the permission of the Author.
EXTRACTION, COMPOSITION
AND S OME OF THE
PHY S IC AL AND CHEMICAL PROPERTIES
OF
COMPONENTS OF D I ETARY F I BRE
by
War r en Oe smond Ho l l owa y
A the s i s pr e sented in par ti a l ful f i lment o f the r equi r ements for the d eg r e e 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 oub l ed i f one confe s s e s to not knowi ng wha t one does n o t know . Wha t one knows i s then r a i sed beyond the suspi c i o n to which i t i s expo s ed when one c la im s to know wha t one d o e s n o t know . "
Schopen hauer
iii
ACKNOWLEDGEMENTS
I wi s h to acknowledge the fo l l owing people and organ i sations that have a s s i s ted in the preparation of thi s the s i s . The chi e f s upervi sor Profe s so r E . L . Ri chard s a nd the second s upe rvi sor Or J. Le l i ev r e and the ac ting supervi sor or M . Ta yl o r , a l l f rom the De pa r tm e n t of Food Te chno l ogy ; a l so Appl ied Bi ochemi s try Di vi sion , Depa r tme n t of
Sc i e n ti f i c and Indu s tr ia l 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 pro
j
e c t to be und e r take n .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 tur e and Fi she r i e s , Lev i n , and Appl ied Bi ochemi stry Divi s i o n for suppl ying s ampl 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 , pa r ticul ar l y 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 s i o n Spec trome ter f o r mul ti pl e me ta l analys i s ; Mr o . Hopc ro f t and Mr R . Be nnett o f the e lec tron micro s c opy unit and Mr P . C . van Oi ng e n en o f the wo rkshop .He lp f ul s ug g e s tions a nd contributions we r e r ec e 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 a cc e s s to a c ompute r i sed nonlinear c urve- f i tting procedur e , 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 omete r .
Th anks to Mr G . C . Ar nold of the Mathema ti c s Depa r tment for d i s c u s s i ons on the i n te rpr e tation of the s ta ti s tical pr ocedures used i n s tud yi ng the r e l a tionship betwe e n fibre c om po s i tion and m e ta l bind i ng .
I am a l s o i nd eb ted to the s ta f f a nd s tud ents of the Fa cu l ty of Technol ogy for advice , comme n ts and referenc e s , pa r ti c u l a r l y 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 ti ng to locate chem i c a l s and equ i pm e n t and Mr Alg er for manufactur ing a cutting tool tha t e n ab 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 runn i ng the g raphi c s prog ram , to m y wi f e fo r typing the d r a f t s , a nd to Mi s s D . J . Ro sva l l for typing the
final sc ript of thi s the s i s .
T A B L E 0 F C 0 N T E N T S
ACKNOWL EDGEMENTS TABLE OF CONTENTS
L I S T OF FIGU RES AND TABL ES ABSTRACT
INTRODUCTION
P URPOS E OF •rHI S STUDY P ART I
I s o l ation and De te rm i n a tion of the Chem i c al Compo s i tion of some o f the Fi bre Components o f Fr u i ts , Vege tab l e s , Pastur e Grasses and Whe a t Br an
I n troduc tion
Ma te r i a l s and Me thod s Re sul ts
D i scus s i o n P ART I I
Stud y o f the vi s c o s i ty o f Whe a t Bran Hem i c e l luloses
In trod uc tion
Ma te r ia l s and Me thods Re sul ts
Di s c u s s i o n
-, -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 og i cal Chang e s i n Whea t Bran a s a resul t o f po lymer extraction
In troduc tion
Ma te r ials and Me thod s
Re sul ts Di scuss ion
P ART IV
Me tal Ad sorption to Wa te r So l ub l e and I n so l uble Fi b r e s a nd from Fr ui ts , Vege table s , Br an and G r a s s e s
In trod uc tion
Ma te r ials and Me thod s Re s u l ts
Di s c u s s i o n P ART V
Zinc Ad so rption to Whe a t Br an , i ts Fib r e Componen ts and t o Ph yta te
I n tr oduc tion
Ma te r ia l s and Me thod s Re s ul ts
Di scussion
G ENERAL DISCUSS ION C ONCLUS IONS
REFERENCES APPENDICES
4 5 49
5 2 6 4
6 7 69 7 4 8 8
9 9 1 04 1 09 1 2 3 1 3 4 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
Fi g u r e 1 :
F i g u r e 2 : F i g u r e 3 :
Tab l e I :
Tab l e I I :
Tab l e I I I :
Fi g u r e 4 :
F i g u r e 5 :
F i g u r e 6 :
Tab l e IV :
Tab l e V :
F i g ur e 7 :
F i g ur e 8 : F i g u r e 9 A to D :
Flow diag r am o f the five stag e s o f the i nvestig a ti o n s i n thi s the s i s .
Seque nce o f extrac tion of fibre c omponen ts . Separ ati o n o f s ug ar s through a colum n packed wi th 3% ECNS S-M at 19 0 ° C u s i ng a Hewl e tt Packard 5 8 4 0A Ga s Chromatog raph .
Yield in g r am s o f dried polym e r s f rom the s tarting m a te r ia l .
Compo s i ti o n o f i solated fibres from frui ts , v eg e tabl e s , g ra s s e s and wheat b r an .
Re g ions o f New zeal and where the food s a nd g ras se s used for ' fibre ' ex tr ac ti o n
Pag e No s
9 2 0
2 1
2 2
2 3
we re 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 b e fore and afte r d e l i g n i f i c a tion .
Ki nemati c v i s c o s i ty of wheat b r an hem i c e l l u l o s e a f te r solub i l i sation and r ecovery f rom N-m e thyl-morpholine-N-ox id e . Limi ti ng v i sc os i ty numbers o f the
hem i c e l l ul o s e s e xtrac ted in alk a l i b e fore and a f te r d e l i g ni fication and a f te r recovery f rom N-methyl - m o rpholi ne-N-ox id e .
Re lative vi scos i ty o f nond e l ig n i f ied
hem ic e l l u l o s e 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 ox a l a te and bran ex a l k a l i ob se rved at 3 8 0
m ag n i f i c a ti o n b y l ight m i c ro s c opy .
3 4
3 7
38
3 9
4 0
4 8 5 0
5 5
Figure 1 0 A to D :
F i g u r e 1 1 A a nd B : F i g u r e 1 2 A to D : Tab l e VI :
Tab l e VI I :
F i g u r e 1 3 : F i g u r e 1 4 :
Figure 1 5 :
Tab l e VI I I :
T a b l e I X :
Tab l e X :
Tabl e X I :
Tab l 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 oxa l a te and bran ex alka l i ob se rved by l i g h t
microscopy .
Whe at b ran a f te r DMSO and chl o r am i n e- T , observed b y l ight m i c roscopy .
Whe at bran a f ter DMSO and chl oram i ne-T , obse rved by e l ectron microscopy .
Yi e ld s o f hem i c e l l u l o se obta in ed by alka l i a nd 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
hemi c e l l u l o se extracted from whe a t bran wi th alka l i a nd DMSO.
Amicon MP S - 1 m i c ropar ti ti on un i t .
Pe rcent z i n c bound to wheat b r an hem i c e l l ul ose over a pH r an g e 2 . 1 2 to 8 . 3 .
Percen t zinc bound to wheat b ran hem i c e l lulose 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 whea t b r an hemi c e l lulose .
Re liab i l i ty s tudy from ten an a l ys i s of the meta l s i n the sta ndard 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 MP S - 1 micropo r ti o n s ys tem .
Me tal conte n t ( � g/mg dry we ight) o f wa te r solub l e ( S F ) and wa te r insolub l e ( I F ) fibres f rom frui t , veg e tab l e , bran and g ra s se s .
Mean me ta l conte n t ( � gjmg d r y weig h t ) o f frui t , veg e tab l e , whe a t b ran and g r a s s f i b res .
Chang e s i n m e ta l content ( � g/mg d r y we ig h t ) of water solubl e ( S F ) and in solub l e fibres ( I F ) f rom frui ts , veg e tab l e s , bran and g r as s e s a f te r
5 7
5 9
6 0
6 2
6 3 7 3
7 6
7 7
7 8
7 9
80
8 1
8 2
m ixing with a ' s tand ard ' me ta l solution . 8 3
Bound me ta l conte n t ( � g/mg d r y we ig ht) o f f r ui t , vege tab l e , whe a t bran and g ra s s fibres
a f te r mix ing wi th 's tand ard' 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 d r y we ight) of wa te r sol ub l e ( S F ) and i n s o l uble fibre ( I F ) a f te r mixing wi th 's tand ard' me ta l soluti o n .
Mean , standard d ev i a tion (!Jg/m g ) and T values of bound me ta l s from ma tched pa i r ed fibre s , b e fore and a f te r mixing with ' s ta ndard ' me ta l
soluti o n .
Pe rcen t nond i a l ysable me ta l conte n t o f the water solub l e and i n solub l e f i b re s .
Tab l e XVI I I : Tota l ion- b i nd ing capa c i ty ( IJM/ g ) o f fibres extr ac ted from frui ts , vege ta bl e s , bran and g rasse s .
Fig ur e 1 6 :
Tab l e X I X :
Tab l e X X
Tab l e XXI :
F i g ur 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 fo r the pr eparati o n o f l ignin .
Compo s i ti o n o f the cold wa te r soluble fibre and the pur i fied water solub l e fibre from whe a t b r an .
Yields o f the wa te r solub l e f i b r e s and l ig noc e l lu l ose from wheat b r an .
L ig n i n conte n t of wheat b ran , l ignoc e l lul ose and hem i c e l l u l ose .
Zinc b i nd i ng to whe a t bran , i ts fibre c omponen ts and ph yta te .
Scatc hard pl o ts o f zinc binding to whe a t b ran , i ts f i b r e c omponen ts and ph yta te . Zinc b ind ing capac i tie s , l i g and 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 a nd phyta te .
i x
84A
8 5
86
87
1 06
1 1 1
112
1 1 3
11 4
11 8
1 2 2
c omponen ts from �lant c e l l wa l l s a r e d e sc r ibed i n thi s the s i s . Th e c hemi c a l c omposi tion o f the extrac ted polym e r s a nd the mo rpho l og i c a l and ph ys i c al chang e s occurr ing i n wheat b r an a t var ious s tages o f a n e x trac ti o n se quenc e a n d the m e ta l bi nd ing capac i ties of the e x tr ac ts we r e d e te rmi ned .
A s e quenti a l ex tr ac ti o n procedur e us ing wa te r , am ylase , oxalate and a lka l i ( b e fore and a f ter d e l ign i fi c ati o n ) 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 te r i n so lub l e fibres from bean , c abbag e , l e ttuc e , toma to , peach , pumpki n , k um er a , o n i o n , pear , whe a t b r an , luc erne , c l over and ryegr a s s to b e ob ta i ned . The wate r s o l ub l e fibres we re shown to be c omposed
p r ed om i n an tl y of arab i n o s e , g a l a c to se and ur onic ac i d , whereas the wa te r i n so lub l e fib r e s c o n ta i n ed mainly a r ab inose and x ylose .
The v i scosi ti e s o f the alkali solub l e f i b r e s e x tr ac ted f rom whe a t b ra n , b e fo re and afte r chlor i te d e l igni fic a ti on , a n d a f ter
s o lub i l i s a tion in N-me thyl-mo rpho l i n e-N-ox ide we r e d e te rmined . Th e a r ab inoxyl an ex trac ted be f o re d el i g n i f i c a ti o n , yi e ld of 7 . 9 g / 1 0 0 g , had a l imi ti ng v i sc os i ty num be r of 2 2 0 . 6 m lj g , whe r eas the
a r ab inoxyl an ex trac ted a f te r c hl o r i te 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 ti ng v i sc osi ty numbe r o f 7 4 . 2 m lj g . When the s o lvent N-me thyl -morpho l in e-N-ox ide had been used to d i s solve the n o nd e l i g n i fied arab i noxyl an , a consid er ab 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 a ti on ) o f whe a t b ran , enables a l e s s d eg raded arab inox y l an
to be ex tr ac ted in ad e qua te yie lds . The use o f
N-me thyl-morpho l in e-N-ox ide a s a so lven t for a r abinoxylan res ul ted in e x te n s i ve d eg r ada ti o n .
2
The struc tur al chang e s in whe a t b ran a t e ac h s ta g e o f the
e x tr ac ti o n sequence and when d i me thyl sulphoxide ( DMSO) wa s s ub s ti tuted f o r alka l i we r e ob s e rved usi ng light and scanning e l e c tron 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 r emov ed afte r the amyl ase tr eatmen t. Alka l i r emoved cell wa l l ma te r i a l pred om i n an tl y f rom the a l e uro ne laye r . DM S O wa s not an e f f ic ient e x tra c to r o f
a r ab i noxyl ans from c e l l wa l l s , a yi e ld o f o nl y 0 . 4 % being ob tained and the a l e ur o ne c e l l wa l l s r em a i n i ng intac t . The a r abi noxyl an , e x tr ac ted wi th DM SO, had a hi g he r ferul i c ac id and ace tyl conten t than the
arab i noxyl an extrac ted wi th a lka l i .
The i n te r ac tions o f f ib re s wi th m e ta l ions ( c oppe r , i r o n , z i nc , c al c ium , po ta s s i um , m ag n e s i um , mang anese and sod i um ) us i ng
c onc en tr a tions tha t would be expec ted in the h um an sma l l bowe l a f te r a ' typi c a l ' me a l we r e i nv e stigate d . It wa s found tha t the wa te r solub l e f ib r e s b o und more c oppe r , i r o n and zinc than the wa te r i n so l ub l e
f i b re s . Th e c opper , i ro n 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 mang anese . The water i ns o l ub l e f i b res ( hemi c e l lul ose s ) conta i n ed a highe r c a l c ium conte n t than the s o l ub l e f i b r e s ( pec ti n 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 tha n c a l c i um to hem i c e l lul ose . Po s s i b l y d iv a l en t c al c ium ions pl a y a r o l e i n s tabi l i s ing the hem i c el lulose c omponents of pl a n t ce l l wa l l s .
b ran , i ts componen ts a nd to ph yta te we re d e te rm i n ed . Zi nc b i nd i ng c apa c i t i e s ( � M/g dry we ight o f pl ant ma te r ia l ) i n o rd e r of magni tud e we r e ; ph yta te ( 6 5 8 2
±
192 ) , DMSO solub l e hem i c e l lulose ( 5 0 8 9±
9 2 1 ) , wa te r s o l ub l e fibre ( 4 0 3 8 ± 2 1 6 ) , c e l l wa l l s ( 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 4 0 . 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 ( 1 6 7 . 7 ± 1 2 . 7 ) , b r an ex ox a l a te ( 1 4 8 . 3 ± 5 0 . 0 ) , b r an ex e thanol ( 1 42 . 3±
4 . 4 ) and cellul ose ( 5 7 . 4 ± 5 . 3 ) .The wa ter so lub l e f i br e , f r actionated us i ng ammonium s ul pha te , c ompo sed pr ed omi n a n tl y o f a r ab i nose ( 2 4 . 0 % ) , g a l ac tose ( 2 0 . 3 % ) , xylose
( 1 8 . 6 % ) , mannose ( 1 6 . 2 % ) , g l uc os e ( 1 0 . 9 % ) and r hamnose ( 6 . 0 % ) , bound z i nc more strong ly than ph yta te or the DMSO h em i c e l l ul ose . The Sc a tc hard pl o ts of z i nc b ind ing to phyta te and to the fibre s , e x c ept f o r the water solub l e fibre s , we r e con c ave and marked ly nonl i n ear , 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 i ng to the wa te r s o l ub l e fibres showed well pro no unced maxim um , i nd i c a ti ng the b i nd i ng m ec han i sm i s by po s i tive cooper a tivi ty .
Pa r 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 c hem ic al compo s i tio n o f d i f fe r en t type s of f i b r e s f rom bean , c abbag e , s we e t potato , le ttuc e , o nion , peach , pe ar ,
pumpki n , toma to , whe a t b ran , whi te c l ove r , l ucerne and ryeg r a s s . Th i s s tud y ha s been pub l i shed i n the Jour na l o f the Sc ie nce 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 und e r taken to inve stig a te the v i sc o s i ty o f hemi c e l lulose s ob ta ined u s i ng the e x tr ac tion
procedure and a f te 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 b een pub l i shed i n Ca rb ohyd rate Re se arch 1 98 5 , 1 4 3 : 2 7 1 -2 7 4 (see Appe nd ix G ) .
Par t I I I d e sc r ib e s s t ud i e s undertaken to ob s e rve the mo rpho l og i cal s tr uc tur e o f whe a t b ran , c hang e s occurr ing dur ing the e x tr ac ti o n
s eque nce and influenc e o f DM SO when sub s ti tuted f o r a l ka l i .
Pa r t IV d e sc r ibe s studi e s o n b i nd ing o f me ta l s to wa te r so l 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 r ibe s a more d e ta i l ed stud y o f zi nc b in d ing to whe a t b ra n , i ts fibre c ompo nen t s , a n d t o 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 a nd a s ummar y o f the c o nc lu s ions .
