1998,76, 1998,76, 39-48 39-48
S c F oo oo d A gr gr i
Divi Divisi sion on of Bota Botany ny an Zool Zoolog ogy, y, Aust Austra rali lian an Nati Nation onal al Univ Univer ersi sity ty Canb Canber erra ra (Received 23 Septem September ber 1996;revi 1996;revised sed versio versio receiv received ed 10Apri 1997;acce 1997;accepte pte
AC
0200 0200 Aust Austra rali li
24 Jun 1997) 1997)
simp simple le picr picrat at pape pape ki meth method od wa deve develo lope pe fo th semi semi A bs bs lr lr ac ac t quantitative determination of the cyanog cyanogeni enicp cpote otenti ntial al for cassav cassav flour. flour. The (absor (absorbedi bedi smal vial adde adde
filterpap filterpaper er disc)to disc)to 100 mgof Hour Hour +0·5 ml wate waterpla rplace ce in rectan rectangleof gleof yellowp yellowpicr icratepape atepape attach attached ed to plasti plasticstrip cstrip was
an the vial vial cappe
an lef over overni nigh ght. t. The yellowpap yellowpaper er chan change ge colou colou
towardsbrow towardsbrownand nand its colourwascompare colourwascomparedwiththa dwiththa of standardcolourcard standardcolourcard with with 10 shad shades es of colo colour ur whic whic
corr corres espo pond nded ed with with cyan cyanog ogen enic ic pote potent ntia ials ls of 0-
equivalentsI<:g-1lour(ppm).To obtain 800 mg HCN equivalentsI<:g-1lour(ppm).To
more accurate measure ofcyanogenicpotential(±20%) ofcyanogenicpotential(±20%) the paperwas eluted with waterand waterand theabsorb-
an ncc e from from
e as as ur ur e a t 5 1 n m h e c y yaa n no o ge ge n nii c p ot ot e t ia ia J cali calibr brat atio io grap graph. h. Th meth method odol olog og is. avai availa labl bl
method may be use in the fieldby fieldby
relati relativel velyuns yunskil killedp ledpers erson. on. The mor
accu accura rate te meth method od requ requir ires es spec spectr trop opho hoto tome mete te an labora laborator tories ies in develo developin pin countr countries ies i { 2 ; ' 1998 SCI. S c F ou ou d A gr gr i
76
p m w a e te te r i ne ne d in ki form. Th simp simple le
is suit suitab able le fo us in simp simple le
39-4 (1998) (1998)
Ke word words: s: cass cassav av flou flour; r; cass cassav av root roots; s; cyan cyanog ogen enic ic pote potent ntia ial; l; cyan cyanid ide; e; picr picrat at pape paper; r; simp simple le ki meth method od semi semiqu quan anti tita tati tive ve meth method od
Th cyan cyanog ogen enic ic pote potent ntia ia of cass cassav av root root an leav leaves es 1000p 1000ppm pm CN qu va le ts kg fr sh eigh eight) t) (Coo (Cooke ke an de la Cruz Cruz 1982 1982 Bradbury 1991 1991 Boka Bokang ng 1994 1994a,b). a,b). It is gene genera rall ll cons consid ider ered ed that that cass cassav av root root that that cont contai ai 100ppm HC quiv quiv lent lent shou should ld proc proces esse se to educ educ he cyan cyanog ogen enic ic pote potent ntia ial, l, befo before re us fo huma huma cons consum umpption. tion. Som tradit tradition ional al proces processin sin method method develo developed ped in Sout Sout Amer Americ ic an West West Afri Africa ca remo remove ve near nearly ly al th cyan cyanog ogen en from from cass cassav av prod produc ucts ts bu othe othe meth method od such such as thos thos used used fo cass cassav av flou flou prod produc ucti tion on in East East Afri Africa ca an Indo Indone nesi si redu reduce ce bu do no elim elimin inat at th cyan cyanog ogen en pres presen en (Nam (Nambi bisa sa an Sund Sundar ares esan an 1985 1985 Bainbridge et a119 a11994 94 Mlingi Mlingi 1995). 1995). Whil Whilst st ther ther ar fe repo report rt of pois poison onin in an deat deat
INTRODUCTION Cassava (Man (Manih ihot ot escu escule lent nta) a) is th st pl food food or than than 00 illi illion on eopl eopl in th ro ic an of ho ar very very poor poor (Coc (Coc 1985 1985).T ).The he leav leaves es an root root ofthe plan plan cont contai ai cyan cyanog ogen enic ic gluc glucos osid id (lin (linam amar arin in an smal smal amou amount nt of lota lotaus ustr tral alin in (met (methy hy lina linama mari rin) n) Th lina linama mari ri is read readil il hydr hydrol olys ysed ed to gluc glucos os an acet aceton on cyan cyanoh ohyd ydri ri in th pres presen ence ce of th enzy enzyme me lina linama mara rase se cyan cyanoh ohyd ydri ri deco decomp mpos oses es rapi rapidl dl in neut neutra ra or alka alkali line ne condit condition ion libera liberatin tin hydrog hydrogen en cyanid cyanid and cyanid cyanid ion mari mari
ceto ceto
cyan cyan hydr hydrin in
hydr hydr ge
cyan cyanid id
nd
cassava cassava sample. sample.
(Mlingi 1992 Akintonw Akintonw 1994; 1994; Cliff Cliff 1994), 1994), ther ther ar seve severa ra diso disord rder er whic whic have have been been asso associ ciat ated ed witb witb regu regula la inta intake ke of subsub-le leth thal al quan quanti titi ties es of cyan cyanoogens gens Thes Thes incl includ ud (1 goit goitre re an cret cretin inis is whic whic ar ue to od ne ef ci ncy, ncy, bu ma exac exacer erba ba ed by
To whomcorrespondenceshould be addressed. On
sabb sabbat atic ical al leav leav fro
Scho School ol of Biol Biolog ogic ical al Scie Scienc nces es Th
Nation National al Univer Universit sit of Singap Singapore ore Ken Ridge, Ridge, Singap Singapore ore 119260,RepublicofSingapore. RepublicofSingapore. :c 1998SCI.
39
S c F oo oo d A gr gr i
0022-5142/98/$17.50. 0022-5142/98/$17.50.Printedin Printedin Great Britain
40
~e
al at 1994), an (3) konzo, f or me r ca le ep s pa st i p ar ap ar e i s a n i rr ev er si bl e p as t a ra l
Bradbury
Delange
(Osuntokun
d em i
(Howlett
Materials
9 90 ; B an e
ed
za
(9 Co
cyanogens.
Lo is
O , U SA , T et r a s ( 4 4 '- me th y e ne bi s an copper (II) ethy acetoacetate ch
N,N -dimethylaniline
fr
dis-
ri
Iinamarase
mari
(Brime
es
e t a 11 98 3
cyanogens si
ci
ic
in ctiv te
f ro m
li ar rasc
an c ya n d e c al or i e tr ic a l y 1991 Essers et a11993 bury e th od s a va il ab l ( Sa d ry th addition
preparing
of exogenou
( Co o ri er
he an
u bs tr at e re
(3) d e e r i na ti o
97 99
B ra d u r r ev ie we d
of er al B ra d
1978; 1974 Cook 1993, 1994),
lina aras
to catalyse
versity.
ts
hydro-
Quantitative
linamarin,
modified by Bradbury D et er m n at io n papers
second possible solution ra ea es an
et
ra et al (1994)
o f c ya no ge n
as
p o e nt ia l D si n t et r
as
filter paper.
av
ea
Genera method of determinatio usin picrat papers
of cyanogenic potentia
at
1997). an
(w/v) sodium carbonate,
am
at ac
an
h e a mo un t o f c ya no ge n p re se n diobi et a11980
cyanohydri
et aI1991).
as
trie (Yeo
aceton
linamarase.
treate (Bradbury
of cyanogen
re
linamarase
rc
mari
determinatio
i ll ia m
an
Ed ar
1 98 0) ,
at
er
P ic ra t
pa er ki fo
c ya ni d
e te rm i a t o n
c as sa v
41
fl ur
e ve l (see (ug)
ra cyanoge
(1994) wi lo
present.
c ya n g e
ev r, s am pl es .
a bl e
give
he
yellow-orange-brown
compariso
i cr a
m et ho d
cr determinatio
of cyanogen
determined
spectrophotornetrically,
er solu-
at
an
ac an h a t he re fo r
b ee n d on e u si n
ak
1-3
24 p ic ra t p ap e oped afte
p ic ra t
p a e rs .
cy
en
eq
e ve lo pe d o r s l w ly . It wa full develat 30'C and 22 at 20'C a n w a s ta bl e ug
HCN
in
re io
rt
1994 bu
TABLE Comparison ofpicrate an tetr base method fo cyanogen in cassav flou Property
Detection limit Eas
(Il
HeN)
of observatio
Spee of test Stabilit ofcolou over time
i cr at e
Tetr
m et ho d
Abou 0· lo
ro
more difficul to se at 30 o ve rn ig h a t 20'C S ta bl e t o 48
base method
Abou 0· Whit to blu easier se
drop of Elutio of colour from pape an spectrometric determinatio at .510om Stabilit of papeTS on (1 Storag in dark at room temperatur (2) S to ra g
d ar k a t 3rC ( 3) S to ra g o n b en c i n a b (4 Exposure to sunlight chemicals
Very satisfactory
Not possible
Stable fo mont with no darkening' mon ths" slight darkening after Slight darkenin after month Slight darkenin after-1 mont
Lose sensitivity
Picri acid explosiv when t ab l i n f il te r p ap e o r alkaline solution"
Tetr
dry,
in
months
Lose sensitivit in week Lose sensitivit in week G re e a ft e 2 0 m i a n Losessensitivity base is carcinogen
Studie quantitatively se Fi 2. Picrat solution stored f o 1 4 th an papers made from it compared with papers made from fres picrat solution No change wa observed over 14months (see text), .Ii-
Bradbury
42
co CN
in
ap a lk al in e s o u t o n r ea d l y a va il ab l
quantita iv
KCN,
icrate
ex
imen
we
No
paper.
in
E ff ec t o f e mp er at ur e development
(2)
o f e xp er i e n
o n c ol ou r
To 10 0·5 mI 0·2
·O and 1 0
1 1 lin-
8,
am picrat
pape
developmen
method
an
calibratio
nm
curv
Visual observ
3TC.
Thes
increase
were much an
ea ct
I, i n w hi c
re
c om pa ri -
cr
3TC.
3TC the
as
ug ro 40 ug
e th o
o ve r h i
r an g
o f e mp er at ur es .
comparison
ug
(sample
12),
t ab i i ti e o f p ic ra t with time II
a pe r
an
a lk al i
h;jil!m!lI'l No W1almanNo3M
at 20
eN
Fig
100
40 e an tl ln t
(I<
a nc e
He
e ~u l . .. .
0mm
at
en
paper, whic isapproximately twic th thicknes of th No paper.
2).
1mo
p ic ra t
so ut on
P ic ra t
a pe r k i o r c y n id e d e e r i n t io n
c as sa v f lo u
43
TABLE Ef ec
o f e xp er im en ta l
Sample of flou (Table 3)
t e p er a u r
Temperature
of experiment
re)
on coou
d ev el op me n
510 nm
at
an
ou
analysis
Total cyanid (ppm) by Picrate method"
12
19
12
30
0·029
12
37
0·032
27
19
0·066
27
30
0·086
0·021
0·092
29
19
29
Add
hydroly,i,
39 42
0·193'
87
Q.226'
102
0·234'
37
13 15
79
106
E a c r es ul t w a s t h a ve ra g
o f t hr e e xp er im e nt s case ther were experiment at each temperatur an sample were removed for analysis after 42 hand 19'C th absorbance were 0·193.0·19 an 0·21 respectively wherea at 30 an 37'C th absorbance were essentially c on st an t o ve r t im e In thes
er absorbances
(i
3.
l in ea r r a g e t he r
i s i tt l
d if fe re nc e i n r e p on s
et ee
he ig l ut io n
r an ge ) a n h en c o w r e u lt s ·5 i cr i a ci d ·5 o di u
c ar b n -
Q.12 0.10
0."
that
0.1)6
o l t o p r d uc e p ic ra t
0.'"
a pe r
(l02
000
100
2:00
T im e
1. Slow ageing of
D ev e o pm en t
~1.l0
l el -a y
hatman No 1picrate papers Absorb
nd rd
curve u s in g d i ff er e n
cy a n og e n s t an d ar d s
ances
picrat papers stored in th dark at room temperatur fo long periods. '0
It r e u lt s a gr e
o.
wi hi
ab ut am
a.5mooU'l 1mcnm
11 mQflIh
0.0
Ablcut'lt al
Fig No
KeN
.0
eo
(~g
HeN oquly.
E ac h c ya n g e
t a d ar d ca
100
b s r ba nc e a t 5 1 r u o f w a e r e l a te s o f h a a n picrat papers ofvarious ages from 0· to 11m, plotte agains th amount ofKCN expresse as pg HC
_-_._ -
h ol e r a g e o f c o c en tr at io n
·0 ·0
44
Bradbury
TABLE
Su
ar
o f cyanogens
C ya no ge ni c p ot en ti a
Sample
and hnarnarase
presen
( pp m b y
in cassav Con.ten
flours
Linamarast!'
of cyanogen
(ppm) by a ci d k y dr o y si s Simple
hydrolysis
picrate
picrate;!
Linamarase
Acetone
Free
cyan.ohydrin
(CW,HCN)
Mozamhique
20
14
15
2·7 10
8·4
9·5
2·8
5-4
3·5 (gari)
10
20
16
(gari)
13
10
3·2 6·2
8·0
12 10
14 15
14
5·0
14 5·9
13
15
14
16
4·0 4·4
0·5'
21
40
23
85
1·5
0,7
90
2·0
1·7
91
2·1
2·1
2·2 0,5
[·
54
1·2 7·6
71
62
28
39
28
14
1·6
34
67
25
1·9
3·6
36
23 22
1·5
2·2
5·0 5·8
95
19
0·6
1·6
2·0
1·0
17
25
3-
1·7
J.6 3-4
7·g'
2·3
Indonesia 26 27
50
46
30
29
29
90
86
0·5 0·8
0·13
79
80
0·18
Australia
30
12
3·5
10
7·9
II
1 8- 2
determined
using
in triplicat
were 'scored' by an olde method usin 1 99 2
These
4,7
4·5
S am pl e Th
c or e
e re - c on ve r e d
result wer
in
o lo u
c ha r
se
scale 0 , 1 , 2 ,
h e a b o rb an c
d et e m in e
0·8 0·8
0·82
1·5
2·4
-a
014
b el o
r ep ar e
ro
f r s h KeN s ta nd ar ds .
s im il a t o t ha t s e f o t et r b y h e s em iq ua nt it a i v
as
e th od )
p er s an
t he nc e
0·74
Th
ra nt
o th e
s am pl e
ry an
an
h e c ya no ge n
flour
th correspondin gradient determined with th same cyanogen standard by Essers (1993), using their O'Brien
4·2
8·3
32 33
2·9
el al (1991)
Essers (1993)and ourselve is extraordinary, bu is actually fortuitous becaus th observed gradient
depend on th detailed experimental procedure. Thus fo exam le th icrate meth d. he ap rs re lute in 6·0 reduce to 3· ml then th absorbance an henc th gradient woul double However, sinc th gradient ar similar, it show that th picrat method is at leas as sensitiveas the Konig method.
P ic ra t
a pe r k i f o
c ya n d e
e te r i na t o n
i n c as sa v
45
l ou r
; 5_
..
Acetcvanoly d
au e ff ec t
h um a
( Ml i g i
1992). H o e ve r
f ou r
er
"."~ C Yl ln id e
e lQ or 1I ! r1 1 (~!iII
!-leN
as
eQl.llv_l
Fi 4. Develo st ar curv of absorbance cyanid conten (Ilg HeN equivalents) using whatmen 3MM picrat papers an thre differen cyanogen standard KeN,
aceton cyanohydrin and linamarin
fr linamarase
e xo ge no u th picrat
l in a a ra s method
i n a na l s i
o f f lo u
cr
ac et od
am le
u si n
ag
r ov id in g
n e e xc lu de s r es ul t
b el o
about 18%, me ho
of
s se r
(1993\ th
aceton
12%
( 19 9
cyanohydri gra-
linamarin
w er e d if fe re nt ,
aj
s ou rc e
ls
an es h er e h e f lo u i s v er y c oa rs e ( sa mp le s 21 and 25), ro
ca
hard gari samples (8 and 9, Table 3) er
(1978) method-
at et ai ar
ar e f r e a na l s i
ou
b e useful,
e. ad
by
correlation coefficient
0'98
(r' 96% of
ar O·77 1992). G oo d s tr ai g
re by Essers et al (1993)
picrat
y an og e a n i na ma ra s c on te n detennined by dilferen method
c as sa v
0'59) (r' i ne s a n i mi la r c or re la ti o
vs semiquantitative
ci picrate,
f lo u cr ar
rn
c oe ff i
re
to ppm,
es
46
Bradbury
to brown,
whic
yellow
fresh flou
(ppm).
52
strip (to mm
S ta bi l t y o f l in am ar i kit
s ta nd ar d f o u s
si pl
p ic ra t
i m e d a te l
m ) c u f ro m o ve rh ea d
t ra n p ar e c y
s ea le d w i (25-3TC)
colour at
tial (m of fr
0m
to
h e c ha r
(Fig 5) ri should allo a ti o th ki
ed
a pe r ra
p ra c i ce .
e ve l cyanogenic potentia
Components of ki fo cyanogenic potentia determination
et of cassav
( Fi g 4).
Hour
of Cassav
weighings
flOUI.
flated
linamarase
an
b uf fe r
of fiour. c le a p la st ic , f la t- bo t o me d d.•5
(2) V ia l
21
(w/v)
(w/v]
polyvinylpyrrolidone-IO
1996),
(Yeoh
s cr e
ed
at
(5
(6
ap (m HC
,,----------------------,
s he e s . el
1I0ur (ppm). potentia fl ur pp
vs cyanogenic equivalent
kg-
(8
dard. III
l i n8 0 m _~ 1 l _0 c l L . e .m .p ap .
II
0.'
,.
Lirlampap.30
cy
Linam·Pflp_t(lC}(j
flour.
..,..'--------,.-----------1 ..lum.rln
l id s
pH 0·2 U),
and linamarase. at carbonate) q ua re s o f f il te r a pe r ( Wh at ma n c le a l as t o ve r e a r a s pa re nc y ai
it
(~II !-leN
IIqLiv.)
Fi25. Graphs of absorbance at 51 nm vs linamari conten (J.lgHeN equivalents) for Iinamarin-impregnated filter papers c ti o
ti
CONCLUSION
as
Picrat
pape
ki fo
cyanid
delerminatio
rm
Bradbury
ri
H, Egan o nt en t o f c as sa va .
y an id e
al
Bradbury
flour.
ed
47
in cassaw fiou
a pi d s cr ee n n g a ss a
1994 Improved method
el
P ro ce ed in g
9t
of analysis
I nt er na ti on .a l
Internationa
es be ad ci
m or e q ua nt it at iv e
S ym po -
ociety fo
Tropical Root Crop (ISTRC), Accra, Ghana. Ed Oter F, Hahn K. pp 237-242. Bradbury H, Egan V, 199 Analysis of cyanid
(±20%
ca sa u si n c i h yd ro ly si s S c F oo d A gr k 5 5 2 77 -2 90 . G, r ad b r y r ad bu r
o f c ya n g en i
g lu c s i e s
1994 Comparison of A(_·tl1Hort 375
87-96.
adapted
to cassav
cyano-
field
tubers
s ys te m
a ll ow in g
k in et i
m ea su re me nt s
116.
Brimer 100
e ar s a g
( re vi ew e
by
l li a
of
Anal3 91-94.
Pnytochem
an
E dw ar d ch
1980)
375 L05-
ar
M e g aa r P, Nartey Deterby t h c o p ou nd s a ye r c hr o a -
h r s te n e n o f c y n og e i c I.
i na t o n tography.
Ac
Chern 31789-793.
detailed chemidifficulty.
1994 C a s av a a fe l i n t im e o f a r a n Cliff Mozambique. A ct a u r 375 373-378. 1985 Cock Westview Pre5S,Boulder, CO USA, 10
conten of cassava. Cooke Ia
ACKNOWLEDGEMENTS
2~ 345-352. change in cyanid
1982 Th
Sci
33 269-275. K, Hahn an
F oo d A gr i
r ou gh t
Screening cassava E xp e
A gr i
14
367-372.
1983 Cassav toxicity an thyroid: Delange F. Ahluwalia research an public health issues International Developen
es ar
ener
Delange F . E k c h g en es i
as
an
i od in e
t ta wa ,
0, d ef ic ie nc y
y an o e n
c as s v a
p ro du ct s
REFERENCES An applicatio of modified autoly si method fo th determinatio of cyanid in cassava. In
Aalbersberg
Networ Conference P hi li pp in e i n p re ss . Akintonwa A,
Food
Nutr Res. Lab. Manila Th 1994
375 285-288. Bainbridge Z, Wellings K,
a ta l a n n on Acta
Hort
improved cassav
cyano37
Acta Ho
c as sa v p ro du ct s a n
Van
Ac
37
or
2 89 -
n tr od uc ti o
97-104
new chromogen
SC Food Agric 63 287-296.
FAO/WHO
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1991 Joint g ra mm e C od e A li me nt a i u 4. AO, Rome, Italy.
II
S u p le me n Acta
H ort 375 323-329.
Howlett
om sson
P, Brubaker
R, Mlingi N, Rosling
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1994 Cassav
d is or de rs .
293.
an
Introducin
an da
cs
technologies- in
cyanid
exposure
H, Roslin 1992 Shortcut in cassav ou te r oc es s n g a n r is k d ie ta r c ya ni d e xp o u r i n a ir e 14 137-143. Bokanga 19940 Distribution of cyanogenic potentia in cassava gcrmplasm A c o r 375117-121 B ok an g 1 99 4 r oc e s in g o f c as sa v e a e s f o h u a n consumption. A ct a u r 375 203-207.
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1980
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B io i C he r
HeN Sci 391021-1021 1995
Dissertation
Sweden.
1990
44 2803-2809
Colorimetric method fo determiInd
Cassav
No. 571.
an
processing an ac
ed
dietar
Agric
cyanid
U pp sa l
University"
1992
u tb re a
Bradbury
48
cessed cassav in Tanzania. Nutr Re 12 677-687. a mb is a u nd ar e a n 1 98 5 E l fe c r oc es si n cyanoglucoside conten of cassava. Sci
th
1197-120 3.
enzymi assa fo cyanogen in fres an processe ci Fo
A gr i
5 6 2 7 7 -2 8 9 .
cassava
1993 Evaluation of ri rapi semi-quantitativ assa fo cyanogen in cassava. In: Proceeding oj th Firs Internationa Meetin of th ~T Cassava Biotechnolog Network, M, Centro Internaciona de Agricultur Tropical (CIATI Cali, Colombia PP 39Cl --399 . ri Evaluation
odificatio
an
co pariso
of tw
rapi
assays fo cyanogen in cassava. Sc Food Agri 65 391399. Osuntoku 199 Chroni cyanid intoxication of dietary origin an degenerative neuropathy in Nigerians. Acta Hort 375311-321. 1974 S cr ee ni n f o A cy an o Sadik S, Okerek genesi in Cassav (Technica Bulletin No.4). Internationa Institute for Tropical Agriculture, Ibadan Nigeria,
1994 Regulation of it c Ma h cyanogenesis in cassava. Acta Hart 375 69-77 J, Edward Williams Estimation of cyanid with alkaline picrate. Sc F oo d A gr i 3 1 1 5- 22 . 1989 Kineti properties of ,8-glucosidase from cassava. Phytochemistry Yeah of linamari in 1994 Determinatio cassav using enzyme-sensitised microcentrifuge tubes. Sci 6 4 ' i 31-33. H, Truong
F oo d A gr i
Yeoh mari
in cassav
F oo d C he r
usin
4 7 2 9 5 -2 9 8
1993 Quantitative
cassav
analysis of lina P-glucosidas electrode.
mari indicato pape stri fo th semi-quantitativ mation of linamarin. Biotechno TechnollO 319-322. e th o cassav 262.
esti
f o i so la t n g c as sa v l ea f l in a a ra s u it a l e f o Sc Fo Ag cyanid determination, 75 258-