 8
5 0
5 5
Figure 1 0
A to D :
Figure 1 1
A a nd B :
Figure 1 2
A to D :
Tab l e VI :
Tab l e VI I :
Figure 1 3 :
Figure 1 4 :
Figure 1 5 :
Tabl e VI I I :
Table IX :
Tab l e X :
Tabl e XI :
Tabl e X I I :
Tab l e X I I I :
Tab l e X I V :
Wheat b r an , b r an e x e thanol , b r an ex oxalate
and bran ex alka l i ob served by l i g h t microscopy .
Whe at b ran a f ter DMSO and chloram i n e- T ,
observed b y l ight m i c roscopy .
Wheat bran a f ter DMSO and chl oram ine-T ,
observed by e l ectron microscopy .
Yi e lds o f hemicellulose obta in ed by alka l i
and DMSO e x tr ac ti o n from wh e a t bran .
Composi ti o n o f nond e l igni fied
hemicellulose extracted from whe a t bran wi th alka l i a nd DMSO.
Amicon MP S - 1 micropar ti tion uni t .
Percent zinc bound to wheat b r an hem i c e l l ul ose
over a pH r ange 2 . 1 2 to 8 . 3 .
Percen t zinc bound to wheat b ran hem i c e llulose
wi th d i f f e r e n t mixing times .
In fluen c e o f o smol a l i ty on zinc b i nd ing to
wheat b r an hemi cel lulose .
Re liab i l i ty s tudy from ten ana l ys i s of the
meta l s i n the standard solution and afte r b ind ing t o wheat b ran .
Percen t me ta l rete n tion to the Ami c on MPS-1
micropo r ti o n sys tem .
Me tal conte n t ( �g/mg dry we ight) o f wa ter
solub l e ( SF ) and wa ter insolub l e ( I F ) fibres f rom frui t , veg e tab l e , bran and g ra s se s .
Mean me tal conte n t ( �gjmg d r y weigh t ) o f frui t ,
veg e tab l e , whe at b ran and g r a s s f i b res .
Chang es i n m e ta l content ( �g/mg d r y we ight) of
water solubl e ( SF ) and in solub l e fibres ( I F ) f rom frui ts , vege tables , bran and g ras s e s a f ter
vi ii
5 7
5 9
6 0
62
6 3
7 3
76
7 7
7 8
7 9
80
8 1
8 2
m ixing with a ' s tandard ' me ta l solution . 8 3
Bound me ta l conte n t ( � g/mg d r y we ig ht) o f
frui t , vege table , wheat bran and g ra s s fibres a fter mix ing wi th 's tandard' metal solution . 8 4
Tab l e XV :
Tab l e XV I :
Tab l e XVI I :
Mean bound me ta l conten t ( IJg/mg dry we ight) of wa te r solub l e ( SF ) and insoluble fibre
( I F ) a f te r mixing wi th 'stand ard' me ta l
soluti o n .
Mean , standard dev i a tion (!Jg/mg) and T values
of bound me ta l s from ma tched pa i r ed fibre s , before and after mixing with ' s tandard ' me ta l
solutio n .
Percen t nond i a l ysable me ta l conte n t o f the
water solubl e and insolub le fibre s .
Tab l e XVI I I : Tota l ion- b i nd ing capacity ( IJM/ g ) o f fibres
extr ac ted from frui ts , vege ta bl e s , bran and g rasse s .
Fig ure 1 6 :
Tab l e XIX :
Tab l e X X
Tab l e XXI :
Figur e 1 7
A to K :
F i g ur e 18
A to K :
Tab l e XXI I :
Ex tr ac ti o n sequence for the preparati o n o f
l ignin .
Compo s i tion o f the cold wa ter soluble fibre
and the pur i fied water solub l e fibre from wheat b r an .
Yields o f the wa te r solub l e fibres and
l ignoc e l lu l ose from wheat b r an .
L ignin conte n t of wheat bran , l ignoc e llul ose
and hem i c e l lulose .
Zinc b i nd i ng to wheat bran , i ts fibre
c omponen ts and phyta te .
Scatc hard plots o f zinc binding to whe a t
bran , i ts f i b r e c omponen ts and ph yta te .
Zinc b ind ing capac i tie s , ligand 5 0 % , ( IJM/ g )
and Hi l l coe f f i c i e n ts for whe a t b ran , i ts fibre c ompo nen ts and phyta te .
i x
84A
85
86
87
1 06
1 11
112
11 3
11 4
11 8
1 2 2
A B S T R A C T
The e x trac tion and some o f the chem i c a l and phys ical prope r ti e s o f
c omponen ts from �lant c e l l wal l s a r e d e sc r ibed in thi s thesi s . Th e
c hemi c a l c omposi tion o f the extrac ted polym e r s and the morpholog i c a l
and ph ys i c al chang e s occurr ing in wheat bran a t var ious s tages o f an
e x trac ti on se quenc e and the m e ta l bi nding capac i ties of the e x trac ts
wer e d e te rmined .
A sequential ex tr ac ti o n procedur e us ing wa te r , amylase , oxalate
and alka l i ( before and a f ter del ign i fi c ation) wa s used to i so l a te
c omponen ts o f plan t c e l l wal l s . Th i s enab l ed wa ter soluble and wa ter
i n so lub le fibres from bean , c abbag e , le ttuc e , toma to , peach , pumpki n ,
k um er a , onion, pear , whe a t b r an , luc erne , c l over and ryegra s s to b e
ob ta ined . The wate r solub l e fibres we re shown to be composed
p r ed om i n an tl y of arabinose , galacto se and ur onic aci d , whereas the
wa ter in so lub l e fib r e s conta i n ed mainly a r ab inose and x ylose .
The v i scosi tie s o f the alkali soluble fibres e x tr ac ted from whe a t
b ra n , b e fo re and afte r chlor i te d e l igni fica ti on , a n d after
s o lub i l i s a tion in N-me thyl-mo rpholine-N-ox ide we r e d e te rmined . Th e
a r ab inoxyl an extrac ted be f o re d elignificati o n , yi e ld of 7 . 9 g/1 00 g ,
had a l imi ti ng v i sc os i ty num ber of 2 20 . 6 m lj g , wher eas the
a r ab inoxyl an ex trac ted a f te r c hlorite d e l i g n i fic a ti o n , yield o f 3 . 8
g / 1 00 g , had a l im i ting v i sc osi ty numbe r o f 7 4 . 2 mlj g . When the
s o lvent N-me thyl-morphol in e-N-ox ide had been used to d i ssolve the
n ond eligni fied arab inoxyl an , a considerab l e d ec rease in v i sc o s i ty , to
6 . 3 m lj g , wa s observed . It wa s c onc lud ed tha t d i r ec t extrac ti o n ( no
d el ig n i fic ation ) o f whe a t bran , enables a l e s s deg raded arabinoxylan
to be ex tr ac ted in ade qua te yie lds . The use o f
N-me thyl-morphol in e-N-ox ide a s a so lven t for arabinoxylan res ul ted in
e x te n s i ve d eg rada ti o n .
2
The struc tur al chang e s in wheat b ran a t e ach s ta g e o f the
e x tr ac ti o n sequence and when d imethyl sulphoxide ( DMSO) wa s s ub sti tuted
f o r alka l i we re observed usi ng light and scanning electron m i c ro sc opy .
It wa s shown tha t the commerc i a l l y g round sample o f whe a t b r an
c on ta ined a high propo r tion of s tarch , whi c h was removed afte r the
amyl ase tr eatmen t. Alkal i r emoved cell wa l l ma terial predom i n antl y
f rom the a l e uro ne laye r . DM SO wa s not an e f fic ient ex tra c to r o f
a r ab i noxyl ans from c e l l wa l l s , a yi eld o f o nly 0 . 4 % being ob tained and
the aleur o ne c e l l wal l s r emaining intac t. The arabi noxyl an , e x tr ac ted
wi th DM SO, had a hig her ferul i c ac id and ace tyl conten t than the
arabinoxyl an extrac ted wi th a lkal i .
The i n te r ac tions o f f ib re s wi th m e ta l ions ( coppe r , i r o n , z i nc ,
c al c ium , po ta s s i um , m agnesium , mang anese and sod i um ) us i ng
c oncen tr a tions tha t would be expec ted in the human sma l l bowe l afte r a
' typical' mea l were i nve stigate d . It wa s found tha t the wa te r solub l e
f ib res bound more c oppe r , i r o n and zinc than the wa ter insolub l e
f i b re s . Th e c opper , i ron and z i nc binding occ ur r ed wi th a
d i s placemen t o f c a l c i um , mag ne s i um and manganese . The water i nsolub l e
f i b res ( hemi c el lul ose s ) contained a highe r c a l cium conte n t than the
s o l ub l e f i b r e s ( pec tin s ) . Af te r ac id tre a tm en t , sod ium wa s b ound
preferenti a l l y r a ther than c a l c i um to hem i c e l lul ose . Po ssibly
d iv a l en t c al c ium ions pl a y a role in s tabi l i s ing the hem i c el lulose
c omponents of pla n t ce l l wa l l s .
3
The b i nd ing capac i ti e s and mechan i sms o f zinc bind i ng to whe a t
b ran , i ts componen ts and to phyta te we re d e te rmined . Zi nc b i nd i ng
c apaciti e s ( � M/g dry we ight o f plant ma te r ia l ) i n o rd e r of magni tud e
we r e ; phyta te ( 6 5 8 2 ± 192 ) , DMSO solub l e hem i c e llulose ( 5 0 8 9 ± 9 2 1 ) ,
wa ter soluble fibre ( 4 0 3 8 ± 2 1 6 ) , cell wal ls ( 1 0 1 2 . 6 ± 19 3 ) ,
l ig noc el lulose ( 5 1 0 ± 4 1 . 9 ) , c old wa ter solub l e fibre ( 4 40 . 0 ± 1 5 . 3 ) ,
a lk a l i solub l e hem i c e l lul ose ( 2 2 7 . 9 ± 6 1 . 4 ) , b r an ( 16 7 . 7 ± 1 2 . 7 ) , b r an
ex ox a l a te ( 14 8 . 3 ± 50 . 0 ) , b r an ex e thanol ( 1 42 . 3 ± 4 . 4 ) and cellul ose
( 5 7 . 4 ± 5 . 3 ) .
The wa ter solubl e f i br e , f ractionated us i ng ammonium s ul pha te ,
c omposed pr ed omin a n tl y o f arab inose ( 2 4 . 0 % ) , g a l ac tose ( 2 0 . 3 % ) , xylose
( 1 8 . 6% ) , mannose ( 1 6 . 2% ) , g l uc ose ( 10 . 9% ) and rhamnose ( 6 . 0% ) , bound
z i nc more strong ly than phyta te or the DMSO hem i c e l l ul ose . The
Sc a tchard pl o ts of z i nc b ind ing to phyta te and to the fibre s , except
f o r the water solubl e fibre s , we re concave and marked ly nonl inear ,
sugg e s ti ng tha t the b i nd ing mechani sm i s by n eg a tive c oope r a tivi ty o r
s i te he terog enei ty . Th e Sc a tc ha rd pl ots o f z i nc bind ing to the wa ter
s o l uble fibres showed well pronounced maximum , i nd i c a ti ng the b i nd i ng
m echan i sm i s by pos i tive coopera tivi ty .
Par t I o f thi s the si s d e sc r ibes the stud i e s under taken to i so l ate
a nd de te rm i n e the chem ic al compos i tio n o f d i f fe r ent type s of fibres
f rom bean , c abbag e , s we e t potato , le ttuc e , o nion , peach , pe ar ,
pumpki n , toma to , whea t b ran , whi te c l ove r , l ucerne and ryeg r a s s . Th i s
s tudy ha s been pub l i shed i n the Jour nal o f the Sc ience o f FOod and
Ag r iculture 198 3 , 3 4 : 1 2 36-1240 ( see Appendix F ) .
4
P a r t I I o f thi s the s i s d e sc r ib e s stud i e s undertaken to inve stig a te
the v i scosi ty o f hemi c e l lulose s ob ta ined using the e x tr ac tion
procedure and a fte r so lub i l i sa ti o n in N-m e thyl-mo rpho l ine- N-oxid e .
Th e wo rk has been publ i shed i n Ca rbohyd rate Re search 1 98 5 , 1 4 3 :
2 7 1 -2 7 4 ( see Append ix G ) .
Par t I I I d e sc rib e s s t ud i e s undertaken to obs erve the mo rpho l og ical
s tr uc tur e o f wheat b ran , chang e s occurr ing dur ing the e x tr ac ti o n
s equence and influenc e o f DMSO when sub s ti tuted f o r a l ka l i .
Pa r t IV d e scr ibe s studi e s o n b i nd ing o f me ta l s to wa ter sol ub l e
and i n so l ub l e fibres from frui ts , veg e tabl e s , b r an and g r as se s .
Par t V d e sc ribe s a more d e ta i l ed stud y o f zi nc b in d ing to wheat
b ran , i ts fibre c ompo nen t s , and to ph yta te .
The the s i s conc l ud e s wi th a g eneral d i scus s i on o f the f i nd i ng s and
a s ummary o f the conc lu s ions .
I N T R 0 D U C T I 0 N
The qua lity of food is dependen t on a high content o f
nutr i tionally desirab le componen ts and low levels o f detr imen tal
s ub s tances . The fibre c omponent of plant foods has had i ts status
c hanged from tha t o f an undesirabl e componen t, that reduced the
baking qual i ty of flour , a ffected food texture and did no t make a
significan t contributio n to the caloric intake, to a very desirable
c omponen t that c an prevent maj o r health d i so rders .
In r esponse to the c onsumer s ' demand fo r whi te flour , processes
were developed which l owered the fibre c ontent of the diet of We stern
popul ations ( Trowell , 1 9 7 3 ) . It has been observed tha t over the l ast
o ne hundred years , the incidence o f certa in type s o f d i seases have
increased ( Trowe l l , 1 97 3 ) . A hypo thesi s has been advanced relating
the increased incidence o f cer ta in non-in fec tious di seases of unknown
aeteology , to a r educ tion in the in take o f pl ant fibre in the diet
( Burki tt, 1 97 2 , Burki tt et al . 1 97 2 ) . In c ontrast to the possible
advan tag es o f the consumption of high fibre die ts , there is the
5
possib i l i ty that i t may lead to m ineral de fic ienc ies o f calcium , i ron
and zinc ( Reinhold , et al, lY�l).
Despi te thi s apparen t confl ict , as ye t unr esolved , fibre in foods
has become the basi s o f new and g rowing indus tr ies i n the We st . Of ten
wi th apparen t di sreg ard for the type of fibre , from pea hul l s
( Co l l in s � al . 1 98 2 ) to l in seed meal ( Duboi s , 1 97 8 ) being added back
to foods to increase the ir ' fibre ' content.
Die tary fibre has so far eluded a simple o r single defin i tion. It
may be best consid ered in terms of a concept proposed by the
6
hypothe sis tha t cer ta in d i seases are l inked to fibre-deple ted d ie ts
( Cleave , 195 6 , Trowe l l , 1 97 6 , Burki tt, 1 97 2b) .
Die tary fibre was initially considered to be the remnants o f pl an t
c el l wal l s tha t are re si stant to hyd rolys i s by the al imen tary enzymes
o f man and are ther efore presen t in the ileum , but may be par tly
h yd rolysed by the bac teria in the colon ( Trowe ll , 197 4 ) . Trowe l l
( 1 97 4 ) , van Soest and McQueen ( 1 9 7 3 ) consider ed tha t the maj or
components of d ie tary fibre we r e cellulose , hemicellulose and l ignin .
Howeve r Southgate e t al . (1976 ) , Jenkins � a l . (1976 ) and Spi ller and
Shipl ey ( 1 977 ) considered tha t pec tin , muc i n , gum s , waxes and cell
wall bound pro teins should also be included . Therefore the defin i tion
of d ie tary fibre was expanded to include the sto rage polysaccharides .
Thus dietary fibre wa s rede fined as the plant polysaccha r ides and
l ignin which are resi stan t to hydrolys i s by the d ig estive enzymes o f
m an ( Trowell e t a l . 1 97 6 ) . Southgate � al . ( 1 97 6 ) considered tha t
"thi s definition i s e s sen tially a phi losophical one and the term
appl ies to all the indigestible polysaccha r ides and lignin tha t may be
imag ined to reach the larg e intestine" . If thi s phi losophical
approach to the defin i tion o f d ie tary fibre is accepted , it then
d enies the possibi l i ty of dete rm ining the chemical iden ti ty of d ie tary
f ibre , for i t is no t pos sible to measur e compounds imag ined to have
r eached the beginn ing of the large in te stin e . However , in o rder to
substantia te the d i e tary fibre hypothesi s , i t is necessary to iden ti f y
the plan t cell wal l components tha t reach the sta r t o f the l arge
inte stine . One of the imped iments in achievi ng thi s obj ec tive has
been the lack of e stabl i shed procedures by whi ch plant cel l wa lls and
7
the i r c on s t i t ue n t s may be de t e rmi ned . E a r l i e r s t udi e s p l a c ed the
empha s i s on the me a s u rement of c e l l u l o se , hem i c e l l u l o se , p e c t i n and
l i g n i n ( Va n Soe s t and McQue e n , 1 9 73; S o u thga t e , 1 9 6 9; Ho l l oway, e t a l
1 9 7 7) . Th i s exp e r i men t a l approach i s l im i t ed , howe ve r , a s i t h a s n o t
ena b l ed t h e c omp o s i t i on o f the wa t e r s o l ub l e a nd wa t e r i n s o l ub l e
p o l y s a c c h a r ide s p r e s e n t i n d i f f e r ent f o ods t o b e de t e rmi ned , n o r h a s
i t e n a b l ed the inve s t iga t i on o f the phy s i c a l a n d chemi c a l prop e r t i e s
o f p l an t c e l l wa l l s and t he i r c on s t i t uen t s t o b e unde r t aken .
Purpose o f thi s Study
Thi s study wa s under taken in r esponse to the need fo r :
(1) Fur ther defi n i ti on o f the type s o f polysaccharides pr esent i n a
var iety of foods and fe eds .
{2 ) Inve stigation o f d i fferen t method s of extr ac tion o f pl an t cell
wal l polymer s and prepara tion of whole cell wal ls .
( 3 ) To determine the b inding of meta l s to d i f feren t types o f fib re s
from a var ie ty o f foods and feeds .
( 4 ) To quanti ta te the zinc bind ing proper tie s o f wheat bran , i ts
i n tact c el l wal l s , c ell wal l componen ts and phytate .
8
A f l ow d iag ram showing the various s tages i n the investigation o f
the extr ac tion , compo si tion and metal binding o f c omponents o f d ie tary
f ibre is shown in figure 1 .
0'1
Figure I : Flow d iag ram showing the var ious stages o f the extr ac tion, compo si tion and some o f the physical and chemical properties o f componen ts of dietary fibre .
I Par t I I
Vi scosi ty of whea t bran hem icelluloses
Par t I
Extrac tion and analysis of fibre from frui ts , veg etables, bran and
I Par t I I I
Struc ture of wheat bran at var ious s tages of extrac tion sequence
g rasses
I Par t IV
Metal binding to wa ter soluble and insoluble fibres from frui ts , vegetable s , bran and g rasses
I Par t v
Zinc bind ing to whea t bran and i ts components
10
Par t I
Isolati on and dete rmination o f the chemical composi tion o f some o f
the f ib re componen ts o f frui ts, vege tables , pasture g rasse s and
wheat b ran .
In troduction
Thi s sec tion of the thesis wa s initi a ted in the Applied
Bi ochem i stry Divi sion o f DS IR in 1980 . The experimental procedure s
f o r extraction were devi sed and pr el iminary experimen ts were c arried
out in tha t e stabli shment. The entire wo rk wa s compl eted and
submi tted for publ ication at the end of 1982 . Thi s par t o f the thesi s
wa s wr i tten at tha t tim e . Appendix D of thi s thesi s reviews thi s work
in l ight of subsequent publications as do e s the d i s c u s s ion in P a r t I .
Studies of the geog raphical and chronol og i c al inc idence of
non-in fec tious d i seases of the intestinal trac t resul ted in the fibre
h ypo thesis being proposed ( Burki tt, 197 3 ) . OVer the last 100 year s
there have been many chang es in we ster n Societies tha t may have an
influence on the human intestinal tr act. These include a decrease in
physical activi ty , i ncrease in c igarette smoking and stress associated
wi th urbanisa tion . Changes i n the diet are as soc ia ted wi th an
inc reased calorie intake in the fo rm of fa ts , s imple sugar s and
pro te in s and a correspond ing decreased cons umption of cereals and
vegetabl e s ( Walker , 197 4 ) . The decreased fibre conten t of cereals
used in deve loped socie ties can be traced to the changes i n milling
technology wi th the introduction of steel rol ler mills at the end o f
l ast centur y ( Trowe l l , 1973 ) .
11
I ni ti a l l y the fibre hypo thes i s l aid stre s s o n the dec reased whe a t
b ran c onte n t o f the we s tern d i e t ( Burki tt, 1973). However , many
Soc ie ti e s tha t have a l ow incidence of the ' fi b re tle f i c iency di se a se s '
have trad i tionall y had a low c er eal consumption and high c ontent o f
f rui ts and veg e tab l e s i n their d i e ts ( Ea s twood e t � · 1974).
Plan t c e l l wal l s have been s ug g e s ted to be a maj or c omponen t o f
d ie tary f ib r e ( Tr owe l l , 1974). Th e s truc tur e and chemi s tr y o f plan t
c el l wal ls has been r eviewed by Alber she im (1973)1 Aspinal l
(1 980 ) , Rog e r s and Perkins (1968), Pr es ton (1974), Se lvend ran (1983),
Monro et al . (1976), �Ne i l e t �. (1984) and Re e s (1977). However
few s tudie s have inv e s ti g a ted the extrac tion and compo s i tion o f
c omponen ts o f c e l l wa l l s from a variety o f frui ts , veg e tab l e s , bran
a nd pa sture g ra s se s to enable c ompa r i sons b e twe e n spec ies to be
und e r taken.
EXtr ac tion of the po lymeric c onsti tuen ts of c el l wa l ls has been
subj ec ted to extensive inve stigati ons see for example i\spinal l (.1982).
In A spinall s' (1982) ,-cvicw of Ct'll wall chemisLry he staLed
t h a t the obj e c tives o f the i so l ati on proc edure s , arc to obtain
po l yme r i c ma t e r i a l in a s h i gh a y i e ld a nd a s c h emi cally pu re
and homogeneous form a s po ssible . So me ti me s the se two obj ec tives may
work in oppo s i tion to each o ther . The ini ti a l probl em s are tho se o f
i so l a tion wi thout d eg rad a tion, t h e n pur i f i c atio n s o t h a t t h e
poly s ac c harid e s a r e ob ta ined f r e e of contam inati ng ma te r ia l s and
f r actionation to g ive s ub s tanc e s tha t a r e c hemi c a l ly and phys i c a l ly
homogenous wi thin acc eptable l imi ts ( Aspinall , 1 9 8 2 ) .
In some instanc e s , a s wi th c aps ul a r polysaccha r id e s from bacte r ia
and exud a te gums , polysacchar ides are r e ad i ly s o lub i l i sed and may
o ften b e s imply i so l a ted in hom og eneous form ( As pi n al l , 1 98 2 ) . In
o the r c a se s , e spec i a l l y cell wal l componen ts , the po l ysac c harid es a r e
presen t i n c l ose a s so c i a tion fo rming a supe rm o l ecul ar c el l wa l l
s truc ture ( Fe nge l , 1 9 76 ) . Gr aded ex trac ti o n c an se lectively
solub i l i se mac romolecular c om ponents of the c e l l wall ( Roger s a nd
Pe rki n s , 1 96 8 ) .
The simpl e s t extrac tion me thod for po l y s ac c har ides i s by using
wate r al one at var ious tempe r a tur e s ( Wi se , 1 94 2 ) . Some
polysaccha r ides may be b roug h t i n to soluti o n b y the use of po l ar
nonaqueous solvents s uch a s d i m e thyl s ulphox i d e , a s i n the extrac ti on
o f s tarch ( Le ach and Sc hoch , 1 96 2 ) and the i so l a tion of
1 2
o- ac e tyl- 4-o-me thyl g lucuronoxyl an s from hardwo od s and of
o- ac e tyl-galac tog lucom annan s f rom some sof twood s ( Bower ing et a l .
1 96 1 ) •
Ex tr ac tion o f pe c ti n s c an b e achi eved by wa ter ex trac tion o r wi th
aqueous s o lutions o f e thylened i am i n e te tr aac e ti c ac id or ammonium
oxa l a te ( Rogers and Pe rki n s , 1 96 8 ) .
Ex tr ac tion o f o ther c e l l wal l polysacchar id es s uch a s c ellulose
and hem ic e l lulose , i s g enerally more di fficul t . Wi lkie ( 1 97 9 )
rev iewed the chem i s try o f pl an t cell wa l l s and the i r hemicellulose s .
He c i ted the s ta teme n t b y No rman ( 1 9 3 7 ) tha t he consid e r s sti l l hol d s
true .
"The chemi stry o f the hem i c e l lul ose s r em a i n s to be
wr i t ten • • • • the ir i so l a tion is b e se t wi th pi tfa l l s and the i r sepa r a tion
and pur i fication wi th uncer ta in ty . The i nv e s tigato r who wi she s to
analyse plan t ma ter i a l for i ts conten t o f c el lul ose or hem i c e l lu l ose
m u s t f i r s t o f a l l be c lear in hi s own mind about the que stions whi c h
the analy s i s i s expec ted to answe r . It can no t be too stro ng ly
emphasi sed tha t ' c el lulos e ' and ' hem i c e l l u l o s e ' are normally
d e te rmi ned as the r esul ta n ts o f certa i n s e t s o f ope r a tions , r a the r
than a s c hemi c al l y de fined s pe c ie s . Th e term hemicellul ose ha s no
un i que d e fin i ti o n i n spi te o f , and ind e ed , in par t because o f , i ts
1 3
l ong usag e . Fo r wo rking purpose s , hem i c e l l u l ose s a r e often impl i c i tl y
o r exp l i c i tly d e f i ned a s the c e l l wal l and i n tercel lul ar
pol y s accharid e s tha t c an be extrac ted by a l k a l i from higher l and- pl a n t
ti s s ue s tha t a r e , o r we r e , l ignified , b u t s i m i lar mater ial c an b e
ex trac ted b y wa te r from the d e l ig ni fied ti s s ue s of g rasse s" .
A review , c onduc ted to d e te rmine the m e thods used by o the r
i nv e s ti g a tors for the i so l a tion of hem i c e l l u l os e s showed tha t the most
c ommon me thod used is extrac tion wi th a lka l i ( Timel l , 1 965 ; Jermyn
and I s herwood , 1 9 5 6 ; Furda , 1 9 79 ; Whi stler et al . 1 9 70 ;
Tha r anathan � a l . 1 9 75 ; Whi s tler and Fe a the r , 1 96 5 ; Bl ake and
Ri c hards , 1 9 70 ; Br i l l oue t e t �· 1 98 2 ; Ma r es and Stone , 1 9 73 ;
Keeg s tr a e t a l . 1 9 73 ; Si egel , 1 962; No r r i s and Preec e , 1 9 3 0 ;
Mac Ar thur and D ' Appolun ia , 1 9 80 ; Mo nro e t a l . 1 9 76 ; Reid and Wi l ki e ,
1 96 � and Sh ibuya a nd Iwa sak i , 1 9 78 ) .
However , only minor quan ti tie s of hem i c e l lul ose s can be r em ov ed
f r om soft wood s by d ir ec t ex traction and general l y a pr e l iminary
d el ig ni fication is r equi r ed to ob ta in ad equa te yield s . Time l l ( 1 96 5 )
pos tula ted tha t hem i c ellul ose may be pro te c ted by the l ignin pre s e n t
i n the se condary c e l l wal l s .
The most freque n tly used mate r ia l for the e x tr ac tion o f
hemic e l lul oses i s holoc e l lul ose , tha t i s pl an t mater ial tha t has b een
s ubj ec ted to d e l ignific ation. A s urvey of pub l i shed procedur e s used
to i so late hem i c ellulose s , from wo od s and plan t food s showed tha t
d el igni fica ti o n o f the m a te r i a l i s c ommonly c arr ied out b e fo re
c ommenc ing the hemi cellul ose extrac ti on ( Timel l , 1 96 5 ; Je rmyn and
Is herwood , 1 9 5 6 ; Bl ake and Richard s , 1 9 70 ; Br i l loue t et al . 1 98 2 ;
S i eg el , 1 962) .
However the severe c ond i tions o f ox id ation ( ozo ne ,
c hl or ine- 2 -aminoethanol , sod i um chl o r i te and chlorine) and e leva ted
temperatur e s and/o r pr e ss ur e n o rmally used for delig n i fi c a tion a r e a
pos s i b l e c ause o f po lysacchar id e deg rad a ti o n ( Timel l , 1 965 ) .
14
The yields o b ta inab l e b y d irect extrac tion from a var ie ty of pl an t
food s , nor the po s sible d eg rad a tion c aused b y d e l ig ni fi c ation o f food
h em i c e l lulose s , d oe s no t appe ar to have b e en d e termined .
Th i s stud y wa s d esig n ed ( s ee appendix D ) to i solate and then to
d e te rm i ne the c hem i c al c ompo s i tion o f s ome o f the c omponen ts o f
d ie ta r y fib re i n frui ts , vegeta ble s , pa stur e grasses and whe a t b ran .
Ma te r ia l s and Me thod s
Sampl ing pr oc edur e and g eneral sampl e tr eatment
The sampl e s s e l e c ted we re l argely d e te rm ined by the ir common
usag e i n New Zeal and . Commercially g rown and marke table frui ts and
v eg etables ( 1 kg f r e sh we ig h t o r a min imum of 3 sampl e s o f e ach
s pe c i e s ) we r e c o l l ec ted by Horticul tur e Ad v i sory O f f icers o f the
15
Mi n i stry o f Ag r icul tur e . The regions o f New ze al and where the food s
and g rasses wer e ob ta ined i s shown in Tab l e I l l . Three pa sto r a l
g ra s s e s ( 1 kg f r e sh we ight o f e ach spec ie s ) we r e ob ta in ed f r o m Appl ied
Biochemi stry Divi sion o f DS IR and wheat b ran (5 kg ) wa s ob ta ined from
Manawa tu Mi l ls Ltd . On arr ival at the l aboratory the sampl es we r e
sepa r a ted from the wa ste , we ighed , then f r e e ze-dr ied to a con stan t d r y
we igh t . Th e d ried mate r i a l wa s g round s o tha t i t would pas s throug h a
4 4 me sh s i eve . Th e homog en i sed sampl e s wer e s tored , a s a dry powd e r ed
produc t , i n a d e ep f reeze t-to•q.
Ex tr ac tion
Al l the e x tr ac tions wer e c arr ied out in d upl ica te . Sample s (5 g )
o f the d r ied food s ( bean , c abbag e , l e ttuc e , tomato , peac h , pumpkin ,
k um e r a , onio n , pear , whe a t b ran and the thr e e g rasse s , lucerne , c l over
and ryeg ra s s ) wer e extrac ted fo r 1 0 hour s in a Soxhl e t wi th 2 00 ml o f
95% e thanol and the e thanol i n solub l e so l id s were then air d r ied .
Oold Water Extr ac tion
wa ter ( 1 50 ml) wa s added to the e thanol i n solub l e sol id s and the
s us pe n sion slowl y s ha ken for 15 hour s . Th e l iquid l ayer wa s d e c an ted
and added to 3 volume s of e thanol . Af ter s tanding 12 hr the r esul ti ng
pr ec ipi ta te s we r e sepa r a ted by c en tr i f ug a ti o n . Th e prec ipi ta te s we r e
wa shed wi th e thanol then d ia l ysed ag ainst r un ning wa ter for 3 6 hour s
aga i n s t d i s ti l l ed wa ter for a f ur ther 2 4 hour s b e fore be ing
f reeze-dr ied . The d r ied e x tr ac ts were we ighed then s tored a t - 2 0 ° C
u n ti l they wer e requ i r ed f o r chemical analys i s .
1 6
Ho t Wa ter EX tr ac tion
Wa ter ( 1 50 m l ) wa s added to the c o ld-wa ter ex tr ac ted residue s .
The suspen sions wer e then he ated to 9 5 ° C f o r 2 hour s . Af te r cool ing
to room tempe r a tur e 20 ml of a lpha am ylase s olution ( Si gma A 6 880 ,
Porc i ne Panc reati c , 5 gj l i tr e- 1 i n phospha te buffer at p7. 0 ) and a few
d rops of toluene we r e added to the s uspensions which wer e then
incub a ted fo r 1 7 hour s a t 3 7 ° C . The s uspensions we r e r e- he a ted and
r e- incub a ted un ti l the KI-I � s po t te st for s ta rch wa s n eg a tive . Th e
s uper natants wer e dec an ted and added to 3 vol um e s o f e thano l . The
r es ul tan t precipi ta te s wer e trea ted in the same wa y as the cold wa ter
extrac ts .
Ox alate EX tr ac tion
To the so l id s r emain ing a fter the ho t wa ter extr ac tio n we r e added
1 00 ml of ammonium oxalate ( 1 0 g l i tr e-1 ) . Th e suspension wa s m i x ed ,
a nd a l l owed to s tand for 9 0 minute s . The s upernatants we r e dec an ted
and pr ec ipi ta ted as fo r the o ther ex tr ac ts .
Alkali EXtrac ti o n
To the oxala te in solub l e ma te r ia l wa s add ed 1 00 m l o f s od i um
hyd roxide ( 1 00 g l i tr e- 1 ) und er n i trog en . Th e suspensions we r e m ix ed
and a l l owed to s ta nd fo r 6 hour s . The aqueous layers we r e d ec an ted
and add ed to 3 volum e s of e thanol . These prec ipi ta te s wer e tr e a ted in
the same wa y a s the pr evious l y ob tained pr ec i pi ta te s .
D e l ig ni f i c a tion
TO the a lkali insolub l e r esidue s were added wa ter ( 1 00 m l ) and
g lac ial ace tic ac id ( 5 m l ) . Th e m i x tur es we r e hea ted to 75 ° C and
sodium chl or i te ( 1 g) wa s added s l owl y every 25 minute s un ti l a to tal
o f 4 g had been added . Af te r a fur ther 90 minute s a t 75 ° C the
m i x tures we r e then cooled , the s upernatants decan ted and d i sc arded .
Th e insolub l e r esidue s we r e then r e- ex trac ted wi th sod ium hyd rox ide
u s ing the pr evious pr oc e s s . Al l the e x tr ac ts , wa ter so lub l e and
a lka l i solub l e we re d i al ysed for 4 8 hours befo re fr eeze- d ryi ng ,
f o l l owed by d ryi ng in a d e s s i c a to r to a constant dry we ight . The
s equenc e used in the e x tr ac ti o n of the po lysaccharid es i s summari sed
i n figur e 2 .
Analys i s
1 7
The c ompo s i tions o f the d ried po lymers wer e dete rmined by
m ea s uring the ind ividual s ug ar s , a fter tr i fluoroac e ti c ac id
h yd rolys i s , by GLC of the a ld i to l ace ta te s ( Jones and Al ber she im ,
1 9 72 ) . The r esul ts of GLC analy s i s o f s ugar s tandard s are shown in
f ig u r e 3 . Th e uronic ac id s we r e measur ed using m- hyd roxyd iphenol
( Bl umenkr an tz and Asboe- Hansen , 1 9 73 ) , ace tyl g roups by the
h yd roxyl am i n e procedur e ( Mc Comb and Mc Cr ead y , 1 9 5 7) , me thoxyl g roups
b y the pe n tane- 2 , 4-dione me thod ( Wood and Sidd i qui , 1 9 71 ) and pro te in
u s ing the Fo l in-Ciocal te u r eage n t ( Sc hacte r l e and Po l l ack , 1 9 73 ) . Al l
the extr ac ti o n procedur e s we r e c arried out i n d upl i c a te , a s we r e al l
the analyse s . Re ported m e an va lue s we r e wi thi n 10 % var i a tion.
The yi e l d s were obtained by d e te rmining the d ry we ig hts ( a f te r
f r e e ze d ryi ng ) o f a l l the e x tr ac ts and expre s sed rel a tive to the d ry
1 8
we ights ( fr eeze d r ied ) o f the s tar ting m a te r ia l . Al l the freeze d ried
ex trac ts we r e s tored in we ig h ed air- tight pla s ti c c onta iner s
i mmed i a te ly a f ter d rying , then a fter we ig hing they we r e s to red a t
- 2 0° C .
Re s ul t s
Fig u r e 3 s hows the separati o n of s ugar s tand ards through a column
packed wi th 3% ECNSS-M at 1 90 ° C u s i ng a Hewl e tt-Packard 5 8 4 0A g as
c hroma tog r aph .
Tab l e I shows the yi e ld in g of fr eeze-dried pol ymer pe r 1 00 g of
fr eeze- d r ie d s tar ting m a te r ial .
Table I I shows the c ompo si ti on of the i solated compo nents of
d ie tary fibre from frui ts , veg e ta b le s , g ra s s e s and whe a t b ran .
Table I I I s hows the r eg ions of New Zealand wher e the food s and
g rasse s , used in thi s study , wer e obta ined .
1 9
Lipid chlorophyll free sugars ( discarded)
Cold water-soluble1 fibre
hot water-soluble1 fibre
20
Food Ethanol extraction
(Wheat) 2 (Bran )
(Bran ex ethanol) 2
Cold water, 15 hr
oxalate-soluble1 fibre Boiling water, 2. 5 hr
alpha amylase
water-insoluble1,3 fibre 1% ammonium
oxalate, 2 hrs Not delignified
waterinsoluble 1 ' fibre delignified
alkali residue
(Bran ex oxalate) 2 10% NaOH, N2
or DMSO
(Bran ex alkali)2 Chlorite 10% NaOH, N2
or Chloramine-T DMSO
(DMSQ-cell wall)2
Figure 2: Sequence used in the extraction of the water soluble and water insoluble fibres from fruits, vegetables, wheat bran and grasses. 1, Precipitated in ethanol, dialysed, freeze dried. 2, Insoluble material subjected to light and scanning electron microscopy. 3, Alkali soluble (hemicellulose) and DMSO soluble (DMSO hemicellulose)
. r::.
., ... . CG • • !O .I ! ;:, . �CO! i • • 18 ja; 1 .. , ;:, ... •
• � 't:J c • > • ii :C' •
� 0 ii �: -;. )( E - • 0 M £ • • • • -0 (1\ • 0 •
� 0 ;; 0 0 ·� c. � c • z:
'lt • ... Cl • � 2 Cl � ('J \l)
,.. � C(l
. ; CL Q ....
i '·" .._)
'
Fi gure 3 : S e paration of su gar s throu gh a co lumn packed with 3 %
ECNSS-M at 190•c u s in g a H e w lett Packar d 58 40A gas
chromatograph.
Table I
Mean yields in g of freeze-dried polymer per 100 g of freeze-dried
starting material
Food Cold
water
Hot
Water
oxalate
Soluble
Hemicellulose
before
Hemicellulose
after
22
Soluble Soluble delignification delignification
Beans 1. 0 4. 0 1 • 1 3. 7 1. 3
Cabbage 1. 5 1. 3 2. 4 4. 1 0. 6
Kumeras 0. 5 4. 3 4. 3 11 • 0 1 • 5
Lettuce 2.0 2. 1 3. 3 3. 7 1 • 0
Onion 1. 4 2. 8 2. 9 2.0 0. 7
Peach 2. 1 3. 3 1 • 3 3. 9 2. 5
Pear 0. 8 2.4 1. 8 2. 2 1 • 9
Pumpkin 0. 9 3. 7 3. 6 1 • 1
Tomato 3.5 0. 8 0. 7 2. 8 0.5
Wheat Bran 0.5 1 • 1 0. 8 7. 9 3. 8
Feeds
Clover 0. 9 2. 0 5. 0 4. 0 2. 4
Lucerne 1. 3 2. 0 3. 2 6. 2 2. 7
Ryegrass 0. 8 0. 5 1. 4 11 • 3 2. 5
23 TABLE If.
COMPOS IT I ON OF THE ISOLATED COMPONENTS OF DIETARY FIBRE FROH FRUITS.
VEGETABLES. GRASSES AND WHEAT BRAN, ( me an va l u e s)
(EXPRESSED AS A % OF THE ANALYSED COMPONENTS) Urooic
Food Fraction Rhamnose Arabinose XyloscMaonosc Galactose Glucose acid M�b.o.Jtyl Acetyl Protei a Beo.n Cold water 0.6 10.3 1.7 1.3 40.9 �-� 27.6 3.0 0.� 8.6
(Phauolw Hot water 7.2 6.5 0.7 0.4 23.5 l.4 49.5 1.4 1.1 6.1 .,lroru) Oxalate S.4 8.9 0.3 0.0 24.1 13.1 33.0 ).0 l.S 10.7
NaOH 1.0 2.1 21.9 4. I 12.3 "· 7 14.4 1.0 3.3 24.2 NaOH (o!ter 4.4 11.7 :J.a 0.0 6.1 18.4 5.3 1.9 2.4 25.2
dclii"illcation) Cabbaac Cold watct 0.8 4.2 0.3 0.4 6.1 1.1 71.5 1.0 0.2 14.3 (BrauiCG Hot water 3.3 16.3 0.7 0.3 12.7 0.0 53.6 2.1 0.6 10.3
olnucetJ, Vil:'. o�alnte 4.2 22.0 0.9 0.2 17.S 2.2 40.4 l.S 0.4 10.7 Capitatoll) NaOH 1.8 11.0 13.8 S.B 24.1 17.6 13.0 1.0 3.0 8.9
NaOH (after 0.4 4.2 29.S 2.7 1.0 18.0 7.7 3.0 1.1 25.3 dclignification)
Sweet potato Cold waur 3.9 14.0 o.s 1.9 2.5.6 6.8 15.9 9 . 7 0.5 21.3 (fpo,.ora Hot water 0.9 0.3 2.0 0.1 2.3 82.4 1.7 1.6 1.1 0.7 botatas) Oxalate 0.4 3.1 0.1 0.3 3.2 72.6 13.S 0.7 3.1 2.9
N:.OH 0.0 0.8 2.3 0.2 0.7 90.6 2.6 O.J 1.2 1.2 No OH (:.ftcr 0.0 1.7 24.4 0.0 0.0 55.0 a.a 1.3 0.4 8.4
clcli:;nific:.tion) Lcttuc.a Cold watU 2.4 10.2 1.1 2.8 2.5.6 5.5 45.3 1.1 0.3 S.6
(L.Dctua� Hot water 6.9 5.1 0.4 0.4 IS.5 0.0 58.3 4.4 0.9 7.5 uulvt�, Oulatc 7.5 s. 7 0.3 1.3 10.1 S.7 5S.1 3.1 0.6 10.1 var. Yate:slilkc) NaOH 1.6 19.7 23.3 8.2 0.') 0.0 24 9 1.'1 1.9 18.·'·
NaOH (after (;.J 2.2 �.& 3.8 8.3 16.-L 7.6 1.6 0.3 18.8 c1clisoi&:ation)
Onion Cold water 0.8 1.9 3.2 7.7 21.0 13.7 9.1 11.3 0.4 23.4 (Aflwm Hot water 0.3 0.6 0.6 9.3 0.0 40.2 27.4 11.2 0.0 10.3 ctpa) Oxalate 3.3 3.& 0.7 0.0 49.3 0.8 ll.S 2.7 0.7 8.0
NaOH 0.7 9.9 12.4 1.1 24.4 36.S 8.0 1.1 0.0 5.3 NaOH (;Utcr 0.0 1.3 31.9 2.6 7.5 46.3 S.l J.O 0.3 4.0
clcli�:nitic:ation) Pc:ICb (Prwt.a Cold W:ltcr 0.9 10.9 1.3 0.7 8.S 2.4 54.3 8.S 0.7 12.0
ptrsica) Hot water 5.0 42.6 2.4 0.6 11.9 1.7 23.6 8.7 1.1 2.4 Oxalate 2.7 31.6 1.2 0.4 7.S 3.3 44.1 2.7 1.6 4.9 NaOH 1.8 9.4 18.3 4.2 11.9 19.6 9.S 1.3 2.S 21.4 NllOH (afte1" 0.1 2.7 7S.S 0.0 4.1 3.6 6. 7 1.3 0.2 S.1
delisnification) Pear (Pyms Cold wat<r 2.S 23.1 1.7 0.6 23.S j_Q 27.6 5.8 0.4 9.1
comm:mis) Hot w:�.ter 1.7 4S.I 1.3 0.2 12.2 4.2 21.3 9.1 0.8 4.0 Oxillatc 2.3 SO. I lA 0.4 15.4 3.7 19.9 2.1 0.4 4.3 NaOH 1.6 17.2 19.8 3.8 13.4 27.7 8.3 0.7 1.7 j.S NaOH (after 0.0 4.2 80.6 0.0 2.6 1.9 4.2 1.$ 0.0 · 5.1
d�liKnificnion) Pumpkin Cold water 0.0 18.4 0.5 0.7 43.0 0.0 17.6 3.2 0.3 16.3
(Cucwrbita Hot water 0.0 10.3 0.5 0.5 17.$ j_J pt-pulmaximn) OJ<alatc 3.3 4.6 0.4 0.0 I4.S 5.2 58.9 7.3 0.4 5.2
NaOH 0.0 0.0 14.2 IS.S 32.7 7.2 12.S 2.6 (1.8 14.j NaOH (after o.u 3.9 ;.9 0.0 19.8 3u.2 17.S 4.0 o.a 19.8
deli�itication) Tomato Cold water I. I 5.8 0.7 0.1 12.1 0.0 69.6 1.3 0.1 8.6
(Lycoper>icon Hot wato:r 1.7 11.9 2.2 0.4 14.S 2.2 41.7 3.0 6.7 15.8 tscuJt"fU,I) Ox:.late 1.6 1$.4 1.6 0.8 14.6 2.4 26.8 5.7 1.6 29.3
NaOH 0.0 3. 7 27.0 12.1 6.9 31.9 9.2 O.j 0.5 1.2 NaOH (after 0.0 6.6 35.7 7.7 7.0 16.4 10.8 2.1 0.7 12.9
dcliiiJ1ifiC:J.tioa) Whc-•t bran Cold """ter 0.3 23.5 29.9 1.7 7.2 33.1 0.6 0.1 0.4 3.3
(Triticum Hot water 0.0 20.8 39.0 1.1 2.7 33.1 0.6 0.1 0.2 2.S autivum) O�:J.l:ltc 0.0 12.9 40.1 0.0 0.0 38.7 1.9 0.2 0.6 S.6
No.OH. 0.0 31.9 60.1 0.3 2.1 4.0 0.7 0.1 0.1 0.9 NaOH (at\er 0.0 29.4 61.8 0.0 0.8 $.1 1.2 0.1 0.$ 0.5
delianification) Wbite clover Cold water 3.1 9.8 10.9 0.7 15.3 5.4 20.0 3.4 9.5 22.0
(Trifolium Hot water 7.0 10.5 S.l 0.6 15.0 2.5 40.3 2.3 1.9 14.8 rcptru) Oxalate 2.1 3.9 0.4 0.0 5.0 6.4 49.$ 2.8 1.4 28.5
NaOH 8.1 28.4 27.2 0.0 3.0 0.0 19.0 1.2 1.6 11.5 NaOH (after 0.1 3.3 S0.2 5.6 S.4 13.3 6.4 1.5 0.3 13.3
clclignification) Lucerne Cold water 1.9 &.9 0.8 0.4 11.1 4.1 29.7 1.9 &.5 32.8
(Mrdicaro Hot water 9.8 11.6 1.9 0.4 14.0 4.3 42.$ 1.4 0.9 13.2 satiN) Oulato S.2 9.4 1.0 0.0 7.1 1.0 64.9 1.6 0.0 9.7
NaOH 2.0 2.8 53.1 4.4 6.1 11.5 10.9 0.8 0.5 1.9 No.OH (after 2.4 26.9 39.1 11.9 0.0 2.7 9.S 2.0 0.4 5.1
clclignific:uion) Ryearass Cold water 2.0 9.4 2.5 16.3 12.6 23.0 11.9 3.2 4.9 14.3
(Loliwrt Hot water 2.0 7.2 2. 7 0.7 8.6 1.9 24.3 6.4 S.4 3S.1 percrrrrr) Oxalate 1.3 8.1 2.6 0.9 9.8 7.7 41.$ 2.1 2.1 23.9
NaOH 0.2 14.3 S2.1 0.3 j .1 10.5 IO.S 0.7 1.2 4.9 N:.OH (after 0.2 10.1 63.9 0.0 4.8 9.9 4.4 0.4 0.3 5.9
c1cliaailie.ttion)
2 4
Table III
Regions of New Zealand where the foods and grasses, used for fibre
extraction, were grown. All food samples were picked by Ministry of
Agriculture and Fisheries, Horticultural Advisory Officer's in a fresh
state, and at the stage of ripeness normally used for human
consumption.
Name
Bean
Cabbage
Sweet potato
Lettuce
Onion
Peach
Pear
Pu mpkin
Tomato
Wheat bran
White clover
Lucerne
Ryegrass
Area grown and collected
Horticultural Research Centre, Levin.
Horticultural Research Centre, Levin.
Horticultural Research Centre, Pukekohe.
Horticultural Research Centre, Levin.
Horticultural Research Centre, Pukekohe.
Horticultural Research Centre, Hastings.
Horticultural Research Centre, Hastings.
Horticultural Research Centre, Pukekohe.
Horticultural Research Centre, Levin.
Manawatu Mills, Palmerston North.
Grasslands, DSIR, Palmerston North.
Grasslands, DSIR, Palmerston NOrth.
Grasslands , DSIR , Palmerston North.
25
Di sc u s s i on
The y i e l d s o f t h e p o l yme r s s o l u b l e in c o l d wa t e r , h o t wa t e r ,
oxa l a t e and so d i um h y d r o x i d e ( be f o r e and a f t e r de l i gn i f i c a t i on ) a r e
shown i n Tab l e I . Th e h i ghe s t y i e l d s o f t h e wa t e r s o l ub l e
po l y s a c c h a r i d e s we re o b t a i ne d a f t e r t r e a tm e n t o f the p l a n t ma t e r i a l
wi t h amy l a s e and e x t r a c t ions i n t o hot wa t e r . The grea t e s t y i e l d s o f
hem i c e l l u l o s e s ( non-ce l l u l o s i c po l y s a cch a r i d e s s o l u b l e i n s o d i um
hydr o x i de ) f o r a l l t he f o o d s and f e e d s we r e ob t a ined be f o r e
de l i gn i f i c a t i on . Th i s i s in c o n t ra s t t o s t u d i e s o n t h e e x t rac t i on o f
hem i c e l l u l o s e f r om wo od . Gene r a l l y woo d r e qu i r e s de l i gn i f i c a t i on
be f o r e any quant i t i e s o f hemi c e l l u l o s e c a n be e x t ra c t e d ( Time l l ,
1 9 64) .
The wa t e r s o l ub l e p o l yme r s a re comp o s e d p re dominan t l y o f t h e
suga r s a r a b i n o s e , ga l a c t o se a n d uron i c a c i d . T h e amount o f p r o t e i n
pre s e n t i n the wa t e r s o l ub l e po l yme r s va r i e d f rom 3% ( bran ) t o 35%
( ryegr a s s ) a s shown in Tab l e I I . The h i gh l e ve l s of u r on i c a c i d in
the wa t e r s o l ub l e as we l l as the oxa l a t e s o l ub l e f ra c t i on s c on f i rms
that me t ho d s of a na l y s i s for the uron i c a c i d c o n t en t of f o o d s shou l d
inc l u d e the wa t e r and oxa l a t e s o l ub l e f r a c t i ons ( Ho l l oway e t a l .
1 9 83) . The me thoxy l a n d a c e t y l groups p r e s e n t a l s o s howe d
cons i de r ab l e va r i a t i on . Th i s l a rge v a r i a t i o n i n con t e n t mak e s i t
di f f i c u l t t o s ub s t an t i a t e t h e f i nd ings o f S e l ve ndran � � · ( 1 9 7 9)
that oxa l a t e s o l ub l e p o l y s a c c ha r i de s have a l owe r me thoxy l con t en t .
Pos s i b l y t h i s i s due t o the d ive r s e mon o c o t a n d d i co t y l e donous p l an t
food groups ana l y s e d . On i on h a d t h e h i ghe s t l eve l o f me t ho xy l
group s a t 2 2 . 5 pe r c e n t ; in c on t r a s t whe a t bran had t h e
l owe s t a t 0 . 4 p e r c en t . I t ha s b e e n sugge s t e d t h a t h i gh me thoxy l
2 6
pec ti n s are importan t in reduc ing serum cholesterol levels ( Mokady ,
1 97 3 ) so tha t onions could po ssibly be an interesting source o f high
me thoxyl pectins fo r die tary s tud ie s .
Th e water insoluble fibre c omponents were composed predominantly
of the pentose sugars arab inose and xyl ose . The xylose content of the
polym er s , in many cases , increased marked ly after delig n i fication,
possibly due to loss of arabinose side chain s. The pro te in c ontent o f
the wa ter insoluble polyme r s v ar ied from 0 . 5 percen t i n wh e a t b ran to
2 5 percen t in beans . The prote in i s l i kely to be the hyd roxyproline
r ich pro te in o f the cell wall s ( McNe il e t �. 1 984 ) .
Other inve stiga tors have also endeavoured to isolate and/or
quan ti ta te pl ant cell wall componen ts ( Southgate et �· 1 97 8 and James
and Theander , 1 98 1 ) . se quen tial extrac tion for die tary f ibre analysi s
has been r eported , by among o the r s , Jam e s and Theander ( 1 98 1 ) , Stevens
and se lvendran ( 1 984 A ,B) , Asp et al . ( 1 98 3 ) . Isolation o f cell wall
componen ts for c ompos i tional stud ie s inc lud e the study b y Aspinall and
Fanous ( 1 984 ) . Us ing stepwi se ex trac tion o f cell wa l l m a te r ials from
apple s using wa ter , ammoni um oxalate and di lute alka l i , three
polysaccharides we r e obtained . The y we r e found to be a highly
b ranched a-L-arab ino fur anan , a l inear 4- linked a-D-galac turonan wi th
a-L-arab inofuran an subunits and a fuc ogalactoxyloglucan . The
struc tur al features o f apple and ci trus pec tic substance s were stud ied
by De Vr ie s � �· ( 1 984 ) . Us ing alcoho l insoluble sol ids ( De Vr ies
et al . ( 1 9 8 4 ) they frac tionated the po lysaccharides by ion-exchange
chromatography and gel fi l tr a ti o n . Th e y found tha t the pe c tin
contained rhamnose , arabinose , g alacto se , g lucose and galac turonic
acid residue s , xyl ose wa s presen t only a t l ow levels . En zym ati c
d eg r adation o f the pec tin showed tha t n eutral sugar side chains wer e
present in ' hairy ' reg ions i . e . blocks o f neutral sugar side chains
( De Vr ies � al . 1984) . '!he cell wall ma te r ial from parenchym a tous
tis sue of the pod s of Fr ench and Runner beans were ex tr ac ted from we t
b all-mi lled ti ssue s wi th 1% aqueous d im e thyl sulphoxide ( O ' Ne i l l and
Se lvend ran , 1980) . '!he r esul ts of tha t stud y are similar to o ther
sequential methods o f extrac tion. However the more m i ld treatm en t
used by O ' Ne i l l and Se lvend ran ( 1980) resul ted i n predominan tly
g al ac turonic ac id frac tions being ob ta ined . Presumably the less
2 7
soluble hemicellulose s we r e no t able to be extensively ex trac ted using
dime thyl sulphoxide .
The hemicellulose s present in both d ic o t and monoco t c e l l wal l s
a r e c omposed of xyl og lucans and xylans ( �Ne i l � al . 1984) . In
d icots x ylog luc ans pr edominate , whereas xyl ans consti tute the maj or
hemicellulose o f monocots ( McNe il et �· 1984) . But both types o f
polysaccharides hav e been shown to be prese n t in monocots and d icots .
Xylog luc ans being found in many d i ffe rent d ic ots includ ing mung and
r unner beans , pota to and in the cell wal l s o f the monocots o f r ic e ,
o ats , barley and bamboo ( �Ne il et al . 1984) . The struc ture o f c el l
wal l xyl og lucan has been r eviewed extensi vel y ( McNe il � al . 1979) .
The basic str uc ture of thi s cell wall po lyme r consi sts o f a backb one
of B-4-linked-D- g lucosyl residue s wi th D-xyl osyl side chains a- l inked
to 0-6 of some of the g lucos yl residue s , o- ac e tyl g roups and
g alac tosyl-containing side c hains also have b een shown to occur ( McNe i l
� al . 1979) . The basic str uc ture o f the xylans has been reviewed by
Wi lkie ( 1 97 9 ) . Al l known x yl ans c o ns i s t o f a backbone o f 8 - 4 - l inked
x y l o s yl residue s wi th various s ide c ha in s a ttached through 0 - 2 or 0 - 3
o f the xyl os yl residue s ( Wi lkie , 1 9 7 9 ) . Bo th xylog luc an s and xylans
28
probably hav e a struc tur al role in plant c ell wa l l s , s ome o r a l l be ing
h yd rog en bonded to cellulose , as evidenced by the r equi r em e n t fo r
s trong alka l i for the ir extr ac tion ( Mc Ne i l e t �. 1 984 ) .
The integ r a ti o n of the accumul a ted knowl edg e o f the c hem i c al
s t r uc tur e o f c el l wal l s has b een r ev iewed by inc lud ing MC Ne i l e t �·
( 1 9 7 9 ) , AS pi n a l l ( 1 98 0 ) , Knee and Ba r tl e y ( 1 98 1 ) and MC Ne il e t a l .
( 1 98 4 ) . The c ompl exi ty a nd d iversi ty o f pl an t cell wa l l componen ts
has r e s ul ted i n d i f ficulty i n postul a ti ng compr ehen sive pl an t c e l l
wal l models . Wi th the d evelopment o f new analytical tool s such a s NMR
a nd HPLCjma s s spec trom e try the po s s ib i l i ty of f ur ther d evelopm ents i n
thi s field , par tic ul arl y the study o f whol e c e l l wa l l s , wou l d appear
to be advan tag eous . Th i s i s d i scus s ed f ur ther in Append ix D .
The high g lucose conte n t i n the wa ter , oxa l a te and sod ium
h yd roxide ex tr ac ts from the swe e t po ta to , kum e r a ( Ipomoea b e ta ta s ) may
b e due to amyl ase r esi s ta n t s tarc h . Eng lys t e t a l . ( 1 98 3 ) r epo r ted
tha t the conte n t of amyl a s e r esi sta n t s tarch wa s between 0 and 3 % in a
wid e var ie ty o f foods . However thi s amyl ase r e si stan t s tarch ha s been
propo sed to be a c omponent o f d i e ta r y f i b re ( Eng lys t , 1 98 3 ) . A
f ur ther frac tio na tion o f the hem i c e l lul ose from wheat b ran wa s
und e r taken . Th e r esul ts o f thi s s tudy a r e d i scussed in the g ener a l
d i scussion and r e sul ts shown in append ix A and B .
The extr ac tion and c ompo si tion o f the f i b r e c ompo nen ts from
f rui ts , grasse s and vege tables in thi s study i nd icate tha t they a r e
compo sed of a high propo r ti o n o f wa ter soluble polym e r s c ompo sed
p r ed om inantly of a r abinose , g a l actose and uronic ac id s , wher eas the
f ib r e c ompo s i ti o n of whe a t b ran is mainly wa ter inso l uble polym e r s o f
a r ab inose and xyl ose . The se r e sul ts a r e s im i l ar to tha t found by
The ander and AmQn (1 979), howeve r the f r ac tions i n ���� s tud y we r e
g en e r a l l y higher in g lucose and lowe r in uronic ac id . Thi s i s
pre sumably due to d i s simi l a r s ta r ti ng m a te r ia l s and the d i f ferent
e x tr ac tion pr ocedur e . However the r es ul ts o f b o th the se stud i e s show
the r e i s c onsider able d i f fe r enc e in the solub i l i ty and chem ical
compo s i tion o f the fibre c omponen ts . Th i s would s uppo r t the
h ypo the s i s tha t the greate r wa te r a f f i n i ty o f the fibre c omponen ts o f
frui ts and veg e tables m a y be a s soc ia ted wi th g e l form a ti o n wi thi n the
small bowe l thereby a f fec ting the absorptio n o f o ther nutr ien ts such
as g luc ose ( Jenkins et a l . 1976). Th i s would accoun t for the sl owed
g lucose absorption hence r ed uc ed in sul in r equi r emen ts i n d iabe ti c
pa ti en ts o n high frui t and v eg e tab l e d ie ts ( Do ug las s , 1975). The
f i b re of whe a t b r an be ing less wa ter sol ub l e appears to b e also l e s s
e a s i ly d eg r aded in the upper i n te stinal trac t ( Hollowa y e t �· 1978).
Whe a t b r an fibre may exer t i ts m aj or in fluence in the colonic
env i ro nm en t r a the r than tha t of the sma l l bowe l .
Th e r es ul ts o f thi s stud y a nd thos e o f Theander and Aman (1979)
and Se lvend r an � al . (1979) s ugge s t tha t o ne type o f e x tr ac ti o n
scheme should no t be appl ied to a l l type s o f pla n t food s . Th e
v ar iabi l i ty i n c e l l type s a n d chem i c al compo si tion, par ti cul a r l y the
high s ta rch conta ining food s , m ake s qua n ti ta tion of d ie ta r y f i b re
d i fficul t bec a use each s te p r em oves an inde finable amoun t o f
29
polysaccha r i d e ( Se lvend r an e t a l . 1 97 9 ) . Th e r e is c onsiderable
3 0
a ppeal in us i ng o ne extr ac tion scheme fo r the quanti ta tive analys i s o f
f ibre in food s t o ob ta in suppo rt fo r the epidem iological s tud ie s , s uch
a s applying the Southg a te ( 1 96 9 ) procedur e o f seque n ti a l extrac tio n or
the removal of nonfibre c omponents by d e terg ents as i n the van So e s t
m e thod ( Van So e s t and Wi n e , 1 97 6 ) . However the se proc ed ur e s c an o n l y
g ive a general measure o f the f i b r e c onten t o f foods . Po s si b l y wha t
i s r equi r ed i s a more d e ta i l ed f r ac ti o n a ti o n o f d i f fe r en t pl an t foods
to i solate and d e termine componen ts o f fibre tha t a r e ' ac tive' i n
i n fl uenc ing d i se ase pr even tion and d o no t adversely a f fec t o the r food
c omponen ts .
The next s e c tion , Par t I I , wa s unde r taken to inve stig a te the
influence o f the d el ig ni f i c a tion pro c ed u r e on the vi scos i ty o f
h emic ellul ose s e x tr ac ted from o n e o f the foods used i n thi s s tud y .
31
PART I I
S t udy o f the Vi scosi ty o f Whe at Br an Hemi c ellulose s
In traduc tion
Me thod s o f extr ac ti o n o f polymer ic consti tuents o f pl an t c e l l
wal l s have been subj ec ted to ex ten sive inve stig ations , s e e f o r exampl e
the wo rk of Wh,stler �i1965 ; Whi stler and Ri chard s , 1970 ; Ri ng and
Se l vend r an , 1980 ) and the r eview by As pinal l ( 1 982) . On e o f the
pr incipal d i f ficul tie s tha t sti l l remains i s the s e l ec tive
s o l ub i l i sa tion of the hem i cellulose s . Th e i r i so l a ti o n from woody
t i s sue s i s usually pr ec ed ed by d e l ignific a tion to ob ta i n an
a pprec iab l e yield ( Wh i stler and Be Mi l ler , 1963). HOwever , even in
woody ti s s ue there is the possib i l i ty tha t d el ig n i fi c a tion may c ause
d epolymer i sa tion o f the po ly s accha r ide s . ( G l aud em an s and Timel l ,
1957 ) . Th i s procedur e , however , i s s ti l l wid e l y used in the i so l a ti o n
o f hemi c e l lul ose s from feed s ( As pinall and Mc: Gr a th , 1966) and food s
( Br il lonet e t al . 1982). As shown in Pa r t I o f thi s the s i s
h em icelluloses from foods a nd feed s c an b e ob ta i ned , using alka l i , i n
adequa te yi e ld s , wi thout pr ior d e l ig n i fi c a tion .
Alka l i , whi l e be ing the m os t c ommonly used reag e n t for obta i n ing
hem i c el lul o se s , does have d i sadvan tag e s . Th e y have been r eviewed by
Whi s tler and BeMi l ler (195 8) and As pinall (1959). As pi n al l (1959)
c onsider ed tha t ther e a r e four type s of r eac tio n , r esul ting from the
use of a l ka l i , which may c ause m od i fic a tion o f the hem i c e l lul ose s a s
they occur i n the pl an t ; (1 ) d e- e ste r i fi c a ti o n o f pa r ti a l l y acyl a ted
polysaccha r id e s , ( 2 ) c hem i c al d eg rad a tion i n i ti a ted a t r educ ing
g roups , ( 3 ) a lkaline hyd roly s i s of g lyc o s i d ic l inkag e s , and ( 4 ) the
b re aki ng of c hemical bond s b e tween hem i c e l lul ose s and other c e l l wal l
c omponen ts .
The influence the se r eactions have in d eg rading hemicellulose
d ur ing ex trac tio n was s tud ied by As pi n a l l e t al . ( 1 96 1 ) . The
d eg radation o f xylans by d ilute alkal i at r oom temper a tur e wa s
f o l l owed by the anal ys i s o f the l ow molec u l ar we ight acids fo rmed and
the d e termi nati on of chang e s i n xyl an molec u l ar we ight s . The
c onc lusion from thi s stud y was tha t under the cond i tions norm al l y
3 2
employed for the i r extrac tion from pl an t ti s s ue s , alkal ine d eg r ad a tion
of x yl ans i s s l ow and proceeds only from the r educ ing end- g roups .
Prolonged alka l i e x trac ti o n ( 2 5 d a ys ) led to a 2 0 per c en t d im i nution
o f the molecular we ight o f the xylan . Th e y a l so repo r ted tha t
a lkaline d eg r ad a tion c an be m i n i m i sed by r educ ing l a te n t a ld ehyd ic
end- groups wi th po ta s s i um borohyd r id e .
However , po s sibly one o f the g reat d i sadvan tages i n alka l in e
extrac tion o f hem i c e l l ul ose s , i s tha t the n a tur ally occurr ing e ster
g roups a r e saponi fied ( Whi s tler , 1 960 ) . Other solven ts for e x tr ac ti o n
have a l so been used . Dim e thyl s ul foxid e ( DMSO) h a s been used to
e x tr ac t g luc uro noxylan wi th a 1 7 per c en t a c e tyl conte n t ( Bouveng
e t a l . 1 95 9 ) . DMSO ha s a l s o been used in s tud i e s on the d i str ibution
o f the o- ace tyl g roups in pine g lucomannan ( Ke r ne e t �. 1 97 5 ) .
However , DMSO g enerally enab l e s c e l l wa l l po l ysaccharides to b e
extr ac ted only i n a l ow y i e ld ( Tim e l l , 1 96 0 ) , po s sibly bec ause the
l ig n in- hemicel lul ose l inkag e s a r e s tabl e i n DMSO ( Mo r r i so n , 1 9 7 3 ) .
The d i f ferences i n c ompo s i tio n , yie ld s and phys i cal prope r ti e s o f
hemicel luloses extr a c ted from foods , by a lka l i a nd DMSO d o no t appear
to have b een d e term i n ed .
A new solven t N-me thylmorphol i ne-N-ox id e ( NMMNO ) wa s r epo r ted by
Jo seleau e t al . 1 98 1 , as a ' r emarkable solven t' for c er tain
polysacchar id es ( As pi n al l , 1 98 2 ) . Th i s solven t has been r epo r ted to
have b e en used to d i s solve c e l lul ose nond eg r ad a tively ( Chan zy e t a l .
1 97 9 ) and whole c e l l wa l l s ( Jo se l eau , e t a l . 1 98 1 ) . Its use a s a
s olven t for hemic ellulose s h a s not been r e po r ted in the sc ien ti fic
l i te r a tur e .
3 3
Th e i solation o f hem i c e l lu l ose s , obta ined i n the l east d eg raded
f o rm , wo uld enable appropr i a te samples fo r f ur ther stud ie s o f the
prope r ti e s of d i e ta r y f ibre to b e underta ke n .
Po s sible degrad a ti o n o f whe a t bran hem ic e l l ulose s i solated before
a nd a fter delignific a ti o n and a f ter solub i l i sa ti on in NMMNO . Ca n b e
d e termin ed b y measur ing the limi ting v i scosi ty n umber . Th e l im i ting
v i scos i ty num ber(�)( in tr in s i c v i scosi ty) ha s b een shown to be r el a ted to
the molec ular we ight ( m ) by the g ener al equa tio n
where K and a are empir i c al constants for a g iv e n po l ymer- solvent
s ys tem ( Gr eenwood , 1 96 4 ) .
Ma te r i a l s and Me thod s
Syn the si s o f N-Me thyl-Mo rphol ine-N-Ox ide
The so lve n t NMMNO wa s no t ava ilable in New ze al and at the
c ommenc ement o f thi s stud y , i t wa s the r e fo r e s yn thesi sed using the
proc edur e d e s c r ibed in the pa te n t appl i c a tion ( Johnso n , 1 96 9 ) .
N-me thyl-morpho l ine wa s ox ided us i ng hyd rogen pe roxid e , excess
peroxide be ing r em oved by the use o f cata l a se . The
N-m e thyl- morphol ine-N-ox ide wa s then ob ta i n ed from a benzene- wa ter
a zeotrope by pr ec i pi ta tion i n ace tone , d ried in a d e s s i c a to r , and the
m e l ting po i n t e s tabl i shed to be b e tween 7 4 and 7 6 ° C . Th e s yn the s i s
sequence i s shown i n fig ur e 4 .
Fig ur e 4 : Se que nce of s yn the s i s o f N-m e thyl-morph o l ine- n- ox id e .
John son D . L . 1 96 9 , Br i ti sh Pa te n t No 1 , 1 44 , 04 8 .
N-Me thy!-Mo r pho l ine
H102 2 hr s , 6 7 - 7 2 ° C
Cool overnight
Callase
Be nzene
1 Ac e tone
1
Spo t te s t to confirm
Be n zene - wa ter
aze o trope r emoved
Fi l ter
Dr y
M . P . 7 4 - 7 6
N-Methyl-morphol ine-N-ox id e
3 4
Pr epa r a tion o f solution o f hemicel lu l ose in N-me thyl-morphol in e-N-ox ide
The s ui tab i l i ty of NMMNO as a nond eg r adative solvent for
hemicel lulose s wa s inve stig a ted by add ing NMMNO ( 1 2 . 5 g) to
nond e l i g nified hemicellulose ( 0 . 5 g ) . As a control , a similar sampl e
o f hem i c e l lulose wa s added to wa ter ( 1 2 . 5 g ) . Bo th sampl e s we re then
he a ted ( 1 20° C ) under nitrog en fo r 3 0 minute s . Dime thyl s ul phox id e
( 5 0 m l ) was then added to each s ampl e . Af te r c ool ing to room
temper atur e both solutions we r e d ial ysed sepa r a te l y against wa ter fo r
4 8 hou r s . The nondialysable c o n tents we r e then freeze- d r ied . The
d ia l y s i s tubing rema ined in ta c t throug hout the d ia l ys i s proc ed ur e ,
thus i nd icating tha t the tub ing wa s no t d eg rad ed by the so lve n t s .
Me a suremen t o f v i scosi ty o f hemic e l lul ose soluti o ns
Samples o f wheat b ran hem i c e l lulose used for v i scosi ty
d e termi n a tions were extr ac ted i n alka l i b e fore and a f ter
d e l ig ni fi ca tion, and after solub i l i sa tion in NMMNO .
3 5
The v i scosi ty measur ements we r e mad e a t 2 5 ° ± 0 . 0 5 ° C us i ng
Cannon- Fe nske s i ze 5 0 vi sc om e te r s in a Tams on TCV 40 wa ter b a th .
So lutions o f the hemicellul ose s ( 2 50 mg i n M NaOH, 2 5 m l ) we r e sti r r ed
under n i trogen for o ne hour , f i l tered throug h number two s i n te r ed
g la s s f i l ter s . The sma l l amoun ts of und i s solved m a ter ial obtained on
f i l tr a ti o n were wa shed twic e wi th e thanol and d r ied a t 1 0 5 ° C . The se
we igh t s we r e sub tr ac ted from the star ting we ights i n order tha t the
final conc en trati ons c ould be c a l cul a ted . The fi l te r ed solutions wer e
then d i luted in f i l tered M NaOH t o make a s e r ie s o f solutions o f 5 . 0 ,
3 . 0 , 1 . 2 5 and 0 . 62 5 mg/m l .
Th e i nd ividua l solutions ( 5 . 0 ml) wer e pl aced into five
v i scome te r s tha t had previous l y b een c leaned and cal ibra ted acc ord ing
to the procedures described by Kr agh ( 1 96 1 ) . Th e l im i ting vi scosi ty
number s we r e d e termined by m e a s ur ing the flow time s o f a l l the
s o l utions to wi thin 0 . 1 o f a sec ond . The v i scosi ty r a ti o for e ach
solution ( V/Vo ) was calcul a ted by d iv id ing the flow time of the
hem ic ellul ose solutions by tha t for the pur e solven t ( M NaOH ) . The
r e sults wer e plo tted a s :
V/Vo -
c
ag ainst c onc en tr a tion ( g/ml) and ex tr apo l a ted to zero
c onc en tr a ti o n .
Ef fec t o f exposur e to a l ka l i o n hemicellulose v i sc osi ty
3 6
The in fluence o f time o f expo sure in alka l i on the v i scos i ty o f
hem i c e l lul ose wa s determined . Hem i c e l l ul ose wa s extr ac ted from whe a t
b ran e x oxa l a te as desc r ib ed in Par t I . Th e fi l te r ed solution o f
h em ic e l lulose wa s introduc ed to the vi scom e te r 2 0 minute s a fter the
add i tion o f a lk a l i to the b ran . The r el a tive v i scos i ty ( V /Vo) wa s
d e te rmined un ti l 1 65 minute s had el apsed .
Re s ul ts
The v i scosi ty o f the whe a t b ran hem icelluloses e x tr ac ted before
a nd a fte r d e l ig ni fic a tion are shown in figure 5 .
The vi sco s i ty of b ran hem i c e l lul ose a fter solub i l i sa tion and
r ecovery f rom NMMNO and the c o n trol sample i s shown in figur e 6 .
Th e l im i ting v i scosi ty number s o f the hem i c e l l u l ose s e x trac ted
b efo r e and a fter delig n i f i c a tion and a fter recovery f rom NMMNO a r e
s hown in Ta b l e IV.
The r el a tive vi sc os i ty o f a lka l i nondeligni fied hem i c e l lulose in
a lka l i wi th time is shown in Ta b l e v.
400
300
200
100
0 __________ ._ ________ ._ ________ �--------�--------_. 0 1 2 3 4
C o n c e n tr a t i o n i n g / m l x 1 0-3
F igure 5 :The V iscos ity o f Wheat Bran Hem i ce l lu lose Extracted
i n A l k a l i Be fore (e) and a f ter De l i gn i f ication (*) .
5
3 7
400
100
1 2 3 4
C o n c e n tr a t i o n i n g / m l x 1 0-3
F i gure 6: The Viscos ity of Wheat Bran Hem i ce l lu lose A fter So lub i l isat i on and Recovery f rom NMMNO (� and Contro l Samp le (*)
5
Table IV
Limi ting v i s c o s i ty number of hemi c e l luloses befo r e and a fter
d elig n i fi c a tion and a fter recover y f rom solub i l i s a ti on in
N-me thyl-morphol ine-N-oxide ( NMMNO )
Tr e a tment o f Hem i c e l lulose
Alka l i extr ac ti o n
Al ka l i extrac tio n then d e l ig n i fied
P r i o r del ign i f i c a ti o n , alka l i extr ac ti o n
Alka l i extrac tio n , wa ter c on trol
Alka l i ex trac ti o n , NMMNO s olution
Vi sc osi ty Number m l/ g
2 2 0 . 6
6 8 . 3
7 4 . 2
8 1 • 9
6 . 3
3 9
Table V
Re l ati ve vi scosi ty o f nondeligni fied hemicellulose in
a lk a l i a s a func tion o f time
Re l a ti ve vi sc osi ty Time ( m inute s )
1 . 8 2 4 2 5
1 . 8 2 3 3 5
1 • 8 2 1 4 5
1 • 81 8 5 5
1 . 8 1 7 85
1 . 8 1 5 95
1 . 8 1 5 1 2 0
1 . 8 1 4 1 65
4 0
4 1
Di scus s i o n
The c hem i c al compo s i tion o f the pol ysaccha r id e s i solated from
wheat bran us i ng alka l i , before and a f ter d e l ig n i fi c ation, s howed only
sma ll d i f ferences i n c ompo si tion a s seen in Par t I, Table I I . The pr e
and po st-d e lig nific a tion hemic e l lu l o s e s we re c omposed predom i n an tl y o f
xyl ose 6 0 . 1 a nd 6 1 . 8% , and arab inose 3 1 . 9 and 2 9 . 4% r e spec tive l y .
Table I i n Par t I shows that the g re a te s t yi e ld o f 7 . 9% wa s ob ta in ed
i n the e x t r ac tion pr ior to d e l ig n i f i c ation and that i n the s ub sequent
e x trac tio n the yie ld was 3 . 8% . De s pi te the s imi lar i ty o f c ompos i ti on
the arab inoxyl ans d i f fe r ed considerably i n the ir vi scosi ty , a s seen in
f igure 5 . Th e vi scosi ty n umber s o f the ar ab inoxylans e x tr ac ted pr ior
to and a f ter d e l igni fication were 2 2 0 . 6 and 7 4 . 2 ml/ g respec tively a s
shown i n Tab l e I V . As the two po l ym e r s a r e o f s imi l ar c ompo s i tion , i t
i s po s s i b l e that the a r abinoxyl an ex tr ac ted a f te r chl or i te
d e l ig n i f i c a ti on may have been deg r aded as indi c a ted by i ts l owe r
vi scosi ty . Th e d e l ig ni ficati o n treatmen t o f the hem i c el lulose ,
e x tr ac ted from whea t b ran pr ior to d e lignific ation, showed a simi l ar
d ec rease i n v i scosi ty.
The c e l l wal l s of wheat b ran c on ta i n two main type s o f
polysaccharides , arab inoxyl ans and c e llulosic polym e r s and the r e i s
evidence tha t pheno l ic e s ter l i nkag e s play a role in hold i ng them
toge ther ( Ri ng and Se lvend ran , 1 980 ) . The lignin- hem i c ellulose e s ter
l inkage ha s b een shown to be alka l i l ab i l e ( Wi lkie , 1 9 7 9 ) . Th e r e fo re
the alka l i tr eatmen t pr ior to chlor i te d e l i g n i fi cation wo uld be
expec ted to have cleaved the acce s s i b l e alka l i sensi tive l inkage s
b e tween the l ignin and ar ab inoxyl an . Ex tr ac tion of more hemi c el lu l ose
4 2
requ i r es fur ther d e l ig ni fi c a tio n . Th e me thod gener ally used for
d el ig nific ation ( Wi lki e , 1 97 9 ) i s the aqueous ace ti c ac id and sod i um
chl o r i te procedur e ( Wh i stler and Be Mi l ler , 1 96 3 ) . The r equi r emen t fo r
f ur ther deligni f i c a ti o n c ould ind i c a te tha t the accessibi li ty o f the
l ig n i n-hemicellu lose l i nkag e to the r e ag e n ts i s prevented by the c lo s e
packing of the po l yme r s , a n d tha t par tial deg r adation of the l i g n i n
and/or the hemic e l lul ose i s r equi r ed to open u p acce s s to the
solub i l i sing reagen t . A m ore l i ke ly po s si b i l i ty i s tha t l inka g e s
b e twe e n l ignin and hem i c e l lulose c onta i n b o nd s o ther than alk a l i
sensi tive ester l i nkag e s ( Sm i th and Har tl e y , 1 98 3 ) . Such a s
i sod i tyrosine , whi c h may fo rm a n inter-po lypeptide c ro s s- l ink ,
pos s i b ly being responsible fo r pl an t c e l l wa l l g lyc opro te in
i n so l ub i l i ty ( Fr y , 1 98 2A ) . Phenolic compo nen ts of s uch as fer ul i c and
P-coum a r ic ac id appe ar to be a s soc iated wi th water insolub l e polym e r s
( Fr y , 1 98 2 B ) . Th e mechani sm i s probably b y f o rm i ng cro s s- l inks t o the
c el l wa l l prote in ( Fr y , 1 98 2A ) . Cros s- l inki ng of c e l lul ose and o ther
c el l wa l l polysacchar ides by 5 -ke to-D-mannuron ic ac id in Sphagnum Mo s s
h a s b een r epo r ted ( Pa i n te r , 1 98 3 ) a s have f e r ul oyla ted pe c ti n s i n
primary cell wa l l s ( Fr y , 1 98 3 ) . It i s po s s ib l e tha t the influence o f
the var ious means o f c ro s s- l inking reduc e the a lka l i solub i l i ty o f the
c e l l wal l polysaccha r id e s .
The par ti a l d eg rad ati o n o f hem i c e l l ul os e , as ind icated by the
r educ ed vi scosi ty a f te r d e l igni ficati o n , s ug g e sts tha t an appropr i a te
s tar ting ma te r i a l fo r the i solation o f hem i c e l lulose s from foods i s
not the c hlor i te d e l ig ni f i ed hol ocel lul ose . As hem icel lul ose c an b e
e x tr a c ted wi thout pr ior d e l ig n i f ic a tion and i n a reaso nably high yi e ld
4 3
from whe a t b ran , wh ich has one o f the hig he s t l ignin contents o f human
foods ( Ho l l oway � a l . 1 97 7 ) d e l ign i f i c a tion appear s to be an
unneces sary a s we l l as und e s i r ab l e step i n preparation o f
polysacchar ides r epresenta tive o f those pr esent in the parent
mate r ia l . If the c h l o r i te d e l ig n i f i c a tion s te p i s used pr ior to
extr ac tio n o f the a r abinoxyl an , appropr ia te ma terial fo r
inve stig ati ons o f molecular prope r ti e s may n o t be obta ined .
The po s sible d e g r adation o f a r abinoxyl an s ex trac ted usi ng alka l i
( As pi n a l l e t al . 1 96 1 ) has n o t been overc ane . Other solven ts such a s
d im e thyl sulphox ide have been used , however o n l y low yield s o f
hem i c e l lu lose have b een obta i n ed ( Goring and Time l l , 1 96 0 , Par t I I I o f
thi s the s i s ) • The r e po r ted u s e o f the n e w s o lvent NMMNO ( Jo se l eau
e t a l . 1 98 1 ) in whi c h c el lul o s e i s solub l e and s tab l e at e l eva ted
tempera ture s appear ed to o f fer a new me thod o f nond eg rad a tive
solub i l i sa tion of h em i c ellul o se s . However as seen i n Table IV the
l im i ting v i scosi ty n um ber of hemice llul o se tha t had been so lub i l i sed
i n NMMNO wa s only 6 . 3 m l/g c ompar ed wi th the c ontrol sample o f
8 1 . 9 mlj g . Th e l owe r l i m i ti ng vi scosi ty number o f the c ontrol s ampl e
c ompared to the hem i c e l lulose tha t had not been hea ted ( f igure 5 )
s uggests tha t some d eg radati o n had occur r ed1 po s sibly due to the
e levated temperatur e ( 1 2 0 ° C f o r 3 0 minute s ) , tha t the proc edure
r ecommended by Jose leau et a l . ( 1 98 1 ) requ i r ed . Howe ver the
d eg r ad a ti o n wa s no t a s e x tensive a s fo r tha t observed for the sampl e
tha t had been d i s solved i n NMMNO . The appl ic a tion of NMMNO a s a
nond eg rad a tive solvent for whe a t b ran ar ab i nox y l an i s no t suppor ted by
this s tud y . The alka l i extr ac tion procedure used to ex tr ac t
nond e l igni f i ed wheat b ran wa s found to r e s ul t i n minimal dec rease i n
r el a tive v i scos i ty as s e e n in Tab l e V, thus s uppo rting the c onclusion
r eached by As pinall e t � · ( 1 96 1 ) tha t under the c ond i tions norma l l y
e mpl oyed fo r the e x tr ac ti on o f xylans from pl an t ti s s ue s , a l ka l ine
d eg r adation is s l ow .
4 4
I t wa s c onc luded from the r es ul ts o f the studie s describ ed in thi s
par t o f the the si s , tha t the least d eg rad ed alka l i soluble
h em icellulose i solated from bran , is tha t ob ta ined pr ior to
d el ig n i fic a ti o n , and tha t the use o f the new solven t
N-methyl-morpho l ine-N-oxide r e s ul ts in ex tensive hemi cellulose
d eg r adation . Ther efo r e the hem i c e l lulose s extracted befo re
d el ignific a ti o n , ob ta i n ed in Par t I , wer e used as samples o f
wa te r insol ub l e f i b r e s f o r the s tud i e s o n me ta l bind ing desc r ib ed i n
Pa r t I V and Par t V o f thi s the si s .
PART I l l
Mo rpholog ic al Chang e s observed i n Whe a t Br an a fter Sequen tial
EX trac tion wi th Ethanol , Wa ter , OX a l a te and Alkali o r
Dime thyl s ul phox id e .
In troduc ti on
45
Stud ie s us i ng who l e foods hav e b een under taken in attempts to
r elate advan tageous o r d e tr ime n ta l prope r ti e s to the i r fibre
c omponents ( Ke l s a y , 1 97 8 ) . This approac h , however , is l im i ted by the
c omplex i ty o f the c omponents and by the pl an t s truc tur e s . Some o f the
c omponents , whi c h may normal ly be d ig e s ted , c ould mask the proper ti e s
o f the fibres i n in v i tro s tud ie s .
There have b een two approache s to prepare m a ter i a l tha t wo uld
enable sc i e n ti f i c s tud ie s to be under taken to substan ti a te or r e f ute
the fibre h ypo the si s . The fir s t a ppr o ac h , a s used in Pa r t I , i s to
i solate po l ym e r i c c ompo nents fr om the c e l l wal l s of food s and to
examine the i r indiv idual prope r ti e s . The o ther approac h i s to prepa r e
plan t m a te r i a l wi th i n ta c t morpho log ical featur e s , s uch a s whol e c e l l
wal l s , and t o investi g a te the ir i n v i tro and i n vivo prope r ti e s and to
c ompare wi th whole food s and i so l a ted cell wal l componen t s .
A me thod o f prepar i ng whol e c e l l wa l l s from whea t endosperm us i ng
a sequence o f g r ind ing , amyl ase and e thanol extrac tion wa s d e sc rib ed
by Mares and Stone ( 1 9 7 3 ) . The i sola tion and ul tras truc ture o f
a le urone c e l l wa l l s h a s a l so been d e sc r ibed b y Bacic and Stone ( 1 98 1 A ) .
Th e ma te r i a l i so l a ted in the se s tud i e s we r e , however , no t used to
4 6
inv e s ti g a te the prope r ti e s o f fib r e . The isolation o f c e l l wa l l
m a te r i a l from g round cabbage by s e quen ti a l e x tr ac tion wi th e thano l ,
trea tmen t wi th pro nase , fol l owed by ex tr ac tion wi th phenol ic- ac e tic
ac id- wa te r and DMSO wa s d e sc r ibed by Stevens and Se lvend r an ( 1 98 0 ) .
The s ame me thod wa s also used to prepare c el l wa ll ma te r ia l from whea t
b ran ( Ri ng and Se lvend ran , 1 98 0 ) . The c e l l wa ll ma te r i a l produc ed
from the se s tud ie s we re us ed for c l in ic a l tr ia l s ( Ste phen and
Cumm i ng s , 1 980 ) .
The impo rtance o f wheat b ran b ecause o f i ts maj or contr ibution to
the d ie tar y fibre intake o f most We ste r n n a ti ons ( Tr owe l l , 1 97 3 )
s ug g e s t s tha t stud i e s us ing thi s m a te r i a l may be the most appropr ia te
for i nve s tiga ting the fibre h ypo the s i s .
The c ompo s i tion o f whe a t b ran h a s been revi ewed b y Sa under s ( 1 97 8 )
and i ts anatomy by ES au ( 1 96 5 ) . Th e main m orphological f e a tur e s o f
whea t b ran ar e shown in Figure 7 . The fib rous proper ti e s o f pl an t
mate r i a l have been r eviewed by Se tte r f i e ld and Ba yl e y ( 1 96 1 ) . However
a c le a r d i s tinc tion be twe en b o ta n i c al f i b re and die tary f i b re ha s , as
yet, no t b een e s tabl i shed . Stud i e s o f c e l l wal l s , the i r struc ture and
chang e s tha t occur when extrac ted , ma y a s si st i n thi s d i stinc ti on
b eing mad e .
Th e stud ie s d e sc ribed i n Pa r t I I I we r e unde r taken to ob serve :
( 1 ) The m orphol ogical struc tur e o f c ommerc ially g round wh e a t b r an .
( 2 ) To ob serve chang e s i n morpho l og ical s t r uc ture afte r wh e a t b ran ha s
b een s ubj e c ted to e thanol , oxal a te and alka l i extrac ti ons .
( 3 ) To o b serve the influenc e o n whe a t b r an , o f DMSO when s ub s ti tuted
4 7
for alka l i extr ac tion .
( 4 ) To d e te rmine the chem i cal compo s i tio n o f a lka l i and DMSO ex trac ted
pol ys accha r id e s .
1 mm
A
s c u t e l l um
Fi gure 7 :
48
s e e d coa t
B
n u c e l l a r e p i d e rm i s
p e r i ca rp
ce l l
J
Caryops i s ( A ) of wheat ( Tr i t i c um ae stivum ) and parts of
its peri carp in longitu dinal s ection ( B ) from Esau ,
196 5 .
Ma te r ia l s and Me thod s
Sampl es 1 o f wheat b ran b e fore and a f te r extrac ti on in e thano l
( r e f l ux ) ho t wa ter , amyl ase , ammonium oxal a te and a lkal i , before and
a fte r chlor i te d e l ig n i fic ati o n , a s i nd i c a ted in figure 2, wer e d r i ed
i n a dessicato r over pho s phorus pentoxide to constant we ig h t .
49
Dime thyl sulpho x id e ex tr ac tion procedur e : To bran ex-ox a l a te 2
i n so lubl e m a te r i a l ( 1 3 8 g ) wa s added DMSO ( 4 0 0 m l ) . The s us pe n s i o n
wa s m i x ed for f i v e hour s on a mechanical shake r , allowed to s tand fo r
1 7 hours a t room temper a tur e , d ecan ted and f i l tered through a n umber
two s i n tered g la s s f i l ter . Th e insolub l e ma terial wa s washed thr ee
tim e s wi th DMSO ( 1 00 m l ) . The so lutions wer e pooled , then added to
four volumes o f 9 5 % e thanol . The r es ul tant prec ipi ta te wa s c o l l e c ted ,
d ialysed for 1 2 hours aga in s t running wa te r , then fr eeze- d r ied . Th e
DMSO i nsolub l e ma te r i a l wa s then delig n i fied usi ng chloram ine-T ( 8 g )
and g lac ial ac e ti c ac id ( 5 m l ) added ( Ga i ll ard , 1 9 5 8 ) . Af ter m i x ing
for two hour s i t wa s d ec an ted , and the d e l i g n i fi c ation procedur e
r epe a ted on the i n so lubl e m a ter ial . The decanted l i quo r wa s
d i scarded . DMSO ( 5 00 m l ) wa s added to the i n so lub l e ma te r i a l , the
suspension wa s m ixed for five hour s on a mechanical shake r , then
a l l owed to s tand for 4 8 hour s . The supe r na ta n t wa s sepa r a ted and
added to four volum e s of 9 5 % e thanol . The r esul tan t pr ec ipi ta te wa s
d ia l ysed for 1 2 hour s again s t water and f r e e ze-dr ied and we ig hed . Th e
DMSO extrac tion procedur e i s shown i n figur e 8 .
* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
1 A l l expe r imen ts we r e carr ied out i n dupl i c a te
2 In solubl e r es id ue a f ter extrac tion wi th ammonium oxalate
Fig ur e 8 : DMSO c e l l wa l l prepa r a tion procedure
l"d
Co ld wa ter fx traction
HO t wa tel amyla se
Ammonium! oxalate
DMSO ( 2 2 hr s , room temp )
soluble
5 0
c e l l wa l l s ( 1 ) DMSO solub l e a r ab inoxylan ( 1 )
m i ld d e lign i fi c a tion u s ing chloram ine-T
( i n so l ub l e )
f o l l owed by
DMSO e x tr ac ti o n
Ce l lwa l l s ( 2 )
So l ub le
( 5 3
arab inoxyl an ( 2 )
The chemi c al compo s i tion o f the extrac t wa s d e te rm ined by GLC a nd
the c olorim e tr ic procedur es a s pr evious ly used in Par t I o f thi s
the si s .
Fe rul ic ac id d e te rm i n a tion
The ferul i c ac id conte n t o f the extrac ted polyme r s wa s d e termined
by a mod i fic a ti o n o f the proc edur e o f Ha r tl e y ( 1 97 1 ) . Sampl e s ( 0 . 2 g )
were oxid i sed wi th n i trobenzene ( 1 . 0 m l ) and 2 m sod ium hyd roxid e ( 1 0
m l ) i n sc rew-capped s ta in l e s s steel tub e s ( 2 5 m l capaci ty i n 3 04
s ta i n l e s s steel ) . The tub e s wer e hea ted ( 1 60 ± 2 ° C ) for three hour s
then the c o n te n ts fi l te r ed throug h num ber one sinte r ed g la s s fi l te r s ,
washed wi th wa ter ( 1 0 m l ) then wi th peroxid e- free e ther ( 2 0 m l ) . Th e
f i l trate and wa shing s we r e then extracted wi th e ther ( 2 x 5 0 m l ) and
the aqueous l aye r wa s then ac id i f i ed wi th c oncen trated hyd rochl o r ic
acid to pH2 . 5 . Sol id sod ium chloride wa s add ed to sa tur a te the
s o lution b e fo r e extrac ti on wi th e ther ( 2 x 60 m l ) . The c ombined
e ther soluti ons we r e d r ied ( anhyd rous sod i um s ulphate ) and the
s o lven t eva po r a ted . The r esidue wa s d i s so lved in ace tone ( 1 . 0 m l ) .
The pheno l ic s we r e sepa r a ted by the thin l ayer c hroma tog raph y o n
5 1
Si l icagel-G pl ate s using toluene : m e thano l : ac e ti c ac id ( 7 9 : 1 4 : 7 ) a s the
s o lven t ( Br and , 1 96 6 ) . Fe rulic ac id s tand ard s ( 2 mg/ml) and s amp l e s
wer e quan ti ta ted us ing a Shimadu z Dua l-Wave l eng th Chrom a tograph
Sc anner , wi th r e fe r ence a t 5 5 6 nm and the s ample at 366 nm us i ng the
proc edure a s d esc r ibed by Yamam o to et a l . ( 1 9 7 6 ) .
Light m i c ro s c opy
Dr ied s amp l e s of a l l the in soluble ma te r ia l s were embedded in
r e sin before sectioning for l ight m i c ro s c opy . Al l the l ight
m i c rog raph s wer e at 380 m ag ni fi c a ti o n .
Sc anning e l e c tron m i c roscopy: The fr eeze-d r ied s ampl e s at var ious
s tages of the ex tr ac tion seque nce ( f igure 2 ) wer e sepa r a te l y g lued on
to an e l ec tron mic rosc ope s tub by s pr inkl ing on to a thi n layer o f
c onduc tive s i lver pain t o r d oub led sided tape . The loose par tic l e s
wer e blown awa y , then the s tub was sputter c o a ted wi th 1 00-2 00A o f
g old i n a Pol aron E5 1 00 s putter coater .
Sc anning e l ec tron mi c rosc opy o f the sampl e s wa s c arr ied out o n a
CWIKSCAN 1 00 f i e ld emi s sion sc anning elec tron m i c ro s c ope operated a t
1 6 KV. Mi c rog raphs we r e ob ta i ned a t 4 0 , 2 0 0 , 4 0 0 , 800 and 1 2 , 000
mag n i fication using Il fo rd PF4 f i lm a t 1 6 s ec onds expo sure time .
Re s ul t s
L i g h t mic ro scopy o f whe a t b ran enabled the starch g ranul e s ,
a l e ur o ne l ayer , cuticul ar l a ye r , i n tac t pe r icarp wi th tube c el l s ,
c ro s s c e l l s and epid erm i s to b e obse rved a s seen in figur e 9A .
Fig ur e 9B shows tha t a fte r r eflux i ng in e thanol the s tarch
g ranul e s are c onsiderab l y r educ ed in amoun t. The conte n ts o f the
a l e urone c e l l s are a l so r educ ed in volume and the n uc le i o f the
a l e urone c e l l s are more e a s i l y observed . The per ic arp i s now more
c omp r e s sed .
Fi g ur e 9C s hows the b ran a f ter sequen ti al ex tr ac tion wi th c old
wa te r , hot wa ter , amyl a se and ho t ammonium oxa l a te . The s tarchy
e nd o s pe rm is no longer eviden t . Some of the conte n ts wi thin the
aleur one l ayer s ti ll remai n . Th e cuticular l a ye r and some o f the
c el l u l ar elemen ts of the per i c arp a r e also s ti l l inta c t. The
e pidermi s howeve r , i s no l onge r attached .
Fig u r e 9D s hows b ran wh i c h ha s been treated wi th alka l i . The
c el l ul ar struc tur e of the a leuro ne l a ye r is no l onger id enti f i able ,
the c e l lular struc tur e o f the per icarp does however r ema in .
5 2
Fig u r e 1 0A shows scanning e lec tron mic roscopy o f g round whea t b ran
wi th s ta rch g r anules .
Fi gur e 1 0B shows a m ore ' open ' s truc tur e , po ssibly the epide rm i s
r emoved , a fter ethanol r e fl ux and a pr epond e r ance o f s tarch g ranul e s .
Fig u r e 1 0C shows the str uc tur e o f b ran a f ter wa ter , amyl a se and
oxal a te treatmen t. At thi s s tag e o f the extrac tion sequence most of
the c el l conte n ts appe ar to have been r emoved .
Fig ur e 1 00 shows tha t a f te r treatment wi th sod i um hyd rox ide no
c ell wal l s remained , but only a nondescript s tr iated m a terial .
Fig u r e 1 1 A shows the m a te r i a l remaining when DMSO wa s sub s ti tuted
for alk a l i . The c e l l wal l s o f the a leurone l aye r , the c ellul ar
e l emen ts o f the per ic arp and the cuti c al l aye r r emain .
5 3
Fig ur e 1 1 B shows tha t a fter d e l ig n i fi c a ti o n us i ng chlor am i ne- T and
r epe a ted DMSO extr ac tion tha t there wa s m i n im a l d e te c tabl e c hange in
the struc ture obse rved .
Fi gur e 1 2A shows the m ic rog raph o f the b r an after DMSO e x tr ac tio n .
Th ere appe ar s to b e o nly c el l wal l m a te r ia l pr e se n t .
Fig ur e s 1 2B and C show tha t a fte r m i ld d e l ig n i f i c a tion and
r epea ted DMSO extr ac tion m i n im a l s truc tur a l change ha s occurr ed except
tha t the he ight of the c e l l wal l s appear to be d im i n i shed .
Fi g ur e 1 2D shows the c el l wal l s o f wheat b r an a t 4 , 0 00
m agni fica ti on.
The yie lds in pe r c en t d ry we ight o f wheat b ran o f the DMS O
s olub l e e x tr ac t and f o r c ompa r i son the alkal i e x trac ts , a r e shown i n
Table VI .
The c om posi tion o f the nond e l i g n i fied alkali and DM SO e x tr ac ted
polymer s a r e shown in Table VI I . The standard proc edure whe�
und e r taking a microscopy s tudy i s to take a l arge number o f
pho tomic rog raph s . From the se a r epr e senta ti ve selection i s made .
Th i s proc edu r e wa s adopted in thi s pr esent s t ud y and the r e fo r e the
r esul ts pr esented a r e typical o f the ti s s ue s examined .
5 4
Light Mi croscopY
Figure 9A : Commercial ly ground w heat bran at 380 magnif ication
Figure 9B : Wheat bran ex ethanol at 3 8 0 magnification
5 5
Light Mi croscopy
Figure 9C : Wheat bran ex oxalate at 380 magnif ication
Fi gure 90 : Wheat bran ex sodium hydroxide at 3 8 0 magnifi cation
; J
I . I
I
5 6
l ..
5 7
Scanning E l e ctron Mi cros copy
Fi gure 1 0 A : Commercially ground wheat bran at 2 0 0 0 magni f i cation
Fi gur e 1 0 B : Wheat bran ex ethanol at 800 magn i f i cation
S c anning Ele ctron Microscopy
Figure 1 0C : Wheat bran ex oxalate at 4 0 0 magnif ication
Fi gure 1 0 0 : Wh eat bran ex alkali at 40 0 magni f i cation
5 8
Light Mi cro scopy
Figure 1 1 A : Wh eat bran ex DMSO at 380 magni f i cation
I
Figure 1 1 B : Wheat bran ex ch loramine-T , DMSO at 3 8 0 magnification
5 9
,
I
Scanning Ele ctron Mi croscopy
Figure 1 2A : Wheat b r an ex DMSO at 40 0 magni f i cation
'
Fi gure 1 2B : Wheat bran ex DMSO at 4 0 0 magnif i cation
� . •
6 0
6 1
Scanning El ectron Mi croscopy
Figure 1 2C : Wheat bran ex chloramine-T , DMSO at 40 0 ma gnification
Figure 1 20 : Wheat bran ex chloramine-T , DMSO at 4 , 0 0 0 magni f i cation
Table VI .
Yie ld s o f hemic ellulose obta in ed by a lka l i and DMSO
e x tr ac tion from whe a t b ran
6 2
Ex tr ac tion Re agen t Yi e ld % Dr y We ight
Sod i um hyd rox ide b e fore d e l igni fic a tion
Sod i um hyd roxide a f ter d e l ig n i fication
D MS O before d e l ig n i fic a tion
DMSO a fter d e l i g n i fica tion
7 . 4
3 . 8
0 . 4
0 . 8
M �
EX tr ac tion
Re ag ent
---
Alka l i
DMSO
Table VI I
Compo si tion o f nond e l ig ni fied hem i c e l lul ose extrac ted from whe a t b ran wi th alka l i and DMSO
( expressed as % of the ana l ysed componen ts)
Rhamnose Ar ab inose Xylose Ma nnose Ga lac tose Glucose Uronic Me thoxyl Ace tyl Pro te in
a c id
0 3 1 . 9 60 . 1 0 . 3 2 . 1 4 . 0 0 . 7 0 . 1 0 . 1 0 . 5
0 2 6 . 3 5 1 . 9 0 . 4 0 . 8 1 6 . 1 1 • 1 0 . 2 1 . 6 1 • 6
Fe ru l i c
ac id
1 2 . 0
1 4 . 0
64
Di scus sion
Th i s study of the in solub l e po r tions o f b ran ha s e n ab l ed the
i n f l uence o f the maj or s te ps o f the extrac tion sequence used in Pa r t I
to b e observed .
In the c ommercial s ampl e o f whe a t b ran the pr edom i n an t featur e s
obser ved wer e the cellul ar c ompo nen ts a nd the s tarchy endosperm . Th e
m o rphol og ical s truc tur es c an b e ide n ti f i ed as d e sc rib ed in figure 7 .
Re fl ux i ng in e thanol appear ed to c ompre s s the pe r ic arp , po s sibly by
r emoving tightly bound wa te r , and reduc tion of the amoun t of s tarchy
e nd o sperm granul es wa s ob served . Af te r treatment wi th wa ter , amyl a se
and ammonium oxalate , the s tarch had been r emoved , c on f i rming the
n eg a ti ve res ul t s o f the KI- I4 s po t te s t . Some o f the c e l l contents o f
the a l eurone l a yer and the c e l lu l ar elements o f the pe r ic arp r em a in a t
thi s s tage o f the extraction sequenc e . The e pidermi s , i s however , n o
l ong er a ttached . Af te r alka l i e x tr ac tion i t wa s no t po s s ible to
iden ti f y any c e l l struc tur e o f t he aleurone l a ye r . Th u s the alka l i
tr ea tm e n t appe ared to have r em ov ed c e l l wal l m a te r ia l pr ed om inantly
f rom the aleurone l ayer . Th i s observation s ug g ests tha t the alka l i
s olub l e c e l l wal l polysac c ha r id e s o f the al eurone a r e l e s s s trong ly
b ound , po s sibly due to a l ower l ig nin/phenol ic c onte n t .
Th e s ub s ti tution of al kal i b y DMSO resulted in the c e l l wa l l
s truc tur es be ing reta ined . Th e c e l lu l ar conten ts , however , appear to
b e r ed uced . Af ter mild d elign i f ication us i ng chloramine-T and
r e- e x tr ac tion in DMSO the c e l l wa l l s o f the a l eurone , tube and c ro s s
c el l s a ppear to be the only s tr uc tur al elements r emaining .
65
The procedure us ing DMSO to prepa r e c e l l wa lls a s r e po r ted in thi s
t he si s , and the proced ure s used by Mar e s and Stone ( 1 9 7 3 ) , Ba c ic and
Stone ( 1 98 1 A ) and by Se lvand ran et a l . ( 1 980 ) are di f f i c u l t to
c ompare , a s the resul ts o f the d if fe rent me thod s have n o t b een
s ubj ec ted to the same eva lua tion procedure s by sc ann ing e l ec tron
m ic roscopy of the c e l l wa l ls . However Bac ic and Stone ' s ( 1 98 1 A ) s tud y
d id show a high mag n i fica tion elec trom i c rograph of s tr ia ted c e l l wa l l s
o f s im i l ar appearanc e to tho se shown i n Fig ur e 1 20 . Th e c hem i c al
c ompo si tion o f the ce l l wal l s h a s however been repo rted ( Ma r e s and
Stone , 1 97 3 ; Ring and Se lvend ran , 1 98 0 ; Ba c ic and Stone 1 98 1 B ) .
The r e i s g e n e r al ag reement tha t the c e l l wal l s o f b r an are c ompo sed
predominantly o f arabinoxyl ans a nd cel lulose .
The l ow y i e lds o f the DMSO e x tr ac ted ar ab inoxyl an s ( 0 . 4 a nd 0 . 8 %
o f the d r y we ight o f b r an ) suppor t the obser v a tion tha t c e l l wal l s o f
whea t bran ar e predominantly r e ta ined in DMSO e x tr ac tio n . Thi s i s i n
c ontrast to the d i sinte g r a ti o n o f the c ell wa l ls i n alka l i , whe n 7 . 4%
o f the b r an d r y we ight i s r emoved , pr i o r to d e l igni fic a ti o n .
The two po l ymers , e x tr ac ted by alkal i and DMSO, r e spec tive l y , a r e
s im i l ar , wi th respect to monosaccha r id e , pro te in and ferul ic ac id
conten t , b o th po lymers be ing composed pr edom i n an tly o f a r ab in o se and
x yl ose ( Ta b l e VI I ) . Th e r e wa s however sma l l d i f ference s , i n tha t the
DMSO extr ac t had lower l ev e l s of a r ab i n o se , xylose and g al ac to s e and
higher l ev e l s of g lucose , ace tyl , f e r ul i c ac id and pro te in than the
a lka l i e x tr ac t . Th e r ela tive ac e tyl conte n ts o f the DMSO and a l ka l i
e x tr ac ts a r e c onsi stent wi th the finding s o f Timell ( 1 96 0 ) tha t alka l i
e x tr ac ti o n r educe s the acetyl conte n t o f polysac char id e s . Th e l ower
y i e ld of the DMSO solub l e po lysaccha r ide i s probab l y the r esul t o f
n onc l eavag e o f the alka l i sen si tive bond s .
As pi n a l l ( 1 98 2 ) s ta ted tha t i t i s extreme ly unl ikely tha t
d i ffe r en t me thods o f i so l a ti o n r esul t in the same po l ymer be ing
e xtr ac ted from the c omplex of c e l l wal l s . The r esul ts obta ined in
thi s stud y would confi rm tha t observation.
Th e s i g ni fic ance o f the sma l l d i f ferences i n compo si tio n i n the
d i f fe r en t pr epar a tions o f f i br e s i s not known . Samples o f the
v arious fibre pr epar a tions a nd the c e l l wal l s obtained in the se
s tud ie s were us ed to inves t i g a te r e l a tionsh i ps between the
m o rpho logi c al and chem i c al s truc tur e s and the b i nding of z i nc i s
d e sc ribed i n Par t V o f thi s the si s .
66
67
PART IV
Me ta l adso rption to the wa te r so lub l e and wa ter inso lub l e fibres3
f rom frui ts , veg e table s , b ran and g rasses
In traduc tion
The pr esence of a c ompo ne n t o r o f a nutr ien t i n a particul ar food
o r d i e t d oe s not necessa r i l y indica te i ts b ioav a i l ab i l i ty ( O ' De l l ,
1 96 9 ) . Th e tr adi tional analys i s o f food contents tabul a ted in Food
Ta b l e s g iv e s no indicati on o f the ava i l ab i l i ty o f nutr ients presen t .
Th e r e i s some evid enc e tha t hum an s o n marg inal m i n e r al intake s m a y
b ec ome d e f i c ient i n some e s s e n ti a l e l ements ( Ke lsay e t a l . 1 9 7 9 ) ,
par ticular l y a fter pr olonged consumption o f unr e fined foods
( I sm a i l- Be ig i e t a l . 1 97 7 ) . The most consi ste n t find ing on the
e f fe c ts o f high i n take s o f unr efined plant foods on mineral
b io ava il ab i l i ty ha s been the r educ tion in calcium ( Mc Cance (:b!l 94:l;
I s m a il- Be ig i � al . 1 97 7 ) , i ro n ( Je nkins e t a l . 1 97 5 ) and zinc
a b so rption ( Da vi e s and Ni ghting ale , 1 9 7 5 ; Re inho ld e t a l . 1 97 4 ) .
The m e ta l binding c apaci ty o f d i e ta r y f ib r e has b een r ev iewed by
Ra s pe r ( 1 98 2 ) , Staub , � �· ( 1 98 3 ) , Roya l Co l leg e of Ph ysi c ian s
( 1 98 0 ) and Ke l say ( 1 978 ) . However , the r el a tive impo r tance o f
d i f fe r en t type s o f fibre s , the mechani sms and s i te s o f m e ta l binding
has not been c ompl etely e luc id a ted .
* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
3 nonc e l lul os ic , non starch po l y s accha r id e s o f ed ible plants i so l a ted by
the procedure s described in Pa r t I .
68
I n mixed d i e ts , mineral s ar e obtained fr om both pl ant and animal
food s . As und ig e s ted plan t m a te r ial pas s e s through the sma l l
i n te s tin e , bound m inera l s may be unavailable f o r abso rption into the
sma l l in te stin e . If the bind i ng capac i ty o f the plan t m a te r i a l i s
unsa tur ated , then the exce s s b i nd ing capac i ty would appe ar to i nc r ease
the l i ke l i hood o f m i nerals from o the r food sources be ing complexed ,
the r eb y r ender ing them unav a i l ab l e fo r ab so rption.
The l ocation of minerals in edible ma tter from pl an ts is o f ten
d i f fe r en t than in me at. Mi ner a l s in plants a r e often c l osely
a s soc i a ted wi th the plant c el l wal l and may be conc er ned wi th the
s truc tural in teg r i ty o f the po l ys a cchar ide s , a s in the s tab i l i s a tion
of the pe c tin struc ture by calc ium ( Ree s , 1 97 7 ) . we ak b ind ing of
c al c i um ions ha s b een observed to occur to monomeric urona te s , the
c alcium binding pr oper ti e s o f the polyuronate s are the r e fo r e a s s um ed
to b e due to thei r polymeri c natur e , and the d if ferenc e s b e tween them
to be c aused by d i f ferences i n the ir ste rochemi stry ( Kahn e t al . ,
1 96 8 ) . It ha s b een suggested tha t the s te r ic arrang em e n t o f the fixed
g roups o n the pol ym er should be more impo r ta n t wi th mul tiva l e n t
c atio n s than wi th monov alent c a ti ons ( Ha ug and Sm id sr�d 1 97 0 ) .
Bo und m e ta l s may a l so occ ur on o the r c e l l wal l consti tue n ts . The
c arboxyl g roups o f l ig nin have been suggested to be the s i te s o f
mag ne s i um bind i ng ( Jone s , 1 97 8 ) . Mo rr i s and El lis ( 1 9 7 6 ) repo r ted
tha t i ro n i s bound to phyta te i n wheat b ran . Ce real s al so c o nta in
high c o ncentr a tions of othe r e l em en ts ( Stee le , 1 98 1 ) . The r el ative
impor tance o f m e ta l bind i ng by d i f feren t c la s s e s of po l ysac char ides
extr ac ted from di f feren t food s does not appe ar to have been
e stab l i shed .
69
Th i s s tudy wa s und er taken to d etermine the me tal c on tent o f wa ter
s o lub le and in solub l e fib res from f r ui ts , vegetab l e s , b ran and g rasses
a nd to d e te rmine the ir i n te r ac tion wi th a r ang e of me ta l s tha t may
be pr esent in the reg i o n of the i n te stinal tr ac t whe r e c ompe ti tion
b e tween f i b r e s and inte s ti nal ab so rption wo uld occ ur .
Ma te rial s and Me thod s
Cold wa ter solub l e and wa te r insolub l e fibres e x tr ac ted a s
d e sc ribed i n Pa r t I , o f kn own c ompo si tion ( Table I I ) , we r e used for
the b ind ing s t ud i e s r epo r ted in thi s pa r t o f the the s i s .
Standard Zi nc So lution
A series o f zi nc c hloride solutions ( 0 . 2 5 g ZnCl 2 / l ) we re mad e up
i n sod ium ac e ta te - and sod i um barb i ta l buf fer ( 3 . 88 6 g sod ium aceta te
a nd 5 . 8 86 g sod ium barbi ta l/ 1) wi th pH ' s of 8 . 3 0 , 6 . 9 2 , 6 . 08 , 4 . 0 5 ,
3 . 1 5 and 2 . 1 2 obtained b y the add i tion o f 0 . 1 M h yd rochloric ac id .
Al so a ser i e s o f zinc chloride solu tions c on ta in ing 8 . 0 � g/ml of zinc
i n sod ium a c e ta te/ sod ium barb i ta l buf fer at pH 5 . 5 wi th varying
am oun ts of g lucose ( 1 , 2 . 5 , 5 . 0 g / 1 0 0 m l ) and sod ium chl oride ( 0 . 4 5 ,
0 . 9 g/1 00 m l ) wer e prepa r ed . Th e o smolal i ty o f the se so lutions wa s
d e te rmi ned u s i ng an Ad vanced In s truments Osmom e ter ( USA ) .
Stock Mi neral So lution
A s tock solution conta in i ng ( per l i tre) : sod ium chloride
( 8 . 1 68 g ) , po ta s s i um ch loride ( 0 . 38 g ) , calcium chloride ( 0 . 7 8 3 g ) ,
m ag n e s i um chlor ide ( 0 . 2 42 g ) , f e r rous ammonium s ulpha te ( 0 . 3 5 8 g ) ,
z inc c hloride ( 0 . 0 2 5 g ) , cupr ic chlorid e ( 0 . 0 0 2 g ) and manganese
7 0
chl oride ( 0 . 00 1 g ) i n sod i um ac e ta te/ sod i um barb i tal/h yd rochl oric ac id
( 3 52 . 0 ml o f 0 . 1 m HCl ) buf f e r at pH 5 . 5 wa s mad e . The c oncen tr a tion
of the me ta l s used fo r thi s so luti o n were c a l culated to be
approxima te ly the s ame as wo uld be expec ted to occur in the r egion of
the i n te stinal trac t ( sma ll bowe l , prox ima l j ej unum ) where c ompe ti tion
b e tween fibre ad s o rption and inte s tinal abso rption fo r m e ta l s from a
' typi c a l ' meal would occur . Th e c o ncen tr a tions we r e b ased on the
m easurements by Fb rd tr an et a l . ( 1 966 ) of the me ta l s pr esent i n a
' typi c a l ' me al and a t various r eg io n s o f the in tes tinal tr ac t . The
m agne s i um levels were c alcu l a ted from Guthr ie ( 1 97 5 ) . Th i s m e ta l
solution wa s used a s a ' s tandard ' solution fo r the mul ti ple m e ta l
b ind ing stud ie s .
Bi nd ing Proc edure
The procedur e used fo r the b i nd ing s t ud i e s to sepa r a te the b ound
and free me ta l spec ie s made use of s ix Am i c on MPS- 1 m i c ropar ti tio n
s ystems ( Am icon , 1 980 ) . The appar a tus i s shown i n Figure 1 3 . It
c onsi st s o f a r eservoir c ap , s ample r e servo i r , o- ring , YMT membr an e s ,
c l ips , m embrane s uppor t base and f i l tr a te c up .
Th e wa te r solub l e and i n solub l e fibres ( 0 . 0 1 g) we r e ind ividually
we ighed in to an a s sembled MPS- 1 f i l tr ati on apparatus , and the me ta l
conta in ing solution ( 1 . 0 m l ) wa s then added . Th e apparatus wa s c apped
and i n i ti al l y m ixed by hand for approxim a te l y hal f a m inute , and then
m ix ed on a ver ti c al rota ting pl a te ( 6 0 RPM ) for one hour ( in the
timed m i x i ng exper im en t , mixing tim e s we r e var ied from 0 . 5 to 1 80
minute s ) . Af ter mixing , the MPS-1 s ys tem s we r e c en tr i fug ed using a
f ixed ang le ( 3 4 ° ) SS- 3 4 roto r a t 1 000 RCF ( 4 , 000 RPM ) on a So rval l
centr i fug e . '!he fi l tr a te s ( 0 . 5 t o 0 . 1 m l ) we r e tr an s fe r r ed to
vol um e tr ic fl asks ( 5 . 0 to 1 00 . 0 m l ) , hyd rochl oric ac id ( 2M , 4 . 5 m l )
add ed , and then made up t o v o l um e wi th d i sti l led d e ioni sed wa te r .
Wa ter only wa s used for the z i nc solutions .
7 1
Th e percen tage o f added z i nc bound to whe a t b r an hem i c e l lul ose a t
pH ' s o f 2 . 0 , 3 . 1 5 , 4 . 0 5 , 5 . 0 5 , 6 . 0 5 , 6 . 90 and 8 . 4 5 ; t i m e s o f mixing o f
0 . 5 , 1 , 2 , 3 , 4 , 5 , 1 5 , 2 5 , 6 0 , 1 2 0 and 1 80 minute s ; a nd the in fluence
o f o sm a l a l i ty chang es wi th g lucose and sod i um chl oride from 0 to 2 98
m Osm/kg , we r e determined us ing the various zi nc solutions and the
MPS - 1 f i l tr a tion apparatus .
'!h e percen t r e ten tion o f the 's tock' me ta l solutio n by YMT membran e s
wa s de term i ned in dupl i c a te a n d the r eliab i l i ty o f the analys i s o f the
's tocK s o lution determined on ten sampl e s .
The b ind ing o f meta l s from the ' s tock ' so lution to the fibres wer e
d e te rmi ned i n dupl icate wi th i ndividual sampl e s o f 0 . 1 g fibre and 1 . 0
m l o f meta l solution. '!he wa te r insolub l e fibre from r ye g ra s s wa s
a ls o wa shed wi th hyd rochloric ac id ( 0 . 1 M) for five m inutes , then
wa shed wi th d i sti l led d e ioni s ed wa ter un ti l neutral , b e fo r e b ind ing
wi th the ' stoc k ' so luti o n.
'!he nond i � l ysable m e ta l c o n te n t o f the fibres we r e d e te rmi ned in
the same wa y a s the me ta l b ind i ng , except the ' s t.oc::. k '
r epl ac ed by d i s ti l led d e ioni sed wa ter .
solution wa s
7 2
Analys e s
Zinc Co nte n t
The zinc c o n te n t of the solutions wer e d e termined b y a tomi c
absorption spec trome tr y ( Steele , 1 98 1 ) . Th e sod i um , po ta s si um ,
c al c ium , m agnesi um , i ron, z i nc , c opper and mang anese lev e l s o f the
's tock' solution used fo r eac h run of the b ind ing stud ie s and the i r
f i l tr ate s we re d e te rm ined b y Induc tiv e l y Co upled Argon Plasma ( ICP )
emi s sion spec tro sc op y ( Lee , 1 98 1 ) .
� ta l Co ntent
The m e ta l conte n b o f the fibres were d e te rmined by add ing
conc en tr ated ( A. R. Gr ade) n i tr ic ac id ( 5 . 0 m l ) to the individual
f i b r e s ( 0 . 1 to 0 . 05 g ) . Af ter stand ing for 1 8 hour s the n i tr ic ac id
wa s r emoved by evaporati o n . The nearly d r y r esidue s wer e then
d i sso lved in hyd rochl oric ac id ( 2 . 0 M) and quan ti ta tively tran s f e r r ed
to 5 . 0 m l volum e tr ic flasks . The me ta l conte n ts we re d e te rmined by
ICP .
� ta l Bi nd ing Capa c i ty
Th e m e ta l b ind i ng c apac i ti e s o f the f i b r e s were calcul a ted a s the
s un of the meta l conte n t of the fibre s and the am oun t bound from the
' s toc k ' soluti o n . The to ta l bind ing c apa c i ti e s we re calculated a s
the s um o f c oppe r , i ro n , zinc , c a l c i um , po ta s s i um , m agne s i um ,
manganese and sod i um bind i ng c apac i ty .
.·
Reservo ir Cap -
Y MT Membrane
. F 1ttra re 1
Fi l t rate Cup ---+1
�� m pie Sampfe Reservo 1 r
Membrane Support Base
Ft ltra re . Cap
Centrlfree Mlcropartltlon Unit
Figure 1 3 : Anicon MPS-1 micropartiti on uni t
73
7 4
Re s ul ts
Figure 1 4 shows the per c en tage of zinc b ound to whe a t b ran
h em i c ellulose over a pH r ang e from 2 . 1 2 to 8 . 3 0 , the maximum zinc
b ind ing occurr ing be tween pH 5 and 6 .
Fi gure 1 5 s hows the percentage o f zinc b ound to whe a t b ran
hem i c ellul ose wi th di f fe r en t m i x i ng time s , the maximum zinc b i nd ing
occurr ing a t l e s s than 0 . 5 of a mi nute .
Table VI I I compar e s the i nfluence o f o smo l a l i ty o n the b ind i ng of
z i nc to whe a t b r an hem i c e l lul ose .
Table IX s hows results o f the r el i ab i l i ty s tudy o f 1 0 analys e s o f
the m e ta l s i n the solutio n used be fore and a f ter bind ing to whe a t
b ran .
Table X compares the per c e n tag e r e te n tion o f me ta l s by the Am icon
MPS-1 mi c ropar ti tion s ys tem b y YMT membrane s .
Tabl e XI s hows the m e ta l conte n t ( � g/mg dry we ig ht) of wa ter
s o l ub l e and wa ter insolub l e f i b re s from frui ts , veg e tabl e s , b ran and
g ra s se s .
Table X I I s hows the m e an m e ta l conte n t ( � g/mg dry we ight) o f
wa ter solub l e and wa ter i n so l ub l e f i b r e s from f r ui ts , veg e tab le s , b ran
and g ra s se s .
Table XI I I shows the chang e s i n me ta l conte n t ( � g/mg d ry we igh t)
o f the wa ter solub l e and in solub l e fibres from frui ts , v eg e tab l e s ,
b ran and g r a s s e s a f ter mix ing wi th ' stoc k ' me ta l so luti on .
Tab l e XIV shows the bound m e ta l conte n t ( � gjmg dry we ight) o f
wa ter solub l e and i n so lub le f i b r e s from fr ui t , veg e table s , whea t b ran
and g rasse s a f ter m ix ing wi th a ' s toc k ' m e tal solution.
Table XV s hows the mean bound m e ta l conte n t ( � g/mg d ry we ight) o f
wa ter sol ub l e and inso lub l e f i b r e s from f r ui t s , veg e tabl e s , b ran and
g ra s se s .
Table XVI s hows the mean , s tand ard d evi a tion ( � gjmg ) and T v alue s
o f b ound me ta l s from ma tched pa i r ed fibre s , b e fo re and a fter m i x ing
wi th ' st o c k ' me ta l solution .
Tabl e XVI I shows the percen tag e nond i a l ysable me ta l conte n t o f
the wa ter s o l ub l e fib r e s and i n solub l e fib r e s .
75
Table XVI I I shows the total ion- bind ing capacity ( mM/g d r y we ig ht)
o f wa ter solubl e and i n so lub le fibres from f r ui ts , veg etabl e s , whe a t
b ran and g r a s se s .
7 6
1 0 0
* *
90
* * *
80
"0 70
c :J a
m 80
(.) c
·r-1 !50 N
� c
40 Cl) (.) L Cl) a.. 30
20
1 0
0 2 3 4 5 8 7 8 9
pH
F i g u r e 1 4: P e r c e n t Z i n c B o u n d t o W h e a t B r a n Hem i c e l l u l o s e
Ov er a ph R a n g e f r o m 2 . 1 2 to B . 3 .
100
90
80
"0 70
c :J 0
rn so 0 c
·r-1 50 N
4J c Q) 40
0 c... Q)
0.. 30
20
10
0
77
* *
*
0 5 0 1 0 0 1 5 0 200
T I ME (m i n u t es )
F i g u r e 15: Per c e n t Z i nc Bound t o W h e a t Br an Hem i c e l l u l os e
W i th D i f f e rent T i mes o f M i x i ng .
Ta ble VI I I
In fluence o f o sm olal i ty o f g lucose and sodium chl orid e o n the
adso rptio n o f zi nc ( �g/ml) to whe a t bran hemi c e l lulose a t pH 5 . 5 .
7 8
So lution osmo l a l i ty TO ta l zinc Fr ee zinc Bo und zinc
mosmjkg
% z i nc b ound
g luc o se %
0 1 0 7 . 8 1 • 5 6 . 3 8 0 . 3
1 % 60 7 . 7 1 . 5 6 . 2 so . 1
2 . 5 1 49 7 . 8 1 • 5 6 . 3 8 1 . 4
5 . 0 298 7 . 0 1 • 3 5 . 7 8 1 . 0
% NaCl
0 1 0 7 . 8 1 . 5 6 . 3 80 . 3
0 . 4 5 1 5 5 8 . 4 0 . 4 s . o 95 . 2
0 . 9 2 89 8 . 6 0 . 4 8. 3 9 5 . 7
0\ r--
Tabl e IX
Re l iabi l i ty o f me ta l bind ing analys i s using s tandard solution and afte r mixing wi th wheat b r an
Mean ( n= 1 0 ) meta l conte n t ( � gjml )
� gjml ca l c i um Copper Iron Po ta s s i um Magnes i um Manganese Sod ium Zi nc
S tandard Me an 1 96 . 1 1 • 7 5 2 . 0 2 1 5 . 9 2 9 . 8 1 . 5 4 8 38 . 0 1 2 . 7
s olution s . o . 6 . 5 0 . 1 1 . 2 5 . 6 1 • 2 0 . 2 5 6 . 3 0 . 5 c . v . 3 . 3 6 . 4 2 . 3 2 . 6 4 . 1 1 5 . 5 1 . 2 4 . 1
Fi l tr ate ( mean
o f ten d e te rminati o n s )
a f ter mixing Mean 1 7 2 . 5 2 . 0 1 9 . 7 3 2 5 . 4 64 . 5 3 . 6 484 1 . 3 3 . 7 wi th wheat bran : s . o . 9 . 4 0 . 3 1 • 5 1 1 . 3 2 . 8 0 . 6 67 . 8 0 . 5 ( 1 m l : 0 . 0 5 g ) : c . v . 5 . 4 1 5 . 5 7 . 4 3 . 5 4 . 3 1 6 . 3 1 . 4 1 2 . 3
S . D . = Sta ndard devi a tion
c . v . = Coe f fi c ie n t o f var i a tion
0 CO
Table X
To tal me ta l con te n t o f te s t solution and am oun t a s percentage r e ten tion o f me ta l s by
funi con MP S - 1 m i c ropar ti tion system wi th YMT membranes
Ca l c i um Copper Iron Po ta s s i um Mag nes i um Mang anese Sod i um
To ta l m e ta l
conte n t �g/ml 1 96 . 1 1 . 7 5 2 . 0 2 1 5 . 9 2 9 . 8 1 • 5 4 8 3 8 . 0
Bound meta l
conte n t � g / m l 2 . 9 0 . 3 1 • 5 3 . 0 0 . 3 0 . 2 8 3 . 7
% Bo und 1 . 5 1 5 . 9 2 . 9 1 . 4 0 . 9 1 1 • 1 1 . 7
Zinc
1 2 . 7
1 • 1
8 . 4
'\
..-4 00
Table XI
Me tal content (� gjmg dry we ig ht) of wa ter soluble (SF) and insoluble (IF) fibres from frui ts ,
v eg e tables, bran and g rasse s .
Fibre Source Copper Iron Zi nc Ca lciwn Potassiwn Magnesiwn Mang anese Sodiwn
Bean SF 0 . 3 5 . 7 1 . 2 3 4 . 6 5 . 2 4. 2 0 . 4 1 2 . 7 I F o . 1 9 . 2 0 . 7 1 3 2 . 2 0 . 3 0 . 3 o . 1 3 . 0
Cabbage SF 0 . 6 2 3 .1 4 . 5 1 06 . 5 1 • 2 5 . 0 1 • 5 6 . 7 I F 0 . 2 8 . 3 0 . 5 1 2 6 . 4 0 . 3 0 . 5 0 . 1 3 . 4
Kwne ra SF 0 . 6 1 5 . 6 2 . 8 5 7 . 6 7 . 6 5 . 2 0 . 2 21 . 4 I F 0 . 2 3 . 3 0 . 5 2 6 . 6 1 • 8 2 . 4 0 . 1 29 . 0
Le ttuc e SF 0 . 2 3 . 3 0 . 7 44 . 9 6 . 8 5 . 8 0 . 3 1 2 . 2 I F 0 . 3 1 2 . 0 1 • 5 1 3 7 . 3 0 . 3 2 . 4 0 . 3 2 . 1
Onion SF 0 . 4 2 . 3 1 • 8 7 2 . 4 5 . 4 6 . 7 0 . 6 1 1 • 3 I F 0 . 2 2 2 . 3 1 • 2 119 . 5 0 . 3 0 . 4 0 . 1 4 . 3
Peach SF 0 . 5 1 2 . 5 3 . 3 1 4 . 9 4 . 8 1 • 8 0 . 4 31 . 1 I F 0 . 4 1 7 . 4 1 . 5 203 . 2 0 . 6 0 . 9 0 . 2 4 . 5
Pear SF 0 . 7 1 1 • 4 4 . 0 2 4 . 4 1 2 . 1 3 . 0 0 . 2 1 5 . 2 I F 0 . 2 2 3 . 8 1 . 5 96 . 3 0 . 5 0 . 5 0 . 1 4 . 4
Pumpkin SF 0 . 3 5 . 3 1 . 3 3 0 . 8 6 . 6 4 . 0 0 . 2 1 7 . 5 I F 0 . 2 3 3 . 2 1 • 1 118 . 1 0 . 3 0 . 5 0 . 2 3 . 8
Toma to SF 0 . 2 3 . 3 0 . 7 4 4 . 1 2 . 2 3 . 5 0 . 1 6 . 6 I F 0 . 2 1 5 . 5 2 . 0 1 42 . 8 0 . 4 0 . 5 0 . 2 2 . 9
Whea t bran SF 0 . 1 1 • 2 0 . 2 1 . 0 1 • 0 1 . 0 o . 1 3 . 6 I F 0 . 2 1 5 . 8 0 . 1 7 . 8 2 . 8 0 . 1 q . o 4 . 8
Whi te clover SF 0 . 5 7 . 9 1 • 3 84 . 0 5 . 1 6 . 6 0 . 3 1 3 . 8 I F 0 . 2 3 . 4 0 . 7 4 6 . 3 3 . 2 4 . 2 0 . 1 3 1 . 7
Lucerne SF 0 . 4 6 . 2 1 . 0 7 9 . 5 4 . 8 5 . 2 0 . 3 1 2 . 1 I F 0 . 1 2 . 8 0 . 5 5 2 . 5 1 • 6 2 . 3 0 . 1 6 . 6
Ryeg rass SF 0 . 8 7 . 0 1 . 2 3 4 . 0 6 . 3 4 . 0 0 . 4 1 2 . 8 I F 0 .1 2 . 2 0 . 3 3 6 . 0 1 . 2 , • 7 0 . , 5 . 3
N CXl
Fibre type
Wa ter so l ub l e
f i b r e
Insolub l e
f i b r e
Tab l e X I I
Mean me ta l conte n t ( � g/mg d r y we ight) of frui t , veg e tabl e , whea t bran and g ra s s fibres
Copper Iron Zi nc Calcium Pota s s i um Magne s i um Manganese Sod i um
0 . 4 ±0 . 2 8 . 1 ±6 . 2 1 . 9 ± 1 . 8 4 8 . 4± 3 0 . 2 5 . 3 ±2 . 9 4 . 3 ± 1 . 7 0 . 4± 0 . 3 1 3 . 6± 7 . 0
0 . 2 ± 0 . 1 1 3 . 0±9 . 5 0 . 9 ±0 . 6 9 5 . 8 ± 5 7 . 2 1 . 0 ± 1 . 0 1 • 3± 1 • 2 0 . 1 ± 0 . 1 8 . 1 ± 9 . 9
Tab l e XI I I
� � Chang e s in metal conten t ( ug/mg d ry we ight) of wa te r solub l e ( S F ) and insolub le f i b r e ( IF ) a f ter
mixing wi th ' s tandard ' me ta l soluti o n .
F i b r e Source Coppe r Iron Zinc Ca l c i um Po ta s s i um Ma g ne s i um Ma ng anese Sod ium
Bean SF 0 . 2 2 . 9 0 . 3 -2 1 . 3 1 • 6 0 . 7 -0 . 1 1 2 . 7 I F 0 . 1 1 • 3 0 . 2 1 • 7 1 • 4 0 . 4 -o . o 1 1 • 4
Cabbage S F 0 . 1 3 . 0 0 . 2 - 1 6 . 3 1 . 2 -2 . 0 -0 . 1 1 3 . 3 I F 0 . 1 -0 . 4 -0 . 1 -8 . 5 2 . 1 -0 . 1 o . o 28 . 2
Kum e r a S F 0 . 1 3 . 0 0 . 2 - 3 . 4 -0 . 3 -0 . 1 -0 . 1 -20 . 9 I F o . o 2 . 9 0 . 5 -6 . 2 0 . 5 -0 . 3 -0 . 0 -5 . 1
Le ttuc e SF 0 . 2 3 . 8 0 . 6 - 1 3 . 0 -2 . 8 - 1 . 8 -0 . 2 5 . 4 I F 0 . 1 3 . 4 0 . 7 -2 2 . 6 0 . 9 -0 . 7 -0 . 1 7 . 5
On ion SF 0. 1 3 . 5 0 . 3 -6 . 8 -0 . 2 -0 . 4 -0 . 1 -3 . 0 I F o . o 0 . 7 o . o -5 . 8 1 • 1 -o . o -o . o 7 . 4
Peac h S F 0 . 1 4 . 0 0 . 5 -2 . 5 1 . 3 0 . 2 -0 . 1 3 . 5 I F 0 . 2 2 . 5 0 . 5 2 . 0 1 . 4 0 . 5 -0 . 1 1 6 . 5
Pear SF 0. 1 -7 . 8 0 . 2 -6 . 0 -o . o -0 . 5 -0 . 1 - 1 4 . 7 I F 0 . 1 1 • 2 0 . 2 - 3 . 3 1 • 3 0 . 3 -0 . 1 7 . 5
Pumpki n SF o . o 3 . 6 0 . 2 - 1 1 • 9 - 3 . 9 - 1 • 4 -0 . 1 - 1 3 . 8 I F -0 . 1 1 • 7 -0 . 3 -2 . 2 1 • 3 -0 . 4 -0 . 1 1 3 . 9
Toma to SF 0 . 1 4 . 1 0 . 5 -5 . 2 1 • 3 -0 . 4 -0 . 1 2 1 • 1 I F 0 . 1 3 . 0 0 . 8 -2 . 3 1 • 4 0 . 3 -0 . 1 9 . 9
Wheat Bran SF o . o 4 . 2 1 • 2 1 . 0 1 . 5 1 • 2 -o . o 1 2 . 3 I F 0 . 1 3 . 4 0 . 9 -0 . 2 1 • 5 1 • 5 -o . o a . o
Whi te Clover SF 0. 1 4 . 5 0 . 5 -2 7 . 0 - 1 . a -2 . 8 -0 . 1 - 1 . 3 I F -o . o 3 . 5 0 . 8 - 1 9 . 2 - 1 . 4 2 . 3 -0 . 1 -7 . 8
Lucerne SF 0 . 1 4 . 4 1 • 0 - 2 5 . 7 -0 . 7 - 1 . 4 -0 . 1 1 . 9 I F o . o 1 . 8 0 . 3 - 1 2 . 3 0 . 6 - 1 . 3 o . o 1 6 . 0
R ye g ra s s SF 0. 1 4 . 6 0 . 5 -8 . 6 - 1 . 1 -0 . 5 -0 . 2 5 . 3 I F o . 1 1 . 8 0 . 4 -7 . 1 0 . 7 -0 . 6 o . o 1 1 • 2
\.
Table X I V
"" Bound m e tal conte n t ( � g/mg d r y we ig h t ) of wa ter solub l e ( S F ) and i n so l uble ( I F ) f i b r e s from frui ts , CO v eg e tab l e s , whea t bran and g ra s s e s a f ter mixing wi th a ' s ta ndard ' m e ta l solutio n .
Fibre Sourc e Coppe r Iron Zi nc ca l c i um Po ta s s i um Magnes i um Manganese Sod i um
Bean SF 0 . 5 8 . 5 1 • 5 1 3 . 3 6 . 8 4 . 9 0 . 3 2 5 . 4 I F 0 . 2 1 0 . 6 0 . 9 1 33 . 9 1 • 7 0 . 7 0 . 1 1 4 . 5
Cabbage SF 0 . 7 2 6 . 1 4 . 7 90 . 2 2 . 4 3 . 0 1 . 4 2 0 . 0 I F 0 . 2 7 . 9 0 . 4 1 1 7 . 9 2 . 4 0 . 4 1 • 1 3 2 . 2
Kumera SF 0 . 7 1 8 . 6 3 . 1 54 . 3 7 . 3 5 . 1 0 . 1 0 . 6 I F 0 . 2 6 . 2 1 . 0 20 . 4 2 . 3 2 . 1 o . o 2 3 . 9
Le ttuce SF 0 . 3 7 . 1 1 . 3 3 1 . 0 4 . 0 4 . 0 o . 1 1 7 . 6 I F 0 . 4 1 5 . 4 2 . 2 1 1 4 . 7 1 • 2 1 • 7 0 . 2 9 . 6
On ion SF 0 . 5 5 . 8 2 . 1 65 . 6 5 . 2 6 . 3 0 � 5 8 . 3 I F 0 . 3 2 3 . 0 1 • 3 1 1 3 . 7 1 • 4 0 . 3 0 . 1 1 1 . 8
Peach SF 0 . 6 1 6 . 5 3 . 7 1 2 . 4 6 . 1 1 • 9 0 . 3 3 4 . 5 I F o . s 1 9 . 9 1 . 9 205 . 2 2 . 0 1 • 4 0 . 1 2 1 . 0
Pear SF 0 . 8 3 . 6 4 . 2 1 8 . 4 1 2 . 1 2 . 6 0 . 1 o . s I F 0 . 3 2 4 . 9 1 • 6 9 3 . 0 1 . 8 0 . 8 0 . 1 1 1 . 9
Pumpkin SF 0 . 3 8 . 9 1 • 5 1 8 . 9 2 . 7 2 . 8 0 . 1 3 . 7 I F 0 . 1 3 4 . 9 0 . 8 1 1 6 . 0 1 • 6 o . 1 o . o 1 7 . 7
Toma to SF 0 . 3 7 . 4 1 • 3 38 . 9 3 . 5 3 . 1 o . o 2 7 . 8 I F 0 . 3 1 8 . 5 2 . 8 1 40 . 0 1 • 8 0 . 8 o . 1 1 2 . 7
Whe a t Bran SF 0 . 1 5 . 4 1 • 4 2 . 1 2 . 5 2 . 2 0 . 1 1 5 . 9 I F 0 . 3 1 9 . 3 1 • 0 7 . 6 4 . 4 1 • 6 o . o 1 2 . 8
Whi te Clover SF 0 . 6 1 2 . 4 1 . 8 5 7 . 0 3 . 4 3 . 8 0 . 2 1 2 . 5 I F 0 . 2 6 . 9 1 . 5 27 . 2 1 . 8 6 . 5 o . o 2 3 . 9
Luc erne SF 0 . 6 1 0 . 6 2 . 0 ' 5 3 . 8 4 . 1 3 . 8 0 . 3 1 4 . 0 I F 0 . 1 4 . 6 0 . 8 40. 2 2 . 1 1 . 0 o . 1 2 2 . 6
Ryegrass SF 0 . 9 1 1 • 6 1 • 7 2 5 . 4 5 . 2 3 . 4 0 . 2 1 8 . 1 I F 0 . 2 4 . 1 0 . 7 2 8 . 8 2 . 0 1 • 1 0 . 1 1 6 . 5
( a fter 0 . 1 M ) I F 0 . 1 2 . 4 o . s 3 . 0 3 . 0 0 . 8 o . 1 40 . 8 (HC l wa sh . )
'\
<: ..:::r 00
Table XV
Mean bound me ta l conten t ( � g/mg dry we i g h t ) of frui t , veg e tab l e , whe a t bran and g rass fibre s
a f te r mix i ng wi th 1 s tandard 1 me ta l so lution
F i b re type Copper Iron Zinc Ca l c i um P o ta s s i um Magn e s i um Manganese Sod i um
Food s and g rasses
wa ter solub l e 0 . 5 ±0 . 2 1 1 . 0 ±6 . 3 2 . 3 ± 1 . 2 3 7 . 0± 2 5 . 6 5 . 0± 2 . 7 3 . 6± 1 . 3 0 . 3± 0 . 4 1 5 . 3± 1 0 . 4
In solub l e 0 . 3 ± 0 . 1 1 5 . 1 ±9 . 4 1 . 3 ± 0 . 7 89 . 2 ± 59 . 3 2 . 0 ±0 . 8 1 . 4± 1 . 6 0 . 1 ± 0 . 1 1 7 . 4± 6 . 4
Foods
Wa ter soluble 0 . 5 ±0 . 2 1 0 . 8 ±7 . 2 2 . 5 ± 1 . 3 34 . 5 ± 2 7 . 9 5 . 3 ± 3 . 0 3 . 6± 1 . 5 0 . 3 ± 0 . 4 1 5 . 4± 1 1 . 9
I n solub l e 0 . 3 ± 0 . 1 1 8 . 1 ±8 . 6 1 . 4 ±0 . 7 1 06 . 3 ± 5 7 . 1 2 . 1 ±0 . 9 1 . 0± 0 . 7 0 . 2± 0 . 3 1 6 . 8± 7 . 0
Grasses
wa te r so l ub l e 0 . 7 ±0 . 2 1 1 • 5 ±0 . 9 1 . 8 ±0 . 2 4 5 . 4 ± 1 7 . 4 4 . 2 ± 1 . 0 3 . 7 ±0 . 2 0 . 2± 0 . 0 1 4 . 9 ± 2 . 9
In soluble 0 . 2 ±0 . 1 5 . 2 ±1 . 5 1 . 0 ± 0 . 5 3 2 . 1 ± 7 . 1 1 . 9 ±0 . 1 2 . 9± 3 . 2 0 . 1 ± 0 . 0 2 1 . 0± 4 . 0
Ll"l CO
Table XVI
Mean , s tandard devia tion ( � g/mg ) and T values of bound me ta l s from ma tched pa i r ed fibre s , before and
a f ter mixing wi th 1 s tandard 1 me ta l solution
Fibre Type Copper Iron Zi nc Ca lc ium Pota s sium Magnesium Manganese Sod i um
So l uble fibre X 0 . 1 2 . 9 0 . 5 - 1 1 • 3 -0 . 3 -0 . 7 -0 . 1 1 • 7 s 0 . 1 3 . 3 0 . 3 9 . 0 1 • 7 1 • 1 o . o 1 2 . 3 T 7 . 2 3 . 2 5 . 5 -4 . 5 0 . 6* - 2 . 3 * -9 . 0 0 . 4*
In soluble fibre X 0 . 1 2 . 1 0 . 4 -6 . 6 1 • 0 0 . 2 -o . o 9 . 6
s o . 1 1 • 2 0 . 4 7 . 6 0 . 8 1 • 0 0 . 1 9 . 1 T 3 . 6 6 . 3 3 . 6 - 3 . 2 4 . 3 0 . 6* - 2 . 9 3 . 8
x = mean
S = Standard deviation T = T s ta ti s tic for ma tched pai r s ( Wa lker and Lev , 1 96 9 )
* = no t significant for 1 2 deg rees o f fr eedom , a t 0 . 0 1 < 2 . 68
1.0 CO
Table XV I I
Percentage nondia lysable me ta l conte n t aga inst d i s ti l led-de ioni sed wa ter o f the water solub l e and
i n so l ub l e fibres
Fibre source Copper Iron Zinc Cal cium Pota s s i um Magnesium Manganese Sod i um
Bean SF 9 1 . 6 1 o o . o 9 5 . 5 85 . 7 89 . 1 87 . 5 1 oo . o 5 3 . 2
I F 8 1 • 1 96 . 9 87 . 5 99 . 3 1 oo . o 9 1 . 2 90 . 0 84 . 2
Cabbag e SF 8 5 . 8 9 0 . 9 9 6 . 6 60 . 1 8 3 . 1 3 3 . 1 97 . 5 9 3 . 2
I F 8 3 . 1 8 3 . 6 48 . 9 9 4 . 4 87 . 7 6 1 . 8 96 . 7 7 4 . 2
Peac h S F 88 . 3 98 . 8 9 3 . 5 9 1 . 4 1 oo . o 9 6 . 2 1 oo. 0 8 3 . 9
I F 98 . 3 97 . 5 9 5 . 6 99 . 7 1 oo . 0 1 oo . o 98 . 4 9 2 . 8
Ryeg rass SF 9 1 . 6 9 7 . 5 8 9 . 2 87 . 8 7 5 . 2 87 . 9 9 9 . 8 5 7 . 0
I F 6 2 . 5 8 2 . 7 5 9 . 4 90 . 6 6 1 . 3 70 . 0 96 . 0 6 1 . 8
87
Table XV I I I
TO ta l bound ion conte n t ( mM/g dry weight) o f wa ter solub l e ( S F ) and
i n so l ub l e fibres ( IF ) from f r ui ts , veg e tab l e s , whe a t bran and g r a s se s .
Fib r e Source TO ta l ion conte n t
Be an SF 2 . 0
I F 4 . 4
Cabbag e SF 3 . 9
I F 4 . 6
Kume r a SF 2 . 2
I F 1 . 8
Le ttuc e SF 2 . 0
I F 3 . 7
Onion SF 2 . 5
I F 3 . 8
P e ach SF 2 . 4
I F 6 . 5
P e ar SF 1. 0
I F 3 . 4
P umpkin SF 1 . 0
I F 4 . 4
Toma to SF 2 . 6
I F 4 . 5
Wheat Br an SF 1 . 0
I F 1 . 3
Whi te Clover SF 2 . 5
I F 2 . 2
Lucerne SF 2 . 5
I F 2 . 2
R yeg rass SF 1 . 5
I F 1 . 6
( a fter 0 . 1 M HCl ) I F 2 . 0
...
D i scus sion
Pr e l iminary stud ie s were unde r taken to estab l i sh some o f the
parameters of the meta l binding to fibres .
Fo r thi s stud y i t wa s as sumed tha t wheat b ran hemicellulose was
r epresenta tive of the wa ter inso luble fibres and tha t zinc was
r epresenta tive of all the metal s used in the te st solution. Th i s
a ssumption i s probably val id for the fibres , al though wheat bran
hemicellulose did have an initial zinc content s l ightly lower than
tha t from other foods . However , for the meta l s the e ffec t o f
h yd rolys i s and precipi ta tion o f ferric irons a t higher pH , suggest
tha t it may no t be entirely valid . In thi s study , oxidation o f
ferrous ions were dimini shed b y using fresh solutions . Therefore
h yd rolys i s and prec ipi ta tion probably do no t ser ious l y i nvalidate
thi s in v i tro model sys tem .
Ef fect o f pH
8 8
As shown in Figure 1 4 , the max imum amoun t o f zinc binding to wheat
b ran hemicel lulose occur r ed be tween pH 5 and 6 , which i s wi thin the
r ange of pH 4 to 7 , tha t Fo rd tran and Lockler ( 1 966 ) found in the
proximal j ej unum . Th us the pH of the absorption reg ion o f the
i n te s tinal trac t and tha t a t which maximum zinc b ind ing to thi s fibre
occur s are in the same pH r ange .
Ef fec t of Time
The inve stiga tion o f the time r equired to obtain max imum zinc
b ind ing , shown in Figure 1 5 , i ndicate s tha t equi l ibrium is reached
8 9
wi thin a short time . Th e chang e i n binding from 2 0 t o 1 50 minute s was
less than one per c ent. It had been suggested by Phi l l ips and
Fernandez ( 1 98 1 ) , tha t appropr iate stud i e s of me ta l bind ing to fibres
should be under taken wi th the same time ( greater than 1 20 minute s ) as
tha t occurring in the intestinal tr ac t. It was also pointed out by
Sche inberg ( 1 98 2 ) tha t all binding stud i e s should endeavour to r each
e qui l ibrium be tween the bind ing po lymer and the lig and , so tha t the
l aw o f mass ac tion can be appl ied . The r esul ts ob tained in thi s study
s uggest tha t e quil ibrium between the b inding polymer and the l igand
will be reached be fore the time Phi l l ips and Fe rnandez ( 1 98 1 ) suggest .
However , to incorporate a safe ty margin , a l l the binding stud ies we r e
c arr ied out wi th a mix ing time of 6 0 minute s .
Ef fec t o f OSmolali ty
The influence of osmolal i ty on the percentage of zinc b ound to
wheat bran hemicellulose is shown in Table IX . Inc rease s i n
o sm olal i ty o f g lucose from 1 0 to 2 98 mOsmol/kg had minimal a f fect o n
the b inding of zinc ( 80 . 3 to 8 1 . 0% ) . However , the inc rease o f
o smolal i ty using sodium chlorid e , over the same r ange o f o smolal i ty
i ncreased the binding of zinc from 8 0 . 3 to 9 5 . 7 per cen t .
Phi ll ips and Fernadez ( 1 98 1 ) promoted the concept tha t
i nvestiga tions o f me tal binding to fibre should mimic the cond i tions
of the small bowe l , the o smolal ity of which i s from 2 50 to 3 0 0
mOsmol/kg ( Fo rd tran and Locklear , 1 966 ) . The r esul ts o f thi s study
s ug gest tha t i t is not the o sm olal i ty per � that influences me tal
binding to fibre but the ionic streng th o f the solution . Th i s resul t
90
i s consi sten t wi th the finding s of Smidsr�d and Haug ( 1 968, 1 97 2 ) .
The y found tha t the presence o f neutral substances such as sucrose d id
not i nfluence the calcium b inding properties o f pec ti c ac id . The
increase in zinc binding a t higher ionic streng th may be the r esul t of
an inc reased hyd rodyn amic volume as has been observed by Simdsr�d
( 1 97 0 ) in alginate solutions . Therefore me ta l bind ing to thi s
polysacchar ide is conditional upon the ionic strength of the solution.
Fi l tr a tion Sys tem
The valid i ty of the MPS- 1 f i ltration s ys tems for use in separati ng
bound and free metal spec ies wa s i nve stigated . Th i s was done by
d e te rmining the retention of me tals to the apparatus . It can be seen
f rom Table X tha t only sm all pe rcen tages of the me tals we r e reta ined
by the s ystem . Manganese ( 1 1 . 1 % ) and copper ( 1 5 . 9% ) are exceptions
and their higher retention is probably related to the ir low
c oncen tration in the standard solution ( Mn , 1 . 4 mg/ml and Cu 1 . 7
mg/ml ) . The b indi ng of all the me tals to the filtration membrane and
apparatus could possibly have been reduced by prior saturation.
However a coefficient of var ia tion of less than 1 0% fo r most o f the
meta l s wo uld appear to be wi thin reasonable expe r imen ta l error.
Th e r esul ts from thi s study have been taken to confirm tha t the
MPS-1 m i c rofiltration sys tem does enable the separation o f bound and
free l ig ands ( Hammond e t �. 1 98 0 ; Shah e t al . 1 974 ) . Th i s me thod
has , however , not pr eviously been used to d e termine me ta l binding to
food c omponents . It has the advan tage o f ease o f exper imen tal
procedur e compared wi th the e quiva lent but slowe r me thod of
equi l ibrium dialysis ( Sophianopoulos e t al . 1 97 8 ) .
9 1
The ini tial metal conte n ts o f the two groups o f fibres varied
considerably as seen in Table X I . While the origin of these metals
has not been establi shed it is possible that they were of plant orig in
or derived by a Donnan equi l ibrium from the dialysis wa ter ( which
had a content of calcium , 3 3 . 8 �g/ml , magnesium , 6 . 3 �g/ml , potassium ,
5 . 5 �g/ml , copper , 0 . 84 �g/m l , i ron , 1 . 1 6 �g/ml , mang anese , 0 . 08 �g/ml
and zinc , 2 . 8 1 �g/ml ) that occurred during the polysaccharide
preparati ons as described in Par t I . However , the metals were not
r eadi ly d ialysable against disti lled-deionised wa ter as seen in Table
XVI I , ind icating that the me tals were firmly bound to the water
soluble and insoluble fibres from bean , cabbage , peach and ryegrass .
Thus , whi le the origin of the me tals is no t establi shed i t does
i ndicate a capaci ty of these plant ma terials to firmly bind minerals .
Calcium was the predominant metal species present in both classes
o f fibr e . >'< Generally the wa ter inso luble fibres contained more calcium
than the water soluble fibres . The mechanisms of calcium as sociation
wi thin plant material have not been clearly e lucidated . Pectin ,
b indi ng calcium by the proposed ' egg-box ' mechani sm ( Rees , 1 977 ) has
received prominence as the me tal complexing cell wal l consti tuent
( Davi s et �· 1 980 ) . The modes of interchain association for pec tins
resulting in gels have been investigated using a variety o f
techni que s . Mo r r i s et al . ( 1 980 ) investigated non- ionic interac tions
using circular dichroism and found tha t reduced ge l strengths at low
levels of esteri fication to be due not only to increased charge
densi ty but also from a signi ficant loss tha t the contribution�ester
g roups make to the stabi l i ty o f the pec tin interchain j unctions .
'� The h i gh e s t c a l c i um cont e n t ( "> 8 8% o f t h e to t a l me t a l c o n t e n t ) wa s
o b s e rv e d i n p e a c h wa t e r i n s o l ub l e f i bre .
Interchain associations being stabilised by non-covalent forces
analogous to those in pro tein ter tiary struc tur e . Thi s was confirmed
by Oakenfull and Scott ( 1 984 ) tha t the network of polysaccharide
molecules in gels of high me thoxyl pec tins are s tabili sed by a
c ombination of hydrophobic interac tions and hyd rogen bond s . The
contr ibution from hyd rophobic interac tions to the free energy of
forma ti on of j unction zones being about hal f that from hydrogen
bonding but an essenti al requirement since hyd rogen bonding alone i s
i nsuf ficient to overcome the entropic barrier to gelation, sucrose
acts by s tabi lising the hydrophobic interac tions ( Oakenfull and
scott, 1 984 ) . The elution behaviour of aqueous pec tin solutions i s
9 2
also influenced by reversable aggrega tions as we ll a s the chain length
of i ndividual molecules ( Davi s et al . 1 980 ) . Gidley e t al . ( 1 980 )
sugge s t that there are two modes of pectin inte rchange association .
The first mechanism by interchain chelation o f calcium ' egg-box
bind ing ' ( Powell et �· 1 98 2 ) and the second by non- ionic interchain
associations analogous to those in low water activity gels
( Morris et al . 1 980 ) . The me tal binding characte r i s tics of the high
uronic acid containing polysaccharides have been shown to have
considerable complexi ty ( Gid ley e t al . 1 980 ) . However , the resul ts
f rom thi s study suggest tha t the insoluble fibres , such as the
hemicelluloses , are quanti tatively more significant binders of
c alcium . The lower uronic acid content of the wa ter insoluble fibres
but g reater calcium content than the wa ter soluble fibres , suggests
that di fferent binding mechanisms may be operative in the two
d i fferent groups of polysaccharides .
The si te and func tion o f bound calcium in hemicelluloses has not
been determined . However , the resul ts of the determination of me tal
bind ing to ryegrass hemicel lulose wi th and wi thout ac id treatment,
s howed tha t there was a marked decrease in bound calcium after acid
treatment ( Table XIV ) . It is possible that divalent calcium plays a
s tabi lising role wi thin the insoluble fibres by being a biological
c rosslinking agent which c ontr ibute s to the archi tec ture of the cell
wall .
The application o f the MPS- 1 microfi l tration system to determine
the interac tion of metals wi th the water soluble and insoluble fibres
from frui ts , vegetable s , bran and g rasses showed tha t there was
c onsiderable variation in the amounts of the metal species bound , as
seen in Tables XI I I , XIV, XV and XVI .
9 3
Table XV shows tha t the mean bound me ta l content o f the foods and
the grasses do not subs tantially differ except for the bound calcium
content . The foods had a higher calcium content ( mean 1 06 . 3 ± 5 7 . 1
� g/mg ) assoc iated wi th the insoluble fibre compared wi th the grasses
( 3 2 . 1 ± 7 . 1 �g/mg ) . Thi s can not be expla ined in terms of uronic ac id
content , for the foods had a mean percentage uronic acid content o f
1 0 . 3 ± 6 . 7 · compared wi th the grasses o f 1 3 . 4 ± 4 . 8 per cent of the
hemicel lulose content. Thi s i s sugge stive that a mechani sm other than
uronic acid association wi th calcium is the binding mechani sm .
Howeve r , the results repor ted in this sec tion of the thesis were not
designed to inve stigate the basic bindi ng mechani sms •
It can be seen from Table XV I that there was signi ficantly more
c opper , i ron and zinc bound to both the wa ter soluble and insoluble
94
fibres for the matched paired ex tracts be fore and after mixing wi th
me tal solution. Af ter mixing wi th the ' sto c � ' me ta l
solution there was significantly less calcium and manganese bound to
both classes of fibre , whi le there wa s signif icantly more potassium
and sod ium bound to the insoluble fibre s . At the 0 . 0 1 % leve l there
were no significant changes in the magne sium levels from both classes
o f fibre , however , at the 0 . 025% level there was significant
d i splac ement of magnesium from the water solub le polysaccharides .
Haug and Smidsr�d ( 1 970 ) investig ated the selec tivi ty of
polysaccharides for copper compared to calcium . They conclud ed that
selec tivi ty for copper compared to calcium i s a charac teristi c feature
o f all carboxylate containing polymers and of polyphosphate , whi l e no
such selectivity is observed for sulphated polysaccharides .
They suggested that the binding mechani sm responsible for the
higher affinity of copper relative to calcium ions in
carboxylate containing polyanions is independent o f the presence of
free hyd roxyl groups in the polymer and of the steric arrangements of
the carboxylate groups . Thi s exchange reac tion i s associated wi th an
unfavourable enthalpy change and the driving force of the
copper-calcium exchange reac tion must therefore be a relatively large
posi tive entropic change connec ted wi th the bind ing of copper ions and
l iberation of calcium ions from the polyanion . It seems possible tha t
thi s entropic change is caused by the copper ions having a larger
ordering effect upon the water molecules than calcium ions and tha t
hyd ration of the ions i s destroyed when they are bound to the polym e r .
The difference between carboxylate and sulphate g roups may b e due to
9 5
different e ffec t o f the two types of fixed g roups i n destroying the
hydration o f the copper ions .
The fac t tha t copper-calcium selec tivi ty seems to be caused by a
mechanism different from that governing the selec tivi tie s of alkaline
earth ions ( Haug and Smidsr�d , 1 97 0 ) does not mean that copper ions
cannot be bound to polyuronate s by the same mechanisms as are the
calcium ions . On the contrary , the constancy of copper-calcium
selectivi ty coefficients indicate s tha t , when the struc ture of the
polyuronide allows a preferential bind ing of calcium compared to
magnesium , copper i s also bound preferenti ally , probably by the same
mechanism . In add i tion, the mechanism making the binding of copper to
carboxyla te groups energe tically more favourable than the binding of
calcium is operative .
The affinity of coppe r , i ron and zinc b inding to the water soluble
and inso luble fibres i s in general agreement wi th Mod � al . ( 1 9 8 1 )
who found an affin i ty order of copper , iron and zinc to water and
alkali soluble hemicelluloses from rice . Gregor et �· ( 1 955 ) who
s tud ied the binding of divalent metal ions to polyac rylate , found the
complex formation constant for copper was much higher than for
magnesium , zinc , cobalt , calcium and manganese which decreased in tha t
o rder . A s tudy o f the forma tion constants wi th polyme thacrylate wa s
reported to dec rease in the order copper , cadmium , zinc , cobalt and
manganese ( Mandel and Leyte , 1 964 ) .
Studies of cell wall frac tions from a g rass , Holcus lanatus , have
shown tha t pec tin and lignin complex a large proportion of the
96
calcium , whe reas magnesium i s complexed only by lignin ( Molloy and
Ri chards , 1 9 7 1 ) . The results of thi s present study showed that there
was a signi ficant decrease in calcium and magnesium in the water
soluble po lysacchar ides when mixed wi th the ' stD t. \<. ' me tal solution ,
but no signi ficant decrease in magnesium in the wa ter insolub l e
hemicellulose s . As shown in Par t V the hemicellulose of wheat bran
contains some lignin , and a hemicel lulose- l ignin bond has been
d emonstrated ( Hartley 1 97 8 ) and feruloylated pec tins ( Fr y , 1 98 3 ) .
Possibly the di fference in magnesium binding be tween these two c lasses
may be due to the l ignin content.
The range of total me tal bind ing capaci ties of the fibres was
between 0 . 2mM/g dry we ight for the water soluble fibre from beans to
6 . 5 mM/g dry weight for insoluble fibre from peach ( Table XV I I I ) .
The mean value of the total bound metal content of the fibres , from
the plant species examined , wa s 2 . 0 ± 1 . 0 mM/g dry we ight of wa ter
soluble fibre and 3 . 5 ± 1 . � mM/g dry weight of insoluble fibre . The
higher value of bound me tal content of the water insoluble fibres i s
probably a reflec tion of the high initial calcium content.
The me tal binding capac i ty of the fibres compares favourably wi th
the binding capaci ty o f c ommercial ion-exchange resins , which range
from 0 . 6 to 5 . 0 mM/g dry we ight ( Briti sh Drug Houses Bulleti n , 1 97 7 ) .
The cation-exchange capaci ties o f a v a r i et y of ' whol e ' foods and
of neutral detergent fibres ( Mc Burney e t al . 1 98 3 ) have been
d e te rmined ( McConnel l � al . 1 97 4 ) . They ranged from 0 . 6 mM/g dry
weight of pear to 2 . 0 mM/g dry we ight of carrot . While it may not be
97
val id to compare the ion bind ing capacity of such d iverse samples as
manufac tured resins , fibres and ' whole ' food s , d e termined by a varie ty
o f d i f ferent me thods , the similar ity of the quanti tative values
suggests tha t the total number of binding site s are similar .
The content of copper and zinc was generally higher in the wa ter
soluble fibres ( Table XI and XI I ) . This result i s in agreemen t wi th
the findings obtained in investigations of me tal tolerant grasses
( Turner , 1 97 0 ) , where it was found that pec ta te extrac t, contained
more zinc and copper than other cell wall consti tuents . Re sul ts of
i nvestigations of the mechan i sm of zinc tolerance suggest that there
are different binding si tes for copper and zinc ( Pe terson , 1 969 ) .
The bind ing sites o f the se metals is no t known . It has been
s ugge sted that the meta l binding properties of die tary fibre are
d irec tly related to the uronic acid content ( James � al . 1 97 8 ) .
Al though they obtained a very highly significan t correlation ( P <
0 . 00 1 ) for calcium binding to uronic ac id content an R2 value of only
0 . 4 3 56 was obta ined , indicating tha t over 50% of the variation in
calcium binding was no t attr ibutable to a relationship wi th uronic
acid content. Wi thin the c omplex of plan t cell walls and their
consti tuents there are a number of possible metal binding site s , such
as the carboxyl , phosphate , phenolic , hyd roxyl and carboxyl g roups of
l ig n in and ferulic ac id ( Jone s , 1 978 , and Fr y , 1 98 3 ) , binding to
s i lica present in pec tins ( Schwarz , 1 97 3 ) and in secondary cell wal l
polysaccharides ( Jones , 1 97 H ) , and also mechani sms involving
s truc tural interac tions between polymers can not be excluded .
The quan ti ta tion and mechani sms o f me tal interac tions wi th plant
mater ial , par ticularly food polysaccharides wi th a low uronic ac id
content , do not appear to have been extensively investigated .
The next par t of thi s thesis describes exper iments under taken to
determine the binding capaci ties and mechani sms of zinc interac tion
wi th various fibres from wheat bran and phyta te .
98
99
PART V
Zinc ad sorption to wheat bran , i ts fibre componen ts and to phyta te
In troduc tion
Ef ficient milling technology , resul ting in bran removal and fibre
d eple tion, has been suggested to be a contr ibuting fac tor in the
increased human death rate from a variety of d eg enerative di sease s
( Burki tt, 1 97 2 , 1 97 3 ; Walker , 1 97 4 ; Trowe l l , 1 976 ) .
Bran however , has been shown to be not an entirely benign dietary
component. A relationship between poorly or unrefined cereals in the
aetiology of mineral deple tion wa s shown in an investigation of the
role cereals played in the aetiology of nutr i tional ricke ts , tha t
occurred in Dublin in 1 94 2 , when the extrac tion rate of flour was
i ncreased from 7 0 �1 00 per cent ( Rober tson et al . 1 98 1 ) . It has been
s ugge sted that phyta te is the maj or facto r in reduc ing mineral
b ioavailability ( Re inhold et al . 1 97 3 ) . Phytate ( phytic ac id ,
i nosi tolhexaphosphoric ac id ) , 87 percent of which i s assoc iated wi th
the aleurone layer of bran ( O ' De l l et al . 1 97 2 ) appear s to reduce the
b ioavailabi lity of metals ( McCance and Widdowson , 1 94 2 ) .
Ingestion of phyta te containing foods has been shown to in te r fere
wi th calcium ( McCance and Widdowson , 1 94 2 ) and zinc absorption
( Re inhold � al . 1 97 3 ) . The chemi stry , occurrence and metal bind ing
of phyta te has been recently reviewed by Maga ( 1 98 2 ) , Graf ( 1 98 3 ) and
Oberleas ( 1 98 3 ) .
In stud ie s on the availab i l i ty of calcium and iron wi th respect to
MASSEY UNI VERSI T Y LIBRARY
refining , McCance and eo-wo rkers ( 1 94 2 , 1 948 ) ascribed the
d eleter ious e f fec t of unrefined cereals to the phytate content.
However studies by Re inhold and eo-workers ( 1 97 5 ) investigating the
1 00
b inding of calcium , zinc and iron to var ious wheat breads and wheat
b ran , found tha t even whi te bread and dephytinised bran , bound zinc .
Moreover , phyta te consumed in a pur i fied form i s less effec tive in
d ec r easing the retention of zinc and calcium than when equivalent
amounts are consumed in the form of wholemeal bread ( Re inhold � al . ,
1 97 3 ) . Whi te bread conta i ning neg lig ible amounts o f phytate , also
i n te r feres wi th minera l absorption ( Callender and Malpas , 1 96 8 ) .
Fr� lich and Asp ( 1 980 ) investigated the relati onship between the fibre
c ontent o f wheat flour s obtained from mi l l ing of d i f ferent ex trac tion
r a tes and the assoc i a tion of mineral s . Th e y conclud ed that the
m inerals were mainly assoc iated wi th a wa ter soluble fraction
c ontaining polysaccha r ides and phyta te . The results obtained in Par t
I V o f thi s thesis suggests that there was significant bind ing of the
three me ta l s , copper , iron and zinc . Pr el im inary studies on i ron
b i nd ing were under taken . However , fac i l i ti e s were no t available to
e l im i nate the possibi l i ty of i nsoluble meta l complexes of copper and
i ron being formed by oxidation. Therefore a more detailed study of
only zinc binding was undertaken .
In Cumming s ' ( 1 97 8 ) opin ion there are many questions relati ng to
z inc absorption and fibre that remain unanswe red , fur ther info rmation
is needed particularly on the mineral-binding prope r ties of d i e tary
f ibre as present i n commonly eaten frui ts and vegetables . Fi sher and
Berry ( 1 980 ) on reviewing the present sta te of research on d i e tary
fibre , concluded tha t binding of metals by fibre undoubted ly occur s ,
but tha t the relative impor tance of binding by fibre and of phytate
r emains controversial . As the role of fibre and of phyta te in
r educ ing mineral bioavai labi l i ty i s not clear ( Fi sher and Be r r y ,
1 98 0 ) , the debate o n the importance o f wheat r e fining o n food qua l i ty
( McCance and Widdowson , 1 95 5 ) has as ye t to establish quanti ta tively
the importance of fibre or phytate as the me tal bind ing substance .
1 01
Numerous stud ies have investigated the bind ing of small molecule s
s uch as metals , to macromolecules . A number o f methods for g raphical
and computer assi sted analysis of the binding data have been employed .
The most commonly used g raphical me thods includ e ; Scatchard plo ts
( Sc a tchard , 1 949 ) , Hi l l plots ( Dahlquist , 1 97 8 ) and Klotz plots
( Klotz , 1 98 2 ) . It i s c lear tha t a given se t of binding data contains
the same information, independent of the par ticular method used to
interpret i t . However , certain g raphical methods show some aspec ts o f
thi s information more clearly than o thers .
Sca tchard ( 1 949 ) desc r ibed a procedure for determining the way in
which small molecules may combine wi th mac romolecule s . Th i s procedure
c an enable the determination o f the amoun t , the tightnes s and the
s i te s of binding to be d e termined . The me thod ha s been summar i sed by
Sche inberg ( 1 98 2 ) as follows : if the ini tial probability of bind ing a
molecule of A is the same at each of n si te s or g roups on a
mac romolecule , P , the change in free energ y
( bG ) v for the reac tion
i s zero , i f all the components and species of the reac tion are in
e qui l ibrium when a me asur ement is made ( Scatchard , 1 97 6 ) . If bind ing
the first molecule of A to P has a negligible effect on the tightness
of binding o f the second molecule of A and so on, then
kcA v/ ( n-v )
wher e k i s the intrinsic association constant for the reac tion a t a
1 02
s ing le si te , CA is the concentration of free l igand molecules and v i s
the averag e number o f A molecules bound to each mac romolecule .
A li teral interpre ta tion of Scatchard analysis ( Sche inberg , 1 98 2 )
appl ie s only to the b inding o f small molecules and ions to
m ac romolecules in solution , expres sed in molar terms . A more general
appl icati on has also been appl ied to more complex systems , i nvolving
the suspension of cells or membranes , wi th the concentration expressed
in terms of milligram of po lymer ( We i siger et al . 1 98 2 ) . The
g raphical extrapolati on ( Rosenthal , 1 96 7 ) of the binding parame ters ,
i n e i ther form , from nonl inear Sca tchard Plots can result in
d i fficul tie s in interpr e tation ( N�rby et a l . 1 98 0 ; Pe ters and
Pingoud , 1 98 2 ) such as the over estimation of the number of si te s and
an under-estimation of the binding affinity ( N� rby e t �· 1 98 0 ) .
Wei siger et al . ( 1 98 2 ) employed scatchard Plots for the di splay o f the
data , to indicate the model , i . e . the occurrence of positive
1 03
cooperativi ty , negative cooperativi ty , or the presence of both
posi ti ve and negativi ty , and/or the presence of more than one c lass o f
s i te s .
A means o f investigati ng posi tive and negative cooperativity by
Hi ll Plots was suggested by Dahlquist ( 1 97 8 ) . Al l forms of lig and
bind i ng can be represen ted by a sing le- step interac tion be tween
macromolecules and ligands :
M + nx t MXn
wher e M i s the macromolecule and X is the ligand .
Thi s equi l ibrium can be defined by a phenomenolog ical cons tan t , k ,
s uch that [ MXn] / [ M] [ X ] n = kn .
The apparent value o f K i s the inverse of the half-sa tura ti ng
l igand concentration .
The Hi ll binding equa tion can be linear i sed by taking logari thms :
log [MXn) I (M) n log k + n log x .
The bind ing data may then be plotted as log ( si te s bound ) / ( s i te s
free) versus log ( free l igands ) . The slope o f the region o f hal f
s a turation g ives n , the Hi ll coeffic ient , which i s a measure of the
e x tent of cooperative- in teractions among the po te n tial si te s ( i .e . the
m ean square devia tion of the number of ligands bound at hal f
s a tura ti on) . When posi tive cooperativity i s presen t , the Hi l l
coefficient i s greater than un i t y , and less than uni ty when negative
c ooper a tivi ty is pre sent ( Ko shland � al . 1 966 ; Dahlqui st , 1 97 8 ) .
1 04
The exper iments desc ribed in thi s section were under taken in an
attempt to quan ti ta te zinc bind ing to various type s of wheat bran
fibres and to phyta te . The s trength of binding , and the mechani sms of
i n te r ac tion be tween zinc , fibres and phyta te s were also inve stigated .
Ma terials and Me thods
Sodium phytate obtained from Sigma ( type V ) , sod ium sal t from
corn, and cel lulose from Macherey , Nagel and Co . wer e used .
i ) Fr ac tions from Par t I
The soluble whea t bran frac tions obtained in Par t I , cold
wa ter solub l e polysacchar ide and the alkali sol uble ( non-deligni fied )
hem i c ellulose , were used in thi s study . Be fore us i ng the
hemicellu lose i t was suspended in wa ter and neutral ised wi th 0 . 1 M
h yd rochlor ic ac id , dialysed for eight hour s agains t deionised water
then freeze-dried .
( i i ) Frac tions from Par t I I I
The d r y insoluble wheat bran fractions prepared in Par t I I I were
a lso used ; bran , bran ex e thanol , bran ex oxala te , bran cell wa l l s
( DMSO insoluble residue after chloramine - T ) and DMSO soluble
hemicellulose ( nondeligni fied ) .
( i i i ) Wa ter soluble polysaccharide from bran
A wa ter soluble polysaccharide from wheat bran was also extrac ted
using the procedure of Ne ukom and Markwalder ( 1 97 5 ) . Whea t bran
( 1 00 g) was blended in water ( 5 00 m l ) for 1 0 m inute s in an
* homogeni ser . The contents were centr i fuged ( 2 0 minute s , 760 RCF )
and f i l tered . The solution obta ined wa s then sa turated wi th ammonium
sulpha te , mixed and allowed to stand overnig ht at 4 ° C . The solution
wa s then centr i fuged for 20 minute s at 760 RCF . To the supernatant
wa s added alpha amylase ( 2 g) then dialysed for 1 20 hour s against
d i stil led wa ter . The solution wa s again sa tur a ted wi th ammonium
s ulpha te , allowed to stand overnight at 4 ° C , centri fuged , then
r edialysed for 48 hour s . The volume of the solution was reduced from
1 05
968 m l to 3 50 m l by ro tary evaporation ( 3 2 ° C ) , then freeze-dried . The
c omposi tion of the dry mate r ial wa s determined by the same procedures
as used in Part I .
( iv ) Lignocellulose frac tion
A l ig nocellulose frac tion, from wheat bran , wa s prepared using
m ild extrac tion procedure s to remove non- lignin components . Br an ex
oxalate ( 1 50 g ) , as prepared in Par t I I I , wa s used as the starting
mate r ial . It was wa shed in deionized wa ter , peps i n ( 1 . 5 g ) added in
2 50 m l of 0 . 1 M hydrochloric acid , the mixture wa s shaken for 1 20 hour s
a t 3 0° C , then dialysed and the pepsin procedure r epeated unti l the
s po t test for amino ac ids ( Fe ige l , 1 956 ) wa s neg a tive . The liquid was
d ec an ted and the residue washed in wa ter ( 3 x 500 m l ) . To the residue
was added sodium hyd roxide ( 1 0% ) under ni trogen , mixed and allowed to
s tand for 2 hours . Af ter decan ti ng the l iquid , the residue was wa shed
wi th wa ter ( 3 x 500 m l ) then neutrali sed wi th 0 . 1 M hyd rochloric ac id .
* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
... ( RCF Re lative centr i fugal force , 1 1 . 1 7x (radius in cm) x i
RPM 2 1 oool
Ce l lulasejhemicellulase ( ce llul ase 3 0 , 000 , PFIZER Chemical s ) in
pho sphate buf fer ( pH 2 . 5 ) was then added . The mixture wa s shaken for
1 20 hours then dialysed and the enzyme procedure repeated unti l the
s po t te st for carbohydrate s ( Vogel , 1 95 8 ) in the supernatant wa s
n eg a tive . The brown insoluble residue obtained on decan ting , was
washed twice in water and fi nally in 95% ethanol and dried . The
procedure used is summari sed in Fig ur e 1 6 .
Bran ex oxalate
1 Water wa sh
p
lrsin
�:�� ��s�=�.��e
1 0% sodium hyd roxide
l Ni trogen , 2 hr s wa ter wa sh 3x
Ce l lulasej Hemicellulase
P�ospha te buffer pH 2 . 5
Un ti l spot te st for carbohyd rates negative , followed by a wa ter and 95% e thanol wash
Lig nocel lulose
Figure 1 6 : EX trac tion sequence for the preparati on of lignin .
Lignin determination
The l ignin conte n t of bran , lignocellulose frac tion and the
1 06
hemicelluloses were dete rmined using the 7 2% sulphuric acid procedure
a s desc ribed by Hol loway et al . ( 1 977 ) . To the d ry we ighed mater ial
wa s added 7 2% sulphuric acid , mixed for four hour s , then fi l tered
through No . 2 sintered g lass c ruc ibles , wa shed in water and acetone ,
oven dried , we ighed , then ashed at 5 5 0 ° C .
De te rmination o f zinc bind ing
1 07
The me thod used for de termining the zinc binding to wheat bran and
i ts var ious fibre frac tions and to phyta te was as described in Par t
IV. The membranes used fo r the phyta te studies were UM05 membranes
wi th a molecular weight cut off 500 ( Amicon , 1 980 ) , cut to size to fi t
the MPS- 1 fi l tration systems . The phytate content o f the filtrate wa s
d e te rmined using the me thod of Davie s and Re id ( 1 97 9 ) .
A ser ie s of zinc solutions from 1 . 0 � g/ml to 6 , 000 �g/ml in sodium
ace ta te/sod ium barbital buffer at pH 5 . 5 was made . The fibre free
MPS- 1 f i l tr ate s conta ining unbound zinc were d iluted , when required ,
wi th di sti l l ed deionised wa ter to obtain solutions wi th a
c oncentr ation of zinc be tween 1 . 0 and 50 . 0 � g/ml before measur ing by
a tomic absorption .
An alys i s o f binding resul ts
The resul ts of the bind ing studie s were analysed us ing g raphical
d isplay and Hardman ' s ( 1 98 3 ) adaption of wood ' s version ( Daniel and
Wood , 1 980 ) of the Marquard t maximum neighbourhood method of nonlinear
f i tting program ( Marqua r t , 1 96 3 ) . This For tran program , run on a
Cromenco CS- 2 microcomputer wi th 64K RAM, i s a c ombination of the
Gauss-Newton and steepe st-descent methods ( Daniel and Wood , 1 980 ) .
Da ta , expressed as �moles zinc bound/g of fibre , wer e fi tted to the
following equa tions :
Fo r one si te interac ting model;
Y = Ymax * xn
Lo . sn * xn
Fo r a two s i te interac ting mode l ;
Y = ( Ymax1 * xn 1
(L0 • 5 1 n1 * xn )
( Ymax2 * xn2 )
(L0 • 5 1 n2 xn2 )
where Y = bound zinc , x free zinc , n Hi l l coefficient , Lo . s
l ig and 50% bound .
The minimum least squares value s for zinc bound , max imum zinc
b inding , ligand 50% bound ( l igand concentration requi red for hal f
s a turation) and the Hi l l coeffic ients for the fibres and phytate we r e
d etermined us ing Daniel and woods ( 1 980 ) curve fi tting procedure ( see
Appendix E) •
1 08
Re s ul ts
The composi tion of the cold wa ter soluble polysaccharides and the
po lysaccharide obtained us ing the ammonium sulphate procedur e are
shown in Table XIX.
Table XX shows the yields of the two wa ter soluble fibre ex trac ts
a nd of the l ignocellulose fr ac tion from whea t bran .
Table XXI shows the l ig n in content of wheat bran , l ig nocel lulose
f r ac tion and the hemicelluloses .
Reproduc ibility
1 09
The reproduc ibil i ty study wa s undertaken to asse ss the var iabil i ty
i n the bind ing and the analysis of zinc . In the de termination of the
f r ee zinc l evels from ten binding studies to wheat bran , wi th a zinc
solution of 2 . 0 �g/ml , a coe ffic ien t of var ia tion of 4 . 0% wa s
ob tained .
Binding
The bind ing isothe rms of zinc to wheat bran , i ts various fibres
and to phytate are shown in Figures 1 7A to K. The resul ts of the
b i nd ing experiments , expressed in � M/g of fibre , were plotted against
the total amounts of zinc added to the exper imenta l system s . It was
observed tha t the shape s of the binding i so therms varied consider ably .
Sca tchard plots
Figures 1 8A to K show the Sca tchard plots of zinc binding to wheat
b ran , i ts fibres and to phytate .
1 1 0
Table XXII shows the bind ing capac i tie s , ligand 50% bound and Hi l l
c oe f ficients for zinc binding to wheat bran , i ts fibres and to phyta te .
Table X IX
The percen t composi tion of the cold wa ter soluble fibre and the puri fi ed water soluble fibre from wheat bran
Ex trac tion Rhamnose Arabinose Xylose Mannose Ga lac tose Glucose Uronic Me thoxyl Ace tyl Pro te in procedure ac id
Cold wa ter 0 . 3 2 3 . 5 29 . 9 1 • 7 7 . 2 3 3 . 1 0 . 6 0 . 1 0 . 4 3 . 3 Ammonium sulpha te 6 . 0 24 . 0 1 8 . 6 1 6 . 2 20 . 3 1 0 . 9 0 . 9 0 . 1 0 . 1 2 . 9
1 1 2
Table XX
Yie ld s of the wa ter solub le fibres and lignocel lulose from wheat bran
gm/1 00 g of wheat bran
Cold wa ter soluble extr ac t
Purified wa ter soluble extrac t*
Lignocellulose
o . so
0 . 1 3
7 . 44
* Ex trac ted using the ammonium sulphate procedure of Neukom and
Markwalder ( 1 975 ) .
Table XXI
Lig nin content of wheat b ran , lignin and the hemicellulose s
extracted before and afte r delignification
1 1 3
Fibre type Lignin g/1 00 g dry we ight
Bran
Lignoce l l ulose
Hemicellulose ( not delignified )
Hemicellulose ( deligni fied )
3 . 6
3 0 . 4
1 . 8
0
1 1 4 F i gu r e 1 7: A t o C : Z i n c B i n d i n g t o Whe a t B r a n a n d i ts
Comp o n e n t s i n .t�M/ g, ..
• Bo u n d , a F i tt e d ( 1 , a o n e s i t e i n t e r a c t i n g m o d e l : 2 ' a t w o s i t e i n t e r a c t i n g m o d e l )
200 A . 8r a n 1
Cl • • 0 .......... 150 "'0 c • ::J 0 0 � · CD
100
(.) • c
•M N 0 50 :::E 0 =t 0 •
•
100 2 0 0 3 0 0 4 0 0 500 600 7 0 0
200 B . 1 B r a n e x e t h a n o l
Cl
.......... 150 • • • -o i o 0 0 0 c :J • • 0 CD
100 • (.) 0 c:
-M N
50 0 :::E • =t
0 0 100 200 300 400 500 600 7 0 0
200 c . B r a n e x a x a l a t e 2
Cl
.......... 150 "'0 c: :J 0 CD
100
(.) • • c:
•M • N 50
:::E • =t
•
0 0 100 200 300 400 500 BOO 700
To t a l Z i n c (JJ M)
s o
0) 50
............. -a c 4 0
::J 0
CD 30
(..) c
....-! 20 N
::E � 1 0
700
0) BOO
............. -a 500
c ::J 0 400
CD
(..) 300 c
·rl N 200
::E � 100
400
0)
............. 300 -a c ::J 0
CD 200
(..) c
·rl N
1 0 0 :::E �
. 1 1 5 .
F i gure 17: 0 to F: Zinc Adsorpt ion to Wheat Bran Components
i n JJM/ g� • Bound, o F i tted
D .
�
� •
E .
i
.r� •
F .
• i • 0
( 1 , a o n e s i t e i n t e r a c t i n g m o d �: 2 , a t w o s i te i nt e r a c t i n g m o de l )
•
0 0 •
1 00 0
C e l l u l o s e 1
500
.C e l l W a l l s 2
• 0
1 0 0 0
He m 1 c e l l u l o s e 2
•
2000
T o t a l Z i n c
e
1000
2000
•
{.u M)
•
0 •
3000
1500
3000
•
4000
Cl
' 'C c ::J a CD (.) c
·1""1 N
� �
Cl
' 'C c ::J a CD (.) c -r1 N � :::J.
Cl
' 'C c :::J a CD (.) c
·r-1 N
� =<
BOO
500
400
300
200
100
BOO
500
400
300
200
5000
4000
3000
1 1 6 F i gu r e 17: G to I: Z i nc Adsor p t i o n to Wheat Bran Comp onents
i n .uM/ g� • Bound, o F i tt ed ( 1 , a o n e s i t e i n t e r a c t i n g m o d e l : 2 , a t w o s i t e i n t e r a c t i n g m o d e l )
G . L i gn o c e l l u l o s e 2
0 • •
0 • 0
0 0
. .. 0 •
2000 4000 6000 8000
H . 1 Co l d W a t e r So l ub l e
•
0 0 • 0 • •
•
8
500 1000 1500
I . 1 Pur i f i ed W a t e r So l ub l e
•
e
2000
• 1000 0
o e 0 • "' •
1000 2000 3000 4000 5000 To t a l Z i n c (u M)
�
Cl
' "0 c ::J 0 CD (.) c
·1""'1 N � :::J
Cl
' "0 c ::J a
CD (.) c
·1""'1 N � :::J
5000
4000
3000
2000
1000
10000
8000
6000
4000
2000
1 1 7 F i gure 17: J to K: Zinc Adsorption to · Wheat Br an Components
in uM/ g� • Bound, o f i tted ( 1 , a o n e s i te i nt e r a c t i n g mode l: 2 , a two s ite i n t e r a c t i n g mode l )
J . DMSO Hem i c e l l u l o s e 2
• • 0 •
0 •
25000 50000 75000 100000
K . Phy t a te 1
• 0 0
I •
•
5000 10000 15000 20000 25000 To ta l Z i n c (u M )
4 Fo o t N o t e : Bouf"\o\ 1i nc c o n t e n t
o f f i b r e. t o t a l z i nc m i n u s free z inc in � M / g d r y we ight
i' .. .
9 B . Br a n e x Etha n o l
w * w a: 1J_ 6
a: w > 0 *
� a * ::::> * 0 * * * * CD
0
* * 0 50 100 150 200
a c . Br an ex o x a l at e
w * w a: 1J_ 2
a: w > 0
* 0
1 z ::::> * * 0 * CD *
* * * * *
0 0 25 50 75 1 0 0
.u M Z i n c Bo u n d / · g
1 1 9
F i gure 1 8: 0 to F: Scatchard P l ot s o f Z i n c Adsorpt i on t o
W h e a t Bran Comp o n e n t s *
1 . 5 D . Ce l l u l o s e
lJJ lJJ a: *
* lL 1 a: *
lJJ > 0
0 0 . 5 z ::l *
0 *
CD * * *
0 *
0 20 40 80
8 Ce l l Wa l l s
lJJ lJJ a: lL 4 a: w > 0
� 2 *
::l 0 *
* * CD *
0 0 200 400 BOO 800
8 F ·. Hem i c e l l u l o s e
lJJ lJJ fi lL 4 fi w > 0
0 2 z ::l * 0 CD · * * � *
*
0 * *
0 100 200 :300 400 JJ M Z i n c Boun d / g
LJ.J LJ.J CI Ll.. CI LJ.J > 0 0 z :::J 0 CD
LJ.J w CI Ll.. CI w > 0 0 z :::J 0 (lJ
w w CI Ll.. CI w > 0 0 z :::J 0 (lJ
1 2 0
F i g u r e 18: G t o I : Sc atchard P l o t o f Z i nc Adsor p t i on to W h e a t Bran Comp o n e n t s �
L i g n o c e l l u l o s e
9
� *
* * 0
0 100 200 300 400 500
30 H . C o l d Water So l u b l e
20 * *
*
10
* *
*
100 200 300 400 500
30 I . Pur i f i e d Water So l u b l e
20 * *
* * 10
* *
0 0 1000 2000 3000 4000
JJM Z i nc Bo u n d / g I'
300
w w a: u. 200
a: w > 0 0
1 0 0 z :::J 0 llJ
90
w w a: u. 80
0:: w > 0 0
30 z :J 0 (JJ
F i gure 18: J to K: Scatchard P l o t s o f Z inc Adsorpt i o n
J .
* +e+ *
K .
Wheat Br an Components -!(
DMSO Hemi ce l l u l o se
* 1000 2000 3000 4000
*
Ph y t a t e
2000 4000 6000
.u M Z i nc Bo u n d / g
* Foot No t e ( a s on page 1 1 7 )
1 2 1 to
5000
8000
1 22
Table XXI I
Zinc binding capac i tie s , in � M/g , l igand 50% bind ing and hi l l
coefficients of wheat b ran , i ts various fibre c omponents and of phyta te
Name Bi nding Capa c i ty Lig and 50% Hi l l Coe fficient
Bran 1 67 . 7 ± 1 2 . 7 4 3 . 1 ± 7 . 3 1 . 4 ± 0 . 3
Bran ex e thanol 1 4 2 . 3 ± 4 . 4 1 1 • 4 ± 0 . 8 3 . 0 ± 0 . 6
Bran ex oxalate 1 48 . 3 ± 50 . 0 2 64 . 3 ± 2 1 1 0 . 9 2 ± 0 . 2
Ce llulose 5 7 . 4 ± 5 . 3 1 1 o . 2 ± 32 . 4 0 . 99 ± 0 . 2
Cell walls 1 0 1 2 . 6 ± 1 9 3 765 . 3 ± 327 1 . 02 ± o . 1
Hemicellulose 2 27 . 9 ± 6 1 . 4 3 04 . 4 ± 1 54 0 . 7 ± 0 . 1
Lignocellulose 5 1 0 ± 4 1 . 9 7 26 . 9 ± 1 40 1 . 2 ± 0 . 2
Cold wa ter soluble
fibre 4 40 . 0 ± 1 5 . 3 1 70 . 8 ± 1 5 1 2 . 1 4 ± 0 . 4
Puri fied wa ter
soluble fibre 4038 ± 2 1 6 94 . 6 ± 1 • 1 1 . 65 ± o . 2
DMSO hemicel lul ose 5089 ± 9 2 1 3584 . 3 ± 4200 0 . 5 ± 0 . 8
Phytate 6582 ± 1 92 582 . 4 ± 1 0 1 1 . 2 6 ± o . L.
1 2 3
Di scus sion
A compa r i son o f the c omposi tion of the two wa ter sol ub l e extrac ts
f rom wheat bran , g iven in Table XIX , shows tha t the ammonium s u�pha te
procedure of Neukom and Ma rkwa ld er ( 1 9 7 5 ) gave an ex trac t higher i n ;
r hamnose , mannose , galacto se and g lucose . Bo th me thods of ex trac ti o n ,
cold wa ter and ammonium sulphate , probabl y ex trac t a mix ture o f
polysacchar ides . The h i ghe s t y i e l d s be ing o b t a i n e d f o r t h e c o l d wa t e r
e x t r a c t ( Ta b l e XX ) .
wa ter solub l e arab inoxyl ans have been i s o lated f rom whea t fl our
( Pe r l in , 1 95 1 ) and i t has been shown ( Ewa ld and Per l i n , 1 9 5 9 ) tha t the
arab i no se side cha in s are a posi tive fac to r in solub i l i ty . Low
ferulic ac id conte n t o f arabinoxylans has a l so been a s soc i a ted wi th
water solub i l i ty ( Markwa lder and Ne ukom , 1 97 6 ) . A water so l ub l e
arab i nog alac tan-pepti d e from whea t endosperm ( Fi nche r � a l . 1 9 7 4 ) and
an arabinogalac ta n ( Ne ukom and Markwa ld e r , 1 9 7 5 ) from wheat flour have
been i solated .
The polysaccha r i d e s ob ta ined by the cold wa ter and i �� ammoni um
s ulpha te proced ur e ( he reafte r r e fer red to a s pur i fied wa ter sol ub l e )
wer e found to c on tain low leve l s o f pro te i n , 3 . 3% and 2 . 9%
r espec tive l y . Th i s conte n t i s sim i lar to the 5 . 0% , tha t Neukom and
Markwalder ( 1 97 5 ) ob tained from whea t f l our us ing the ammonium
s ulpha te proced ur e . However , the manno se and xylose conten ts o f the
pur i f i ed wa ter solub l e extr ac t wa .c. conside r ably higher at 1 6 . 2% and
1 8 . 6% respec ti ve l y than the 1 . t d 0 . 7 % tha t Neukom and Markwalder
( 1 97 5 ) obtai ned f r om f l our . Po � · ly a m anno se c onta in i ng
polysacchar ide i s a s soc i a ted pr • u:'· ntly wi th the bran frac tio n
1 24
rather than wi th flour , as has been found in r ice bran by Mod � �·
( 1 97 9 ) .
The resul ts of an evalua tion of the procedure used to prepare the
l ignin enr iched frac tion, by analys ing the products , using the 7 2 %
sulphur ic acid me thod , i s shown i n Table XXI . Whea t bran was found to
c onta in 3 . 6% lignin , thi s i s in ag reement wi th previous ly published
values ( Holloway � al . 1 9 77 ; Southga te et al . 1 976 ) .
As shown in Table XXI the lignin content of the l ignocellulose
f rac tion wa s 30 . 4% , the alkali soluble hemicellulose ex trac ted before
delig nification, 1 . 8% , and the alkali soluble hemicellulose , ex trac ted
a fter delig ni fication , was found to be devoid of lignin .
The enzymatic procedure used in thi s study appears to have enabled
a l ignin enr iched frac tion to be prepared . However , possible chemical
and physical changes wi thin the lignin struc ture cannot be exclud ed .
Me thods o f analysis for thi s complex and variable mater ial appear to
have not reached a suff icient stage of deve lopment to enable deta iled
a ssessmen t o f the valid i ty o f the commonly used 72% sulphuric acid
procedure . I t was therefore concluded that wi thin the lim i ta tions o f
the analys i s procedur e , a l ignin enriched fibre fraction was
s uccessful l y prepared .
The assessment of the analytical errors i nvolved in the
measuremen t of binding of zinc to fibres showed tha t at the l ow r ange
o f zinc concentrations used in the binding studie s , a coefficient o f
variation o f 4 . 0% o f the free zinc level was obtained . An asse ssment
of the variabi l i ty of zinc binding at high concentrations and the
e s t ab l i shme n t t h a t e qu i l i b r i um wa s r e a c h e d w i t h the o t h e r f ib re s ,
wou l d have enab l e d the de t e rm i na t i on o f expe r ime n t a l e r r o r s t o b e
obt a i n e d .
1 2 5
The z i nc b i n d ing d a t a o b t a i n e d i n t h e s e s e r i e s . o f e xp e r imen t s we r e
ana l y s e d i n t e rms o f S c a t cha r d P l o t s , shown in f igu r e s 1 8A t o K .
Unu s u a l b i nd i ng c u rve s we re o b t a i n e d f o r t h e b i nding o f z i nc t o
d i e t a r y f i b r e c omponen t s o f whe a t b r a n . The me a n i ng o f wh i c h i s
unc l e a r , a s imp u r e p o l yme r s we r e u s e d i n t he expe r imen t a l s y s t em .
Henc e , t h e o b s e r v a t ions made a r e t h e s umma t i on o f me t a l b i nd i ng
cha r a c t e r i s t i c s s uch as ion-excha nge , change s in con f i g u r a t i on ,
s p e c i f i c a n d non-spec i f i c b i n d i ng s i t e s o f mu l t i c omp on e n t s y s t ems .
I n i t i a l i n t e rp r e t a t i on was ma d e u s i ng i de a l i s e d sy s t e ms f o r S c a t cha r d
P l o t a na l y s i s . Howeve r t h i s wa s done w i t h c a u t ion a n d w i t h d u e
r e c o gn i t i o n o f t h e cons i de r a b l e c omp l e x i t i e s o f me t a l b i n d i ng
p rope r t i e s o f p l a n t c e l l wa l l c ompone n t s .
A l l t h e b i nd ing da t a o f z i nc t o b ra n , i t s f i b r e f r a c t ions a n d t o
phy t a t e , e xp r e s s e d i n S c a t c h a r d p l o t s , we re ma rke d l y non- l i n e a r
a s s e e n i n f i g u r e 1 8A t o K . T h e c u r va t u r e o f t h e S c a t c h a r d P l o t s
imp l i e s e i t he r t h a t t h e r e i s mo r e than one group o f b i nd i ng s i t e s ,
w i t h e a ch group c h a r a c t e r i s e d b y a d i f f e r e n t i n t r i n s i c a s s oc i a t i on
con s t an t , o r t h a t the b i n d i ng o f e a c h suc c e s s ive mo l e c u l e a l t e r s t h e
a s s oc i a t i o n con s t a n t f o r t h e n e x t b i n d ing mo l ecu l e ( Sc a t c ha r d e t a l
1 9 50 ; S c h e i nbe r g , 1 98 2 ) .
1 26
The sc a tc hard P lots of b r an and bran ex e thanol , as seen in figures 1 8A
and B are complex , po s s ibly i ndicating tha t zinc binding occurred to
more than one componen t . Af ter tr eatmen t wi th wa ter ( see ex tr ac tion
sequence , Figur e 2 ) the shape of the Sc a tcha rd plots changed . Complex
plots showing po si tive and neg a ti ve coope r a ti v i ty have been desc r ib ed l kl•i£, .,r.tJ.
Kowever neg a ti ve coope r a ti v i ty i s the most c ommonly ob served
( Da hlqui s t , 1 97 8 ) . The notab l e exception i s tha t of the i n ter ac tion
o f oxygen wi th haemog lob in ( Dahlqu i s t , 1 9 7 8 ) .
The shape of the sc a tc hard pl ots of the two wa ter so lub l e ex tr ac ts
s ug g e s t posi tive c oope r a ti v i ty as the bind ing mechan i sm ( Da hlqui s t ,
1 97 8 ) . The coope r a ti v i ty be ing more pronounced in the pur i fied wa ter
soluble pol ysacchar id e ( Neukom and Markwald e r , 1 97 5 , ex tr ac ti on
procedur e ) as ind i c a ted by a shi f t in the max imum bind ing along the
abs c i s sa a t hig h d eg r e e s o f sa tur a tion ( Dahlqu i s t , 1 97 8 ) as shown in
Figur es 1 8H and I . The se curve s suggest tha t i n order to b ind the
f i r s t l igand , the energe tic a l l y unfavourabl e s tep of conver sion o f the
polymer from the l ow- a f finity to · the high- a f f i ni ty fo rm is requi r ed .
Subsequent zinc molec ul es bind wi thout the necessi ty of pe r form ing
thi s conver s ion and ��e r e fore have a hig her a f finity.
The sc a tc hard P l o ts o f a l l the b r an frac ti ons , a f ter r emova l of
the wa ter soluble f i b r e , a l l had concave and marke� ly nonlinear Plots
a s seen in Fig ur e s 1 8C , D, E, F , G, J and K . Phenomeno logically thi s
impl i e s that the f i r st zinc molecules b ind wi th a higher a f f in i ty than
subsequen t molecule s . Th i s c ould re sul t from induced changes in the
1 27
other binding si tes resul ting from the first bound ligand . An
a l te rnative explanation i s tha t the archi tec ture of the polymer has a t
l east two distinct environments o f binding site s ( Ber nhard , 1 97 1 ) .
Si te he terogene i ty can not be easi ly distingui shed from negative
c ooperativi ty . However , a qual i ta tive ind icator of the phenomenon of
c ooperativi ty c an be obta ined from Hi ll Plots ( Dahlqu i s t , 1 97 8 ) .
Fur ther experimenta tion would be required to establish tha t
e qui l ibrium had always been reached and tha t the fibre componen ts were
not slowly d issolving or coagulating thereby a l tering the
m acromolecular system .
The Hi ll coef ficients of zinc bind ing to wheat b ran , i ts fibre
frac tions and of phyta te were c alculated using Hardman ' s ( 1 98 3 )
adaption o f the computer program o f Daniel and wood ( 1 980 ) ( See
Appendix E) . The Hi ll Coe f fic ients , shown in Table XXI I , of wheat
b ran and i ts frac tionated components showed considerable var iabil i ty .
The l arge exper imental error associated wi th the de termination o f the
Hi l l coeffic ients and Scatchard plots , which are but the summation of
the various mechanisms o f b inding to the c omplexes o f cell componen ts ,
i t i s not possible to assign definitive binding mechanisms . As i s ,
i nd ic a ted by the similar i ty o f the Hi ll Coefficients obtained for bran
( 1 . 4 ± 0 . 3 ) and for phyta te ( 1 . 3 ± 0 . 2 ) . However , the str ikingly
d i f ferent shapes of the Sca tc hard plots for zinc binding to the
var ious wheat bran frac tions i s sugge stive of di f feren t binding
m echanisms being operative . Whi le i t i s conceded tha t there is
c onsiderable experimenta l error involved in thi s data , the obse rvable
trends of the curves o f the Sc atchard plots a s shown in Figures 1 8A to
1 28
k i s suggestive of di fferent bind ing mechani sm s occurring . It i s
therefore hypo thesi sed that the occurrence of d i fferent shape s o f the
Sc atchard plots of zinc binding to wheat bran fractions , i s indicative
o f d i f feren t binding mechanisms . Th i s hypothe sis wi ll require fur ther
exper imenta tion to establ i sh i ts val id i ty .
Th e use o f Sc atchard Plots to summar i se and analyse bind ing data
has been c r i ti c i sed ( Klotz , 1 982 ) , as the level of maximum binding was
o ften not r eached . He suggested that a semilog plot of the amount
b ound versus the log concentration o f the free ligand has a number of
use ful featur e s ; an in flec tion point a t half-maximum bind ing , the
s- shaped curve is symme tr ic about the inflec tion point and a plateau
i s reached asymptotically as the concentr atio n of the free ligand
approaches very large ( infinite ) val ues , and the slope decrease s to
the plateau value as the maximum bind ing is approached .
Attempts wer e also made to use Hi l l and Klotz plots . However they
d id not reveal any insight into the mechani sms of zinc binding to
wheat bran and i ts fractions , and we r e not reported wi thin thi s
the si s .
In response to Klotz ( 1 98 2 ) cri ti c i sm o f the use of Sca tchard
Plots and hi s advocate of semilog pl ots . Mun son and Rodbard ( 1 98 3 )
s ta ted tha t while transfo rming the d a ta does not change the
info rma tion content, the al tered shape o f the curve results i n a
c orrespond ing change in the shape o f the envelope of uncer tainty
around the curve . Large sca tter occur s in both Scatchard and Klotz
Plots , as one approache s the axi s for the concentration of bound
l ig and , e specially in the presence of significant nonspec i fi c b ind ing .
1 29
In Mun son and Rodbard ' s ( 1 98 3 ) opinio n " In deal ing wi th whol e
ti s sue s and therefore numerous impur i ti e s , a t l arg e free ligand
c oncen tr ati o n s , nonspec i fi c bind ing wi l l inevi tably become larg er than
s pe c i fic sa tur able b ind ing " . Ne ar the uppe r pl a teau reg ion o f the
b ind ing i so therm , s pe c i fic bind ing i s o ften measur ed as a smal l
d i f ferenc e be tween two l arge number s . Henc e , a ttempts to measur e the
max imum ligand bound by s impl y using larg e fr ee- ligand concen tr a tions
m a y be unrel iable and tha t the pre sence o f nons pec i fic bind ing
m agni fies the problem .
Munson and Rodbard ( 1 98 3 ) did however , ag ree wi th Klotz ( 1 98 2 ) ,
tha t e x tr apolation o f Sc a tchard pl ots i s fr aug ht wi th di fficul ti e s ,
and tha t severe probl em s c an ar i s e in ex trapo l a tion o f nonl inear
plots . The y suggested tha t g raphical me thods serve we l l to provide
s ubj ec ti v e , pr eliminar y understand ing of the data and tha t nonlinear
l east- squares curve fi tting procedur es should be used for quan ti ta ti o n
( Munson and Rodbard , 1 98 3 ; Thakur et al . 1 980 ) . We i s iger e t al .
( 1 98 2 ) a l so only used Sc a tchard plots for the d i splay o f the binding
data , the ac tual d e te rm i n a tion o f the bind ing par amete r s being done by
c omputer analys i s .
Acc ord ing ly data ob ta ined from the bind ing o f zinc to bran and i ts
c omponen ts we r e subj ec ted to the non- l inear least- squares proc edure o f
Daniel and wood ( 1 980 ) .
The m a thematical m od e l i n i ti a l l y used wa s tha t o f one i n te r ac ti ng
s i te . As seen in Fig u r e s 1 7 ; A , B, D, H, I and K , whe re thi s m od e l
was used , the calcul a ted value s , shown as o pe n c i r c l e s ( o) , wer e
1 3 0
observed to be i n r easonable ag reement wi th the exper imen ta l value s .
Fo r the zinc bind ing isotherms o f b ran ex oxalate , cell wa l l s , l i gnoce l l u l o s e
a lkal i soluble hem i c ellul ose and DMSO solub l e hemicel lulose , s een in
Figur es 1 7 ; C, E, F,G and J , a two s i te i n te r ac ting model wa s found to
g ive r easo nably close ag reemen t wi th the exper imenta l data .
The fac t tha t reasonably good f i ts b e tween the expe r im e n ta l and
calculated d a ta are ob ta ined , a s seen in figures 1 7A to K, us i ng the se
r ather s imple mathem a tical mod e l s d oes not necessa r i l y mean tha t the se
s imple m od e l s are correc t . It d o e s ind i c a te , however , tha t the se
s a tura ti o n curves per � do no t d emand a t thi s stag e o f s tudy more
c omplex m od el s . However , improvemen ts in r emoving the inhe ren t
expe r im e n tal erro r s in the s ys tem used and the collec tion o f more data
would enhance the probab i l i ty o f e stab l i shing the number o f b inding
s i te s . I f more d a ta on the s a tur a tion curves were ob tained ,
proces s i ng i t , us ing the more powe r ful computer program ' Ma s sac t'
d eveloped by Mc in to sh and Mc in to sh ( 1 980 ) , would enable the te sting o f
m od e l s o f up to two spec i fi c binding si te s and one non- spec i f i c o r low
a f fi n i ty , unsatur abl e s i te , to be d e te rm ined .
The inve stig a tions o f zinc b ind ing to wheat bran , i ts fibre
frac tions and to phyta te provide the an swer to the que stion ; Is i t
f ibre o r phyta te tha t i s the main m e tal bind er? Th i s i n fo rma tion can
be ob ta ined from the r e sul ts of the calculations , shown in Tab l e XXI I ,
o f the max imum amoun t o f zinc binding to wheat bran , i ts var ious fibre
fracti o n s and to phyta te .
Whea t bran , b ran ex e thanol and bran ex oxalate we r e found no t to
1 3 1
bind large amoun ts o f zinc . Their zinc b ind ing capac i ti e s we r e 1 67 ±
1 2 . 7 , 1 42 . 3 ± 4 . 4 and 1 48 . 3 ± 50 . 0 �M/ g r e s pe c tively . The . two
samples , bran and bran ex e thanol , in whi c h the wa ter soluble
c omponen t had no t b een rem oved , a ppear to b i nd zinc more strong ly , a s
i nd ic a ted by the l ow l ig and 5 0 % value s o f 4 3 . 1 ± 7 . 3 and 1 1 . 4 ± 0 . 8
� M/g respective l y . In contr a s t , the bran ex ox ala te sampl e , l ig and
5 0 % value of 264 ± 2 1 1 �M/g , suggest tha t zinc wa s less strong ly
a s soc ia ted than b y the samp l e s in which the wa ter soluble components
had been reta ined . Whi l e the zinc bind ing capac i ti e s o f the se thr e e
s ampl e s are sim i l ar , weaker meta l bind ing i s a ssoc i a ted wi th the
absence of the wa ter soluble frac tio n .
Ce l l ulose ha s been sugge s ted to b e a fac to r i n reduc ing me ta l
b ioava i l ab i l i ty ( Ra nho tr a � a l . 1 97 9 ) . Th e r esul ts from thi s s tud y
i nd ic a te that cel l ulose bound the least zinc at 5 7 . 4 ± 5 . 3 � M/ g . Th e
high l ig and 5 0 % val ue o f 1 1 0 . 2 ± 32 . 4 � M/ g relative to the b i nd ing
c apa c i ty s ugge st tha t the b i nd ing wa s we ak , possibly only s ur fa c e
adsorpti on occurr ing .
The cell wal l prepar a ti o n had a high z i nc b ind ing capac i ty o f
1 0 1 2 . 6 ± 1 9 3 � M/g . The high ligand 50% value o f 7 6 5 . 3 ± 3 2 7 � M/ g a n d
the p o s s i b i l i t y o f mu l t i c omp l ex c omp o s i t i o n , s u g ge s t t h a t wea K l y b o u n d ,
nonsa tur able si te s a r e a s i g n i ficant fea tur e . Consider ing the c e l l
wal l sur face appe a r an c e and wha t appear to b e larg e sur face a r e a , a s
observed in Fig u r e s 1 2C and D , and the mac romolecular struc tur e o f the
wal l s , c ompo sed of l inked po l ym e r s of c e l lulose , hemice llulose and
l ig n in ( Feng e l , 1 97 6 ) , i t would no t be unr easonable to expec t mul tipl e
b ind ing si te s and s i g n i f i c an t nonspec i fi c b i nd ing to occur .
1 32
The wheat bran ar abinoxyl ans ; alka l i sol uble and DMSO solub l e ,
and the l ig nocellulose frac ti on , had d i f ferent zinc b i nd ing
capac i ti e s . The alka li solub l e arabinoxyl an had the l owe s t zinc
b ind ing capac i ty at 2 2 7 . 9 ± 6 1 . 4 �M/ g , l ig nin at 5 1 0 ± 4 1 . 9 � M/ g and
DMSO s o l ub le arabinoxyl an a t 5 08 9 ± 9 2 1 � M/g . The l ow meta l bind ing
capac i ty of the alkali solub l e ar abinoxyl an is consi s te n t wi th the
finding s o f Rend leman and Grobe ( 1 98 2 ) and Mo l loy and Ri chards ( 1 97 1 ) .
The g re a te r zinc bind i ng capa c i ty of the DMSO solub l e po l ymer and the
l ig noce l lu l ose may be the r e s ul t o f a higher phenol ic conte n t .
However , the g reater bind i ng capac i ty wa s n o t observed i n the
l ig noce l lulose . Thi s should no t be taken to impl y tha t a l l l ig n i n s
are r el a tively poor meta l binde r s . The me thod used to pr epare the
frac tio n may have caused d eg r ad a ti on or induc ed chem ical chang e s to
the phe no l ic s . The zinc b ind i ng by all thr ee of the frac tions , were
weak , as i nd icated by the high l i g and 5 0 % value s . Th e b ind ing o f zinc
to the se s ubstanc es may be a r e f l ection of the r e ta ined mac romolecular
s tr uc tur e from wi thi n the plan t c ell wa l l , the linking of the
arab i noxyl ans by phenolics , s uc h a s ferul ic ac id , a s proposed by
Markwa ld e r and Neukom ( 1 97 6 ) , f o rming a c ompl ex three d imensional
s tr uc tur e wi th a large in te r nal and ex te r nal sur face area , wi thi n
whi c h the r e m a y b e mul ti pl e s i te s for meta l en trapme n t . At thi s
j unctur e , the relevance the se po l ymer s may have on m e ta l
b io av ai l ab i l i ty can only be s pe c u l ative . However , i t i s suggested
tha t the se polymers appear no t to hold me ta l s suf f i c ien tly strong ly to
sig n i f i c an tly impede me ta l bioava i l abil i ty .
Th e wa ter soluble c omponen ts from whe a t b r an appe ar to b e the
maj o r m e ta l binding sub stance s . The find i ng s o f Fr � l i c h and As p
( 1 980 ) and of Rend leman and Grobe ( 1 98 2 ) suggest tha t e i ther the
wa ter soluble fibre or phyta te c ould be the pr edom in an t m e ta l bind ing
sub s tance . The r e sul ts from thi s stud y have shown tha t both
sub s tances have a high zinc binding capac i ty . The ' pur i fied '
water solub l e polysaccha r id e bound 4 0 3 8 ± 2 1 6 �M o f zincj g wi th a low
l ig and 5 0 % value o f 9 4 . 6 ± 1 . 1 r e l ative to the bind ing capac i ty .
Ph yta te bound 6 5 82 . 4 ± 1 9 2 � t-1 o f zincjg wi th a l ig and 5 0 % value o f
5 82 . 4 ± 1 0 1 . Ther efore , i t i s conc luded tha t both s ub s tanc e s are
s ig n i f i c an t bind e r s o f zinc , phyta te b ind ing more than the fibre , but
zinc b e ing more s trong ly bound to the wa ter soluble fibr e .
1 3 3
Fo r i n terpr e ta tion o f the zinc bind ing experiments a n ideal i sed
s ys tem ha s been employed . However due to the consider ab l e c ompl exi ty ,
a more d e ta i led interpr e ta ti o n o f the mechan i sm s o f meta l bind ing to
c omponents o f d i e tary f ibre should awa i t fur ther r esearc h .
Gener al Di scus sion
From epi d emiological s tud i e s , it ha s been sugg e sted tha t the
a e te o l og y o f a var ie ty o f d i se ase s such as c arc inoma o f the colon
c ould be related to the qua n ti ty o f non- absorbed pl an t cell wal l
carbohyd rate in the die t ( Burki tt, 1 97 3 ) . The ind ige stible d ie ta r y
c omponents , which as a g ro up are referred to a s ' d ie ta r y fibre' , have
an ex tr emely c ompl ex chemi stry ( ARC/ MRC , 1 97 4 ) . Th i s compl ex i ty has
r es ul te d in fibre be ing chem i c a l l y i ll-de fi ned , thus imped ing the
ver i f i c a tion o r repudia ti o n o f the fibre hypo the s i s .
1 3 4
Po l ysaccha r ides of var ious composi tion , have b een marke ted as
' fi b r e ' and represented as s ui table fo r use in food manufac tur ing o r
a s d ie ta r y s upplemen ts . Ho wever , i t ha s been sug g e s ted tha t all type s
o f fibre may no t be equivalen t ( Trowe l l , 1 97 3 ) . Th i s sug g e s tion i s
s uppor ted b y the finding s i n Par t I o f thi s the si s . It wa s found tha t
o f a l l the spec ie s of pl an t s examined , only from whea t bran wa s the r e
a high propo r ti on o f arab inose and xyl o se conta in ing po l ysacchar id e s
e x tr ac ted wi th wa ter and oxa l a te , whereas the pol ysacc har ides ob ta ined
from f r ui ts and vegetab l e s we r e gener al ly high in pec tic polymer s .
Pr evious s tud ie s have ind icated tha t pec tic po l ymers are diges ted
wi thin the human dig e s ti v e tr ac t befo re the y reach the sta r t o f the
colon ( Hollowa y e t al . 1 97 8 ) . Arabinox ylan s , howe ver , wer e found to
b e r el a tive ly r esi stent to d ig e stion ( Ho l l oway � �· 1 980 ) , thus ,
s uppo r ti ng the h ypo the si s tha t the phys iolog ical in fluence o f d ie ta r y
f ib r e from whea t b ran may b e d i ffe r en t than tha t from frui ts and
v eg e tab les .
Thi s doe s no t impl y tha t the phys ical and chem i c al prope r ti e s o f
pec ti c po l ymers d o no t c on tr ibute to the to ta l prope r ti e s o f ' d ie ta r y
f ibre' , such as slowi ng o r reduc ing in te s tinal abso rptio n o f o ther
n utr ien ts .
Cog n i sance must be taken of the fac t tha t r elatively c rud e
i so l a tion procedur es have been used to ob ta i n plan t c e l l wal l
po lysacchar ides . Thi s has po s s i b l y resul ted i n the pr esence of more
than one population of pol ys accha r id e s be ing present i n a pa r ti c u l ar
sampl e ( e . g . the pr esence o f am yl ase resi stant starch , tha t may have
g iven neg a tive KI- I4 s po t te s t s , par ticular l y in the ho t wa ter and
oxal a te e x tr ac ts ) . The separ ation o f polyd i spe r se m a te r i a l isolated
f rom cell wa l l s can be d i f f i c ul t , par ti cul ar l y if the r e are few
d i s ti ngui shi ng fe atur e s on wh i c h to base a frac tionati ng procedur e ,
a nd the avoidanc e o f phys i c a l or chemi c al mod i f i c a ti o n o f the
polysaccha r id e s .
A pi l o t i nve stiga tion o f hemicellulose frac tionation wa s
under taken us ing a sim i l ar me thod as Maekawa ( 1 97 5 ) for the
f r ac tionati o n o f xylan s on a DEAE-cel lulose column , wi th borate and
a lka l i a s e l utan ts . The r es ul t o f thi s sepa r a tion i s shown fo r the
l igni fied whea t bran hem i c e llulose in append ix A and the
1 35
d el ig n i fied hemic ellulose in append ix B. It wa s observed tha t two
overl apping polysaccha r id e pe aks and thr ee phenolic/pr o te i n pe aks wer e
o b ta ined from the nond e l ig n i fied hemicellulose , wher eas mul ti pl e
pol ysaccha r id e peaks and one phenol ic/prote in peak wa s ob tained after
d e l ig n i f i c a ti on , c onfi rming the po lysacchar ide deg rad a ti on o f
d el ig n i f i c a ti o n . carboh yd r a te anal ys i s by GLC o f the ind ividua l
frac tions showed onl y tha t they we r e arabinoxyl ans ; no o ther
d i stingui shi ng featur es we re d e te c ted .
1 36
'!his me thod o f fr ac tiona tion however , wa s fo und to be
un sati sfac to r y to ob ta in homog enous po l ysaccha r ide frac ti ons from cell
wal l s , to en ab l e fur ther stud ie s to be under taken , due to the l ow
y i e ld s and s l ow f l ow time s ( > 5 2 hour s ) . Please see Par t I Di scuss i o n
and Append ix A and B .
'!he f i b r e ex trac ts ob ta i ned from a l l the foods bound coppe r , i r o n
and zinc , par ti cular l y the wa ter sol ub l e po l ysacchar ides . Thi s
f i nd ing suppor ts the observations made in human and an imal nutr i ti onal
s tud ie s , tha t hig h fibre d ie ts can l e ad to d e fic ienc ie s of i r o n , zinc
( Re inhold � a l . 1 97 3 ) and copper ( Har tmans and Bo sman , 1 97 0 ) .
Ca lcium d e f i c ienc ies have a l so been d e s c r ibed ( Isma i l- Be ig i e t al .
1 977 ) . The mechanism for calcium ' s r educed bioavailab i l i ty i s no t
r ead i l y appar ent from the se exper imen ts , a s calcium wa s d i spl ac ed from
a l l the fibres by coppe r , i ron and zinc . Po s sibly the mechan i sm s
oper ative wi thi n the d i g e s tive tr ac t a r e more c ompl ex than c an b e
observed us ing rathe r simpl e in v i tr o expe r imen tal tec hn i qu e s .
The g re a te r me tal binding capac i ty o f the wa ter so lub l e
polysaccha r id e s , whi c h conta in a hig h uronic ac id conte n t , c ould be
ascribed to the ' egg-box model ' mec han i sm of Rees and hi s c o l l eague s
( Mo r r i s � a l . 1 98 2 ; Thorn e t � · 1 98 2 , Powe l l � al . 1 98 2 and Furd a ,
1 97 9 ) . ca l c ium ions fi tting be twe e n two o r more chains o f
une s te r i fi ed po l ygalac turono s yl resid ue s i n suc h fa shio n tha t the
c alcium ions c he late to the oxyg en a toms o f four galac tur o no s yl
r e sidue s d i s tr ibuted b e tween two g a l ac turonan chain s .
The po s sibil ity of po lysaccha r id e confo rma tional me ta l bind ing
s i te s tha t d o no t involve uronic acid residue s , is suppor ted by the
observa ti o n o f posi tive c ooperativ i ty o f zinc bind ing to the wheat
b ran wa ter soluble pol ysacchar id e . The low uronic ac id conte n t of
thi s po l ys accharide ( l e s s than 1 . 0% ) pr ec lud e s spec i fi c ionic b ind ing
to uronic ac id carboxyl g roups o r intercha in chel ati o n in the manner
proposed for calcium bind ing to pol y-L-guluronate ( Kohn , 1 97 5 ) or
poly-L-g a l ac turonate ( Sm i d s r �d and Haug , 1 97 � ) as the m e ta l bind i ng
m echan i sm .
1 3 7
Othe r mechani sm s o f me ta l bind ing to neutr al po l ysaccha r id e s
wi thin pl an t c e l l wa l l s m a y be quan ti ta tive l y more impo r tan t than to
pec ti n s . The me tal bind ing to hem icel lulose frac ti o n s ( i . e . alka l i
soluble po l ysacchar ide s ) used i n thi s stud y , we re f r e e from calcium
pec ta te . As pecti n s were r emoved in the e x tr ac tion sequence using the
c helating agent ammonium oxalate , as shown in Fi gure 2 and as
e videnc ed by the low uronic ac id conte n t of the alka l i soluble
f r ac tions ( Table I I ) . It woul d have been advan tageous to have
i nvestig a ted the e f fec ts o f o ther chelating agents such as EDTA on
pec tin r em oval and to have used the che l a te solub il i sed
polysaccha r id e s for me ta l bind ing stud ie s . The wa ter soluble
pol ysaccha r id e s we r e used for the me tal bind ing stud y , based on the
observa ti o n of the i r high uro nic acid con ten t and tha t thi s frac tion
had the least possible d eg rad a tive steps appl ied to it. However
ag ainst thi s , it may we l l be j us ti fiably argued tha t the sl ightly
higher me thoxyl con te n t in about hal f the food s o f thi s frac tion ,
would m i ti g a te ag ainst thi s cho ic e . I t would be o f some inter est to
1 38
compare the metal bind ing charac te r i sti c s of the wa ter so lub l e and
oxalate soluble ex trac ts .
The metal bind ing charac te r i stic s o f neutr al sug ar s have been
i nve stigated ( Angyal , 1 980 ) . It ha s been shown tha t reasonab l y s t ro ng
calc ium compl exe s to neutral sugars can occur . The s trong est b i nd ing
occurr ing wi th sug a r s in whi c h the hyd roxyl g roups are in such an
arrang ement tha t the y can form bonds o f e qua l length wi th the c a tio n ,
s uc h a s a n ax ial , e qua torial and axial hyd roxyl g roups o n a
s i x-membered ring ( Ang yal and Mi l l s , 1 97 9 ) . Me thyl a tion of the axial
o r equato r ial hyd roxyl groups r educ e s c omp l ex formation; however when
a me thyl g roup is carr ied by an anomer ic oxyg en atom , as in me th yl
g lyc oside s , the e f fec t on c ompl ex form a ti o n i s less sever e .
P r e sumably because the anom e r ic oxygen a tom c an d raw elec trons from
the r ing oxygen a tom ( Angya l and Mi l l s , 1 97 9 ) . Bi nd ing of o ther
m e ta ls and sugars wa s i nvestigated by Moul i k and Khan ( 1 97 5 ) . Th e y
found tha t reduc ing s ug ar s b i nd more s trong ly than nonreduc ing ones
and tha t the fr ee- energy change i s of the o rd e r o f h yd rogen bond ing .
Moul ick and Khan ( 1 97 5 ) considered the b i nd ing to be elec trosta ti c in
n a tur e whe r e the c arbohyd r ate ac ts as the c en tre o f posi tive charg e s .
Bourne et al . ( 1 97 1 ) found tha t copper ( I I ) complexes were fo rmed
predom inantly to g lyco s ide aldose s and unde r the cond i tions the y used ,
r ed uc i ng sugars ( except D- r ibose , D-xylose and D-gul ose ) did no t .
Ang yal e t al . ( 1 97 4 ) a l so investigated com pl ex fo rma tion o f ald i to l s
wi th m e tal ions i n aqueous solutions b y e l ec trophor e s i s and then b y
NMR. Th e y found tha t c ompl ex fo rmation o f a ld i to l s wi th me ta l ions
occur s at three consec utive oxyg en atoms wh i c h are in a gauche-gauc he
1 39
arr ang emen t . The energy r equi r ed to form thi s arr angemen t , by
rota tion around carbon- carbon bond s , d e te rmin ing the e x te n t of complex
form a ti o n . However for sub s ta n ti a l complex forma tio n to occur ,
concen tr a ti o n s of sug ar s and c a ti ons are higher than us ua l l y found in
l iving organ i sms are required . Mo reover the sug a r s which read i l y form
compl exe s rarely occ ur in Na tur e , tho se whi c h are c ommon d o no t
c omplex we l l . In Ang ya�' ( 1 980 ) view, most o f the wo rk o n c ompl ex
formation wi th cations ha s so far been carr ied out wi th
m ono saccha r id e s , the behaviour o f po lysaccha r ides ha s not ye t been
s ys tem a ti c al l y stud ied . Other po s si b l e mechani sms wh i c h may accoun t
for the h i g h c alcium conte n t ob s erved i n the hemic e l l ul ose frac tions
( I F ) . suc h a s ph ys i c al en tr apm e n t i n the c omplex network o f c e l l
wal l s and / o r by sur face adhe si o n ( Cann ing and Mad ix , 1 98 4 ) may occur .
Fur ther exper imenta tion would be r equ i r ed to confi rm and e s tab l i sh the
d om inan t b ind ing mechanism for c al c i um to the se wa ter in so l ub l e
f rac tions .
The zinc b inding capac i ty o f the whe a t b ran wa ter so luble extr ac t
wa s highe r in the pol ysaccharide f r ac tion tha t had the g reater
proporti o n s of rhamnose , manno se and galac to se . The mechan i sm of zinc
b ind ing to thi s extrac t may involve c onfo rm a tional change s a s ha s b een
observed i n the agg regation o f s o l ub l e ar ab i noxyl an s ( De a e t al . 1 97 3 ;
Bl ake and Ri c hard s , 1 970 ) or agg rega tion o f he terog l yc an s ( De a e t a l .
1 97 7 ) , pos sibly enhanced by the pr e sence o f d ivalen t meta l ions .
A pr e l iminary i nves tig a tion o f the agg r egation o f the wheat bran
water solub l e polysaccharid e , i n the presence o f s od ium and z i nc , wa s
unde r taken ( Appendix C) . Th i s c on f irmed tha t agg reg a ti o n occur r ed
only in the pr esenc e o f the divalent metal ions .
1 40
Fur ther inve stig a ti on s o f the mechan i sm s o f d ivalent meta l
m ed ia ted agg regation o f l ow uronic ac id po lysaccha r id e s could be
unde r ta ken us ing a var ie ty of me thod s suc h a s i c ircular dichr om i sm
( Mo rr i s e t al . 1 97 3 ; Mo r r i s e t al . 1 980 ) , d e te rmination o f c r i ti c a l
chain leng th ( Kahn , 1 97 5 ) , selec tive removal o f var ious sug ar r e s id u e s
s uch as rhamnose , in fluenc e of fe rulic ac id ( Ma rkwa ld er and Ne ukom ,
1 97 6 ) , e f fe c t o f leng th and d i str ibution o f a r abinose side cha in s .
Investigati ons o f the meta l bind ing prope r ti e s o f var ious f i b r e
ex trac ts from food s , r e po r ted in thi s the si s , h a s tend ed to empha s i se
the negative aspec ts o f high fibre d i e ts . However , stud i e s o f the
f i b re ex tr ac ts ind i c a te tha t alka l i ex trac ted hemicellul ose from wheat
b ran may b e a phys i o l og ically ' ac tive ' sub s tance tha t c an r ed uc e
i n te stinal tr ansi t time s ( Hollowa y and Gr e ig , 1 984 ) .
At thi s j unc tur e i t i s not read i l y appa r e n t what contr ibution
the se in v i tro me ta l bind i ng stud ie s , using i solated fibre componen ts ,
wi l l have towards the d evelopment o f ' new f i b r e ' food produc ts in
whi c h the impact of r educ ed bioava i l ab i l i t y of copper , i ron and zinc
a r e m i n im i sed . For f u r t h e r di scu s s i on p l e a s e s e e append i x D .
Conc l usions
1 . Wa ter solub l e fibres ex tr ac ted from bean , cabbag e , kum er a ,
l e ttuc e , onion , pe ach, pe ar , pumpki n , tom a to , whe a t b r an , whi te
c l over , luc erne and ryeg rass contai ned signifi c an tl y more
g alac to se , uronic acid and m e thoxyl g roups than alka l i soluble
fibre s , which conta ined sig n i fi cantly more xyl ose .
2 . Apprec i ab l e yi elds of alkal i sol ub l e fibres c an be ob tained from
food s and g rasse s wi thout pr ior d e l ig ni fi c atio n .
3 . Ch l or i te d e l ignific ati o n and solub i l i sa tion in
N-me thyl-morpholine-N- ox id e r e s ul ts i n ex tensive d eg rad a ti o n o f
a rab inox y l an s .
4 . LOw y i e l d s o f whe a t b r an hem i c e l lulose ar e ob ta ined on ex tr ac tion
wi th d im e thylsulphoxide .
5 . Dur ing ex tr ac tion o f plan t ti s s ue a compl ex tr an si tion o f
m orphol og i c al chang e s , up to the add i tion of a lkal i , l e ft c e l l
wal l s i n tac t . Co ntac t o f pl an t ti s sue wi th alka l i r e s ul ted in
c ompl e te d i sinte g r a tion of c e l l wal l s .
6 . Wa ter solub l e fib r e s from al l the food s bound more zinc than the
wa ter i nsoluble fibre s . Co ppe r b ind ing al so predom i n an tl y
occur r ed on the wa ter solub l e fibres except i n the c ase o f
l e ttuc e , tomato and whe a t b r an . The bind ing occurred wi th a
d i spl acemen t of cal cium , mag ne s i um and m ang ane se .
7 . Phyta te , a wa ter solub l e po lysaccharid e and a DMSO solub l e
arab i noxyl an a r e the maj or zi nc b ind ing componen ts o f whea t b ran .
8 . Zi nc i s b ound more strong ly , b y a mecha n i sm o f po s i tive
c oope r a ti v i t y , to the wa ter soluble pol ysaccha r id e than to the
DMSO s o l ub l e ar ab inoxyl an or to phyta te .
1 4 1
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1 58
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Chrom . 1 1 6 : 29-4 1 •
1 00
Q) '0 ..-i >< 0 ""
'0 ..c:: E ::l
·.-i '0 0
en � ...
0
Q) .j.) (lj � 0
r:Q � 0
.
S UGAR Phenol/Sulphur i c Abs . 4 8 0nm
APPENDIX A
9 0
80
7 0
60
50
40
30
2 0
1 0
0 . 4 0 . 3 0 . 2 0 . 4
P HENOL IC Abs . 320nm
0 . 3 0 . 2
PROTEIN Abs . 28 0nm
FRACTI ON COLLECTION OF WHEAT BRAN HEMICELLULOSE ( NOT DELIGN I F I ED ) AFTER PAS S I NG THROUGH DEAE-C ELLULOSE COLUMN
9 0
8 0
7 0
6 0
5 0
40
3 0
2 0
1 0
0
1 6 7 . .
� Q)
.0 E ::l
z r:: 0
..-i .j.) () (lj �
�
....
0
Cl) � «l S.. 0
a:l ::£ 0
. ....
1 . 5 1 • 0 o . s
SUGAR
Phenol/Sulphur i c Abs . 4 8 0 nrn
APPENDIX B
90
80
70
60
so
40
30
20
1 0
0 . 4
PHENOLI C Abs . 3 2 0 nm
0 . 3
PROTEIN Abs . 2 8 0nm
FRACTION COLLECTION OF WHEAT BRAN HEMl CELLULOS E ( AFTER D EL IGN I FICATION ) PASS ED THROUGH DEAE-CELLULOSE COLUMN
1 6 8
1 00
9 0
8 0
7 0
6 0
s o � Cl)
,!) E ;::l
z 40 c:
0 ·� � <.> «l s..
r.. 3 0
2 0
1 0
0 0 . 2
Append ix C
Investig ation of the in fluenc e of sodium and zinc ions on the
aggregati on o f the wa ter soluble polysacchar ide from wheat bran .
1 6 9
The possibil ity o f me tal induced prec ipi ta tion o f the wa ter
soluble wheat bran polysaccha r ide wa s inve stigated using a 5% solution
of the ammonium sulpha te pr epared polysaccharide as described in Par t
v . one sample of thi s so lution ( 2 . 5 ml ) was added to a solution o f
pH6 . 2 phospha te buffer ( as used i n Par t I I I ) made from 0 . 0 1 2 ionic
streng th sod ium chlorid e ( 0 . 702g/ 1 00 ml) . A s im i l ar sample was a l so
added to a pH6 . 2 phospha te buffer solution made from 0 . 01 2 ionic
s treng th zinc chloride solution ( 0 . 555g/1 00 m l ) .
The solutions were mixed for 1 0 minute s , then allowed to stand for
o ne hour . The pH wa s determ ined and found to have remained constan t .
A whi te fluf fy prec ipi ta te wa s observed i n the zinc sol ution . No
prec ipi ta te wa s observed in the sodium chloride solution.
Th i s brief supplementary stud y would appear to support the
suggestion proposed in Par t V , tha t divalent metal mediation o f
polysaccharide agg regation is a possible mechan ism b y which zinc and
the wheat bran wa ter soluble polysaccharide assoc iate .
The possibility of o ther d ivalent me ta l spec ie s bind ing by a
similar mechan ism is o nly speculative at thi s j unc tur e . I t i s
probable however , tha t magnesium , calcium , manganese , iron and copper
may a l so bind to thi s polysacchar ide . The consequence of reduced
b ioavail ability has , a s ye t, to be investigated .
1 7 0
APPENDI X D
Current a n d Fu t u re Trends i n the F i e l d o f D i e t a ry F i bre Re s e a r c h .
1 . ( a ) As i n d i c a t e d in Part I and I V o f t h i s t h e s i s , t he uncont e n t i o u s
theory a n d expe r imen t a l e v i dence kn own in 1 98 2 c a n b e c l a s s i f i e d
i n t o t h e f o l l owing :
( i ) Re c o gn i t i on o f the p o s s i b l e advan t a g e s o f h i gh f ib r e
d i e t s .
( i i ) Re c o gn i t i on o f the c omp l e x n a t u re o f d i e t a ry f i b r e .
( i i i ) Re c o gn i t i on o f the d i f f i c u l t i e s i n e x i s t i ng m e t h o d s o f
c h em i c a l ana l y s i s o f d i e t a r y f i b r e .
( i v ) Re c o gn i t i on o f the p o s s i b l e c o n t ra- i n d ica t i on s o f h i gh
f i b r e d i e t s .
( b ) Ma t t e r s wh i c h we re r e g a r d e d a s p r o b l ema t i c o r y e t t o b e
s o lved a t t h a t t ime we re :
( i ) I den t i f i c a t i on o f phys i o l o g i c a l l y a c t ive c ompon e n t s
o f d i e t a ry f ib r e .
( i i ) Re s o l u t i on o f the c o n f l i c t o f t h e who l e c e l l wa l l
f oo d c o n c e p t v e r s e s the p o l yme r i c c omp onen t s o f c e l l
wa l l s a s d i e t a ry f ib r e .
( i i i ) Deve l opment o f me t h o d s o f a na l y s i s tha t a c c u ra t e l y re f l e c t s
t h e d i e t a ry f i b r e c on t e n t o f f o o d s .
( iv ) De t e rm i n a t i on o f the s i gni f i c anc e o f me ta l b i n d i n g t o
d i e t a ry f ibre a n d i t s c omp a r i s on w i t h o t h e r f o o d c omp on e n t s
s u c h a s phy t a t � in re duc i n g me t a l b i o ava i l ab i l i t y .
2 . Deve l opme n t i n the f i e l d o f d i e t a r y f i b r e re s e a r c h f r om 1 9 8 2
t o 1 9 8 5 r e l a t i n g t o p l an t c e l l wa l l c ompo s i t i on a n d f i bre an a l y s i s .
( i ) The i s o l a t i on o f p l a n t c e l l wa l l c omponen t s rep o r t e d
i n P a r t I o f t h i s t he s i s c a n b e c omp a r e d w i t h t h a t r e po r t e d
b y S t e v e n s a n d S e lvendran ( 1 9 8 4a a n d b ) . The s e a u t h o r s
u s e d s im i l a r methods o f e x t r a c t i on ( h o t wa t e r , ammo n i um
o xa l a t e a n d a l ka l i ) t o obt a i n c e l l wa l l p o l yme r s f r om
c a r r o t a n d c abbage and a l s o u s e d s im i l a r m e t h o d s o f a na l y s i s
a s r e p o r t e d i n t h i s t h e s i s . Bo t h s t u d i e s ( S t e v e n s a n d
S e l ve n d r a n 1 98 4a and re su l t s shown in t ab l e I I , p . 2 2 )
1 7 1
show tha t cabbage pec t i n i s c omp o s e d p r e dom i nan t l y o f
a r a b i no s e a n d u r on i c a c i d . S t e vens a n d Se l ve n d r an ( 1 984a )
howe v e r deve l op e d t h e i r s t udy f u r t h e r by do i n g a p a r t i a l a c i d
hyd r o l y s i s and m e t h y l a t i on t o show t h a t the ma j o r p ec t i c
p o l y s a c c h a r i d e s h a d rhamn oga l a c t u ronan backb one t o wh i c h a h i gh l y
b r a n c h e d a r a b i nan wa s l i nke d . S im l a r l y the e x t ra c t i on a n d
c omp o s i t i on o f wh e a t b r a n ' f i b r e s ' r e p o r t e d i n th i s the s e s a r e
supp o r t e d b y the r e s u l t s o f the s t udy o n c e r e a l s o b t a i n e d f r om
d i f f e re n t e x t ra c t i on r a t e s ( Nyman e t a l , 1 9 84 ) .
Wh i l e the s e s t u d i e s and o t h e r s ( re v i ew e d by S e l vend ran 1 9 8 4 a n d
McNe i l e t a l , 1 98 4 ) i n d i c a t e c omp o s i t i on a l d i f f e r ence s a n d
s im i l a r i t i e s o f e xt r a c t a b l e c e l l wa l l p o l yme r s a n d h a ve b e e n
u s e d t o p o s t u l a t e c e l l wa l l mode l s ( Ke e g s t r a e t a l , 1 9 7 3 ; Mon r o
e t a l , 1 9 7 6 ) . The i r va l u e i n d e f i n i n g d i e t a r y f ib r e i n i n t a c t
c e l l wa l l s rema i n s p r ob l ema t i ca l . T h e appr o a c h t h a t i s
r e q u i �e d t o be u s e d i s t o inve s t i ga t e c e l l wa l l c ompon e n t s
' in s i t u ' thus a v o i d ing the p o s s i b i l i t y o f d e g r a da t i o n . To e n a b l e
s u c h i nve s t i ga t i o n s t o b e u n d e r t aken , n e w t e chn i q u e s a r e
r e qu i r e d . One s t rong po s s i b i l i t y i s f o r the deve l opmen t a n d
app l i c a t i on o f s o l i d s t a t e NMR t o ' who l e ' p l a n t c e l l wa l l s .
Such an approach wou l d enab l e i n t a c t c e l l wa l l s t o be c omp a r e d
w i t h t h e i r ' i s o l a t e d ' c omponen t s .
( i i ) The deve l opme n t o f m e t h o d s o f ana l y s i s h a s b e en one o f t h e mo s t
a c t i v e a r e a s o f r e s e a rch on d i e t a r y f i b r e a n d i s p o s s i b l y t h e
o n l y a r e a o f s i gn i f i c an t a dvance i n r e c en t t ime s . The r e h a s
b e e n a genera l a d op t i on o f s e quent i a l e n zyma t i c e x t rac t i on o f
f i b r e ( Asp e t a l , 1 9 83 ) , wh i ch h a s b e e n s t an d a r d i s e d i n a n
I n t e rn a t i on a l i n t e r l abora t o r y s t udy ( Pr o s ky e t a l , 1 98 4 ) . Th i s
p r o c e du r e a l s o r emov e s s t a r c h e n zyma t i c l y , h oweve r a h e a t s t ab l e
amy l a s e ( Te rmamy l ) i n c omb i n a t i on w i t h t h e cC - amy l a s e a c t i v i t y
o f p anc r e a t i n app e a r s t o remove a lmo s t a l l o f the s t a r c h i n t h e
f i b r e r e s i d u e s ( Asp e t a l , 1 9 83 ) . Th e app l i c a t i on o f t h e s e
e n z yme s i n the e x p e r imen t a l p r o c e du r e s d e s c r i b e d i n P a r t I
wou l d appear t o have been adva n t a ge ou s , p a r t i c u l a r l y f o r t h e
h i gh s t a rch c o n t a i n i ng f o o d s such a s swe e t p o t a t o . The
1 7 2
po s s i b i l i t y o f mo re e f f i c i ent remova l o f t h e l i p i d c on t e n t
i n h i gh l i p i d c on t a i n i n g f o o d s , w i t h a s o l ve n t such a s
p e t r o l e um e t h e r ( Asp e t a l , 1 983 ) , i n s t e ad o f r e f l ux i n g i n
e t hano l , ma y have a vo i d e d a p o s s i b i l i t y o f amy l a s e re s i s t a n t
l i p i d- s t a rc h c omp l e x e s be i ng f o rme d ( Hanna a n d Le l i ev r e , 1 9 7 5 ) .
D i e t a ry f i b r e c omponent s e x t rac t e d by the p r oc e du r e s d e s c r i b e d
in Pa rt I a n d by the u s e o f p e p s i n a n d panc re a t i n ( A s p e t a l ,
1 9 8 4 ) we re found t o con t a i n a r e s i d u a l p ro t e i n c on t e n t . Thu s
s uppo rt ing t h e sugge s t i on t h a t s ome p r o t e in c ou l d be
c on s i de r e d as a c omp onent of d i e t a r y f i bre ( D i n t z i s , 1 9 8 2 ) .
3 . I n my a s s e ssment t h e s i gn i f i c a n t pub l i c a t i on s on me t a l b in d i ng t o
d i e t a ry f i bre , tha t have b e c ome a va i l ab l e s i n c e th i s t he s i s wa s
wr i t t en a t the end o f 1 9 8 4 , a re a s fo l l ows . O ' De l l ' s 1 9 84 r e v i e w on
the b i oava i l a b i l i t y of t ra c e e l eme nt s , c on s i d e r e d t h a t the
c omp l e x i t i e s of the i n t e r a c t i o n s of t ra c e m i n e ra l s b i o a va i l a b i l i t y
makes t h i s t o p i c a ' re s e a rch f ron t i e r f o r t h e inde f i n i t e f u t u r e ' .
Among t h e a r e a s o f c o n c e r n t h a t he d i s c u s s e d wa s t h e e f f e c t s o f
p h y t a t e , f i bre and o f o xa l i c a c i d o n z i nc b i o ava i l ab i l i t y . But h e
l e f t open the que s t i o n o f the re l a t ive impo r t ance o f the s e
sub s t an c e s o n z inc a b s o rp t i on .
The s i gn i f i c an c e o f whe a t bran on z i nc a b s o rp t i o n i n the human d i e t
wa s inve s t i ga t e d b y F a r a h e t a l ( 1 98 4 ) u s ing z i nc t o l e rance t e s t s a n d
who l e b o dy mon i t o r ing t o m e a s u r e 7 - d a y p e r c e n t a ge r e t e n t i on o f z i n c
i n huma n s . The y conc l ud e d t h a t whe a t bran l e a d s t o a s i gn i f i c a n t
r e duc t i o n in z i nc ab s o rp t i on wh i c h c ou l d e v e n t u a l l y i n duce a s t a t e o f
z i n c de f i c i ency . Wha t t h e y d i d n o t d e t e rm i n e howe v e r wa s the i den t i t y
o f the c ompon e n t o f whe a t b r a n t h a t r e d u c e d z i nc r e t e n t i on .
I t h a s b e e n sugge s t e d t h a t r e duc i ng the p h y t a t e c o n t e n t o f b r a n by
l e avening o f bre a d , i n c r e a s e s z i nc a b s o rp t i on f rom such bre a d s
( Nave r t e t a l , 1 9 85 ) . However i t wa s n o t s h own t h a t a l l or mo s t o f t h e
z i n c i n b r e a d b e c ome s ava i l ab l e a f t e r the phy t i c a c i d c on t e n t i s
r e d u c e d . F o r a s was s h own by the r e s u l t s d e s c r i b e d i n P a r t V o f t h i s
t he s i s , b o th phy t a t e a n d the wa t e r s o l u b l e f i b r e f r om b r a n a r e
s i gn i f i c a n t b i n de r s o f z i n c . N a v e r t e t a l , ( 1 9 8 5 ) d i d n o t d e t e rm i n e
t h e wa t e r s o l u b l e f i b r e c on t e n t o f t h e b re a d s , t h u s t h e y wou l d n o t
h a v e b e e n a b l e t o a s s e s s t he s i g n i f i c a n c e o f t h e f i b r e c o n t e n t o n
z i nc b i oav a i l a b i l i t y .
Th e imp o r t an c e o f b r a n s i n r e d uc i n g m i ne r a l a b s o r p t i on i s a l s o
c o n f i rme d by t h e s t udy o f D i n t z i s e t a l ( 1 98 5 ) . The y i nv e s t i ga t e d
t h e i n f l u e n c e o f b r a n s o n t he e xc r e t i o n o f c o p p e r , i r on , z i n c a n d
c a l c i um . Th i s wa s d o n e b y de t e rm i n i n g t h e m i n e r a l c on t e n t s o f
c o r n b ran , wh e a t b r a n and s o y a b e a n h u l l s b e f o r e and a f t e r p a s s a g e
t h r o u gh t h e human ga s t r o i n t e s t i n a l t r a c t . They f o und t h a t c op p e r ,
i r o n a n d z i n c i n c r e a s e d f r om s t a r t i n g l eve l s b y f a c t o r s f r om t w o
t o f o u r a n d c a l c i um l e ve l s inc r e a s e d t e n f o l d . Th e y howeve r d i d
n o t d e t e rm i n e c h a n g e s i n magne s i um , manga n e s e , s o d i um o r po t a s s i um
l e ve l s . The i n c r e a s e in c a l c i um e x c r e t i on wa s n o t a s s e s s e d i n t e rm s
o f a p o s s i b l e i n c r e a s e d r e l e a s e o f c a l c i um b y t h e d i ge s t i v e t r a c t
s e c r e t i on s .
Th e b i n d i n g o f m i ne r a l s t o f i b r e i s s u p p o r t e d b y t h e f i n d i n g s o f
Wh i t e h e a d e t a l ( 1 98 5 ) . Th i s group i nve s t i ga t e d t h e d i s t r i b u t i o n o f
t e n nu t r i e n t e l em e n t s i n h e rbage . The c e l l wa l l f r a c t i on wa s
p r e p a r e d by m e c h a n i c a l d i s i n t e g r a t i o n o f und r i e d ma t e r i a l a nd t h e
m i n e r a l c o n t e n t s o f c e l l wa l l s w e r e d e t e rm i n e d . T h e y f o und t h a t
c a l c i um c omp r i s e d f r om 1 1 . 3 t o 5 1 . 8% o f t h e t o t a l m i ne ra l c o n t e n t ,
magne s i um 6 . 4 t o 2 7 . 6% , p o t a s s i um 0 . 1 to 1 . 7% and ' s ub s t a n t i a l '
t h o ugh mo r e v a r i a b l e p r o p o r t i o n s o f t h e t r a c e e l eme n t s i r on ,
mangane s e , z i n c a n d c o p p e r we r e a l s o p r e s e n t in t h e c e l l wa l l
f r a c t i on s . The e x t e n t o f wa t e r a n d 80% e t h a n o l s o l u b i l i t y o n t h e
m i n e r a l c o n t e n t o f t he c e l l wa l l s wa s de t e rm i ne d . Th e i r r e s u l t s
s howe d t h a t n o t a l l o f t h e c a l c i um a n d t o a l e s s e r e x t e n t magn e s i um
p r e s e n t i n c e l l wa l l s we r e r e a d i l y e x t r a c t a b l e in wa t e r o r e t h a no l .
Th i s wou l d s u p p o r t t h e f i nd i n g s d e s c r i b e d i n Pa r t I V t h a t c a l c i um i s
a l s o bound t o i n s o l ub l e p 6 l yme r s s u c h a s t h e hemi c e l � u l o s e s . Th e
l oc a t i o n o f t h e i n s o l u b l e m i n e ra l s w a s h owe v e r n o t d e t e rm i n e d b y
Wh i t e h e a d e t a l ( 1 9 8 5 ) . A l so t h e y c o n s i d e r e d t h a t wa t e r - s o l u b i l i t y e qu a t e s w i t h b i o a va i l a b i l i t y . Howe v e r t h e p o s s i b i l i t y o f
non-a b s o r b a b l e c h � l a L i • • g s u b s l a n c e s s u c h a s o x a l a t e , ph y t a t e a n d
' nond i ge s t a b l e ' wa t e r s o l u b l e po l y s a c c h a r i de we r e n o t c on s i d e r e d .
1 n
1 7 4
The b i n d i ng o f m i n e ra l s wa s howeve r c on s i de r e d by Lee a n d
G a rc i a-Lope z ( 1 98 5 ) . They f oun d t h a t i ron , c o p p e r a n d z i nc but n o t
magne s i um w e r e bound by ne u t r a l ( NDF ) a n d a c i d ( ADF ) d e t e r gen t f i b r e
o b t a i n e d f r om P i n t o b e an s . They rep o r t e d t h a t i ron b i n d i n g i n c r e a s e d
w i t h i n c r e a s ing ph , t h i s p r obab l y r e f l e c t s t h e f orma t i on o f t h e i n s o l ub l e
f e r r i c hydrox i d e wh i c h wou l d d e c r e a s e t h e a p p a r e n t ' f r e e ' i r on c on t e n t .
W i t h t h a t r e s e rva t i on t h e i r re s u l t s wou l d s up p o r t my f i n d i ngs o f
c opp e r , i ron and z i nc be ing the m i n e ra l s p r e domina t e l y bound b y f i b r e .
4 . I n c o nc l u s i on , one o f the ma i n d i re c t i on s o f r e c e n t r e s e a rc h i n t h e
f i e l d o f d i e t a r y f i b r e h a s b e e n t owa r d s l oo k i n g in g re a t e r d e t a i l a t
the c ompo s i t i on o f p l a n t c e l l wa l l s . Howeve r , a s y e t i t h a s n o t b e e n
s hown t h a t a n y new o r p o t en t i a l l y unusua l c h em i c a l c on s t i t u e n t o f c e l l
wa l l s o r i t s c omp one n t s c o r re l a t e w i t h t h e phy s i o l og i c a l p ro p e r t i e s o f
d i e t a r y f i bre . Wh i l e i t may be a rgue d t h a t m o r e d e t a i l e d s t ud i e s o f
c e l l wa l l s even t u a l l y l e a d t o a grea t e r u n d e r s t a nd i ng o f d i e t a ry f i b r e ,
t h i s a p p r o a c h i s l im i t e d b e c a u s e the e f f e c t s o f ' d i e t a ry ' f i b r e a r e
phy s i o l og i c a l . The e xpe r imen t a l de t e rm i n a t i on o f t h e phys i o l og i c a l /
p h a rma c o l og i c a l a s p e c t s o f s p e c i f i c c omp o n e n t s o f p l an t f o o d s t h a t a r e
r e s i s t a n t t o d i ge s t i on w i t h in t h e human sma l l b owe l i s a n a s p e c t o f
f ibre r e s e a r c h t h a t h a s b e e n e s s e n t i a l l y i gn o r e d . I t i s t h i s a s p e c t
t h a t f u t u r e r e s e a rch w i l l h ave t o b e o r i e n t a t e d t owa r d s t o e n ab l e t h e
e xpe r iment a l sub s t an t i a t i on o f t h e d i e t a ry f i b r e hyp o t h e s i s .
The w i d e r i n t e r d i s c ip l i n a ry approach t h a t I have t aken , a n d a s
d o c ume n t e d w i t h in th i s t h e s i s , h a s been a n e n d e avour t o c ro s s t h e
i mpe d iment o f t r a d i t i ona l a c a demi c bounda r i e s a n d t o i s o l a t e s p e c i f i c
' de f i ne d ' c omp one n t s o f p l a n t s and by unde r t a k i ng ' i nv i t ro ' e xp e r imen t s
t o de t e rm i n e s ome o f the i r p ro p e r t i e s s u c h a s me t a l b i nd ing , t h a t w i l l
f o rm a b a s i s f o r f u t u r e ' in v i vo ' s t u d i e s . For a t t h e p r e s e n t t ime
we do n o t know wh i c h s pe c i f i c c ompon e n t s a r e r e spon s i b l e f o r t h e
d e s i ra b l e phy s i o l og i c a l a c t i on s such a s f a e c a l b u l k ing , r e t e n t i on o f
wa t e r i n t h e s t oo l , b i n d ing o f b i l e s a l t s a n d a l t e r a t i o n o f i n t e s t i n a l
f l o ra wh i c h a r e a t t r i b u t e d t o d i e t a r y f i b r e . Unt i l t h e s e c omp o n e n t s
a re i d e n t i f i e d , the d i e t a ry f i b r e hyp o t h e s i s c an not b e ve r i f i e d .
APPEND I X E
The n on l i ne a r cu rve f i t t i ng c ompu t e r prog ram u s e d t o c a l c u l a t e
b i n d i n g c a pac i t i e s (pM/ g ) , l i gand 50% v a l u e s ( p o i n t a t wh i c h ha l f
ma x imum b i nd i ng o c c u r s i n pM / g a n d the H i l l c oe f f i c i e n t s f o r t h e
b i n d i n g c u rve s we re c a r r i e d o u t u s ing t h e same compu t e r p r ogram
as d ev e l op e d by Hardman ( 1 98 3 ) . Th i s p r o c e dure is a d eve l opme n t
1 7 5
o f t h e Da n i e l and Wood ' s ( 1 9 8 0 ) me thod that wa s ba s e d on t h e Ma rqua r d t
max imum n e ighbou rhood me thod o f n on l i n e a r c u rve f i t t i ng p rogram
( Ma r qu a r d t , 1 9 63 ) .
To a c c e s s t h e genera l purpo s e s program , o b s e rva t i o n s were
e n t e r e d f r om t h e keyboard o f a Cromemc o ( 6 4K RAM ) t o s e t up d a t a
f i l e s . Z i nc b i nd i ng d a t a , i n t h e form o f t o t a l z i nc ( bound z inc p l u s
f r e e z i n c expre s s e d i n pM Zn / g o f f i bre ) and f r e e z i nc ( t o t a l z i nc
m i n u s b o u n d z in c i n �M / g of f i b r e ) we r e e n t e r e d . The mod e l to be u s e d
f o r t h e ' f i t ' h a d t o b e supp l i e d by t h e u s e r and l i nke d w i t h t h e ma i n
program a n d o t h e r subrou t in e s t o p roduce a c ommand f i l e . The s imp l e s t
mo d e l o f one i n t e rac t ing s i t e wa s a lway s u s e d i n i t i a l l y and t he n t h e
m o r e c omp l e x t wo i n t e r a c t ing s i t e mode l wa s inve s t i ga t e d . I n t h e c a s e
o f z i n c b i nd in g t o bran ex oxa l a t e , c e l lwa l l s , l i g n o c e l l u l o s e and
DMSO h e m i c e l l u l o s e , the two s i t e i n t e r a c t in g mo de l gave l owe r r e s i dua l
va l ue s . Thu s i n d i c a t ing the t wo s i t e mo d e l gave a ' c l o s e r ' f i t t o
t h e e x p e r i men t a l d a t a . The u s e r h a d t o s p e c i f y f o r e a c h r u n t h e
numb e r o f c oe f f i c i en t s i n t h e mo d e l and the n umber o f i n de p e nden t
va r i ab l e s . S t a r t ing gue s s e s w e r e r e qu i re d s i nce t h i s i s an i t e r a t i ve
f i t t i n g me thod . The i n i t i a l e s t i ma t e s d i d n o t have t o be ac cura t e a s
the Ma r q u a r d t p r o c e dure i s s u f f i c i e n t l y robu s t t o o b t a i n t h e ' c o r r e c t '
va l ue . Howeve r p r oc e s s t ime wa s s aved i f c l o s e e s t i ma t e s o f t h e
max imum b in d i n g w e r e e n t e re d . T h e ma i n program ha n d l e d t h e i np u t
c on t r o l p a rame t e r s , in i t i a l e s t ima t e s and d a t a . A subrou t i ne ( GAUS H S )
c o n t ro l l e d the c u rve f i t t ing p r o c e dure by c a l l i ng o t he r s ubrou t i n e s
to c a l c u l a t e e i genva l u e s , c a r r y o u t ma t r ix i n ve r s i on a n d t hen t o
o u t p u t t h e r e s u l t s o f the ' f i t ' . The H i l l c o e f f i c i e n t s a n d b i n d i ng
c a p a c i t i e s we re then checke d b y manua l l y p l o t t ing a n d c a l cu l a t i n g
u s i n g t h e p r o c e d u r e s d e s c r i b e d i n Dah l qu i s t ( 1 9 7 8 ) .
Cop i e s o f t h e c ompu t e r p rogram on d i s k , a r e ava i l a b l e f rom
Ha rdman ( 1 9 8 3 ) and of Wood ' s ve r s i on f r om s e ve ra l c ompu t e r l i b r a r i e s
a s l i s t e d i n D a n i e l and Wo od ( 1 980 ) .
A c opy o f t h e o u t pu t o f z i n c b i n d i n g t o wh e a t bran p u r i f i e d
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