INTRODUCTION
P o eins a e absolu ely necessa y and
i eplaceable o human nou ishmen . Wi hou hem,
build-up and econs uc ion o issues, as well as
o ma ion o p o eins ha ing a ce ain unc ion in
he o ganism (enzymes o p o eins o blood plasma,
nucleic acids and o he s) would be impossible. In
case an o ganism has no o he possibili y, i e en
u ilises p o eins o co e ene gy equi emen s.
P o eins ha e o spli in se e al s ages down o he
smalles s uc u al elemen s – amino acids. Only
hen can hey be u ilised. The composi ion and
quan i y o amino acids ha he body i sel is unable
o o m (essen ial amino acids) a e he c i e ion by
which quali y o p o ein sou ces is assessed. In a
global sense, he lack o p o eins in nou ishmen is
a gene al phenomenon. The p o ein de ici in
indus ially ad anced lands is no so s ong as in
de eloping coun ies, bu biological alue o p o eins
is low e en in highly de eloped coun ies. Fo his
O ien al Jou nal o Chemis y
Vol. 26(3), 781-788 (2010)
Isola ion o
Ama an h
p o eins by lique ac ion wi h enzymes
PAVEL MOKREJS¹, DAGMAR JANACOVA², KAREL KOLOMAZNIK²,
VLADIMIR VASEK² and PETR SVOBODA¹
¹Tomas Ba a Uni e si y, Facul y o Technology, Depa men o Polyme ic Enginee ing,
Nam. TGM 275, 762 72 Zlin, (The Czech Republic).
²Tomas Ba a Uni e si y, Facul y o Applied In o ma ics, Depa men o P ocessing
Con ol and Applied Compu e Science, Nad S anemi 4511, 760 05 Zlin (The Czech Republic).
(Recei ed: Ap il 10, 2010; Accep ed: May 25, 2010)
ABSTRACT
The p esen ed wo k deals wi h possibili ies in isola ing ama an h p o ein. A biochemical
p ocedu e was chosen, consis ing in employing enzymes ac ing speci ically on p o ein deg ada ion.
Expe imen s we e execu ed h ough wo-le el ac o es s, and he in luences unde s udy we e hose
o enzyma ic hyd olysis du a ion (1 and 5 h) and empe a u e (30 and 50 oC) on e iciency o p o ein
hyd olysis. Ama an h lou was mixed wi h wa e in a io 1:20, hea ing o desi ed empe a u e p oceeded
a a a e o 2 oC.min-1, and 0.1 % enzyme (pe mass o lou ) was added. Cen i uga ion yielded a
p o ein hyd olysa e and a solid, polysaccha ide-en iched ac ion.
Key wo ds: Ama an h, enzyme, isola ion, p o ein, p o ein hyd olysa e.
eason, alue o animal p o eins was
disp opo iona ely emphasised in ea lie yea s. A
majo i y o p o eins om animal p oduc s does no
wholly co espond o he equi emen s o human
o ganism ega ding essen ial amino acids con en
and, mo eo e , he in ake o animal p o eins in ood
is always connec ed wi h a high and undesi able
in ake o a s. Fa s o p o ein- ich animal p oduc s
a e mos ly ep esen ed by sa u a ed a s exhibi ing
a demons ably a he ogenic in luence. Vege able
p o eins may be mu ually combined in such manne
ha hey esul in a comple e spec um o
indispensable amino acids. An op imal si ua ion
appea s when man combines bo h ege able and
animal sou ces o p o eins in ood. A highly
in e es ing and un adi ional sou ce o p o ein is
ama an h g ain1,2.
Ama an h, in ea lie imes a deco a i e o
u ili y plan , has la ely been he objec o keen
in e es especially in he ood indus y, as all
782 Mok ejs
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O ien . J. Chem.,
Vol. 26(3), 781-788 (2010)
ama an h componen s (s a ch, p o eins, ib e, a s,
mine al subs ances, colo an s, i amins) somewha
di e om cu en oods u sou ces. In he las 30
yea s, p oduc ion and consump ion o ama an h
ha e eco ded signi ican expansion. Coun ies
belonging o leading p oduce s a e Russia, USA,
Cen al Ame ican coun ies and India. Ama an h
being impo ed o Eu ope comes p edominan ly
om hese coun ies. F om he nu i i e iewpoin
conce ning con en s o speci ic subs ances,
ama an h biomass possesses an ex a-o dina ily
in e es ing composi ion. I con ains as much as 28
% p o eins pe d y ma e , which is up o 3 imes
mo e han in o he ce eals, 50 % saccha ides, 6–
17 % a . An impo an componen o ama an h a
is squalene, whose con en is 8 imes g ea e han
in oli e oil3. Ama an h biomass u he con ains
bio la onoids, na u al pigmen s, mine als (in
pa icula , Ca, Mg, Fe, K), i amins o he B, C and
K amily and o he mino i y componen s4,5. I s calo ic
alue is 1.5 o 3 imes g ea e han ha o o he
ce eals. Ama an h lea es, hanks o hei as e
p ope ies, may be used in simila manne as
ano he ege able, o example, spinach. Ama an h
is hence one o hose p ecious plan s ha a e wholly
o he good – lea es as a ege able and seed as a
ce eal. In o de o ob ain biomass om ama an h in
an ea ly s age o de elopmen , a mul iple c op may
be eckoned wi h, as ama an h ha es ed be o e
s a o bloom epea edly g ows up du ing he yea .
This edisco e ed plan displays p omising economic
po en ial, and possibili ies a e being sough o how
o include i in exis ing ood p oduc s on he one
hand and also o make new p oduc s om i on he
o he 6-7.
Ama an h g ain con ains mo e p o eins
han o he ce eals, an a e age 18 %, while whea
con ains 10 %, ye 9 %, ice 6 % and maize 5.5 %.
As much as 65 % p o ein in ama an h is
concen a ed in he sp ou . P o ein con en a ies
depending on so s o ama an h and condi ions o
hei cul i a ion. Ama an h g ain con ains se e al
p o eins8. Mos o he esul s suppo ha albumins
a e he main ac ion (48.9 o 65 %). Glu elin ac ion
is he second in abundance (22.4 o 42.3 %).
Globulins ep esen he 13.7 o 18.1 % o he seed
p o ein. Mos da a poin ou p olamins as he mino
ac ion wi h alues be ween 1.0 o 3.2 %. The
amoun o he esidue le a e p o ein ex ac ion is
p obably dependen on he bu e s and he ex ac ion
ime9-11. Ama an h p o ein is high-quali y p o ein,
especially ich in essen ial amino acids, and as i
con ains all essen ial amino acids i anks among
ull- alue p o eins. Con en s o lysine and
yp ophane a e compa able o hose ound wi h
animal p o eins¹²
Usually seed p o eins a e isola ed using
sequen ial sol en me hod. Wa e ex ac ion yields
albumins ac ion, globulins a e ex ac ed wi h dilu ed
sal s, glu elins wi h acids o alkalis and inally e hanol
gi es p olamins13. The yield and p opo ions o he
di e en p o ein ac ions depends on he
p epa a ion me hod used. Acco ding o Ma ínez
and Añón ama an h p o ein isola e is p epa ed by
ex ac ion in alkali en i onmen . Flou is suspended
in wa e (10 %, w/ ) and pH adjus ed o 8 o 11
wi h 2 N NaOH. The suspension is s i ed o 30
min a oom empe a u e and hen cen i uged. The
supe na an is adjus ed o pH 5 wi h 2 N HCl and
hen cen i uged. The yield o isola ed p o ein
inc eased wi h ex ac ion pH om app ox. 5 % a
pH 8 o app ox. 12.5 % a pH 11 (14). Simila
ex ac ion and p ecipi a ion echnique we e s udied
o isola e ama an h p o ein15.
Objec i e o wo k
F ac iona ion o ama an h lou has so a
been execu ed by wa e ex ac ion in which s a ch
is washed ou oge he wi h p o ein. P o ein may be
sepa a ed om s a ch only wi h di icul y. Ama an h
p o ein, howe e , con ains a high ac ion o essen ial
amino acids and can be an impo an componen
o unc ional oods u s in he ield o specialised
alimen a y die e ics; i is hus app op ia e o
isola ion. Wi hin he scope o a p ojec ocused on
ob aining p o eins om un adi ional sou ces, we
concen a ed on isola ing he p o eins o ama an h
lou . We ollowed om he assump ion ha p o ein
sepa a ion can be pe o med, beside he al eady
men ioned g adua ed ex ac ion me hods, in wo
ways. The i s consis s in enzyma ic deg ada ion o
polysaccha ides (s a ch), hei lique ac ion in o
soluble glucose and en ichmen o solid phase wi h
ege able p o ein. This echnique was ied al eady
ea lie by applying an enzyma ic p epa a ion o α-
amylase16. A e lique ac ion, acco ding o published
esul s, he solid phase was en iched wi h p o ein
om 15 o 30–39%, depending on condi ions o
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Vol. 26(3), 781-788 (2010)
s a ch hyd olysis. The second me hod, which is he
subjec o his a icle, consis s in hyd olysing
p o eins wi h p o eoly ic enzyme. Wa e -soluble
p o ein hyd olysa e is subsequen ly sepa a ed om
he solid polysaccha ide ac ion by il a ion. Fi e
enzyma ic p epa a ions (p o einases) we e selec ed
o his pu pose.
MATERIAL AND METHODS
Appa a us and equipmen comp ised:
D ie WTB Binde E/B 28 (Ge many), sha s i e
Heidolph RZR1 (Ge many), elec onic balance Ke n
770/GS/GJ (Ge many), wa e ba h HGL W 16
(Ge many), cen i uge He ich Uni e sal 32
(Ge many), mine alisa ion appa a us Hach
Digesdahl (U.S.A.), mu le u nace Nabe he m L
9/S 27 (Ge many), pola ime e K üss P1000
(Ge many), Pa nas-Wagne dis illa ion appa a us,
Soxhle ex ac ion appa a us.
Ama an h lou (g ounded seeds o
Ama an hus hypochond iacus) used in his wo k
was supplied by he AMR Ama an h Company
(H adec K alo e, he Czech Republic); i s
composi ion is p esen ed in Table 1.
D y ma e was de e mined by d ying a
weighed quan i y o sample in glass weighing bo le
a 103±2 oC o 12 hou s and weighing a e cooling.
Ino ganic solid was de e mined by ca e ully
incine a ing a sample o lou in a ce amic c ucible
o e a gas bu ne and hen by annealing a 600 oC
in a mu le u nace and weighing a e cooling. To al
Kjeldahl ni ogen was de e mined by mine alising a
sample o lou by boiling o 30 min (a app ox. 440
oC) in sulphu ic acid wi h added ca alys .
Ni ogenous subs ances we e hus ans o med in o
ammonium sulpha e om which ammonia was
eleased in an alkaline en i onmen , hen s eam
dis illed and de e mined by i a ion. Coa se p o eins
we e de e mined by mul iplying ni ogen con en by
con e sion ac o 5.70 17. Fa was ex ac ed om
he lou sample wi h n-hexane in a Soxhle
ex ac ion appa a us o 4 hou s. A e dis illing o
he sol en and d ying he lask con aining a o 1
hou and cooling, a con en was de e mined by
g a ime y. S a ch con en was de e mined
acco ding o Czech s anda d CSN 56 0512-16 18.
This de e mina ion is based on ans o ming s a ch
in o soluble s a ch by ac ion o dilu ed HCl while
wa m. A e cla i ica ion, soluble s a ch is
de e mined by pola ime y. The me hod o
de e mining ib e consis s in elimina ing
accompanying subs ances om he sample by
hyd olysis in an acid and alkaline medium; a e a
90 min hyd olysis in 1.25 % H2SO4, he undissol ed
solid ac ion was washed wi h wa e and hyd olysed
o ano he 90 min in 1.25 % KOH. Non-hyd olysed
esidue ( ib e), a e washing wi h wa e and d ying
a 103±2 oC o 6 hou s, was weighed19.
Lique ying ama an h p o ein in o soluble
hyd olysa e used 5 speci ic p o einases enzymes
p oduced by deep e men a ion o gene ically
modi ied mic oo ganisms supplied by No ozymes
A/S (Bags ae d, Denma k): Alcalase 2.5 L DX,
Pola zyme 12 T, Espe ase 8.0 L, E e lase 16 L EX,
Sa inase Ul a 16 L.
Hyd olysis o ama an h lou p o eins
p oceeded unde mild eac ion condi ions
( empe a u e, a mosphe ic p essu e, neu al pH)
which we e p oposed and op imised o his pu pose.
The a io o ama an h lou and wa e was 1:20.
Ten g ams lou was weighed o an accu acy o ±1
% in o a 250-ml boiling lask, 200 ml p ehea ed
dis illed wa e o a empe a u e o 22±1 oC was
added and he con en s we e b ie ly s i ed (app ox.
20 sec). The lask con aining mix u e was hen pu
in o a wa e ba h and sha s i e se in o mo ion a
600 pm, wi h simul aneous hea ing a a a e o 2
oC.min-1. On a aining he desi ed empe a u e, 0.1
% enzyme was added (pe lou d y ma e ). A e
a de e mined ime, he mix u e was cen i uged (10
min) a a a e o 6,000 pm. The liquid phase (p o ein
hyd olysa e) and solid cake (polysaccha ide basis
wi h esidually bound p o ein) we e hen analysed
o d y ma e con en and o al Kjeldahl ni ogen.
RESULTS AND DISCUSSION
Lique ying p o eins o ama an h lou
Expe imen s we e pe o med applying
wo-le el ac o es s. We in es iga ed how du a ion
o enzyma ic hyd olysis (1 and 5 hou s) and
empe a u e (30 and 50 oC) a ec ed p o ein
con e sion (%). Pe cen age o p o ein was
calcula ed om he a io o ni ogen con en in
hyd olysa e and ini ial ni ogen con en in ama an h
784 Mok ejs
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Vol. 26(3), 781-788 (2010)
lou , and by mul iplying ha by con e sion
coe icien 5.70. Resul s o lique ying ama an h lou
p o eins a e shown in Fig. 1. Hyd olysis o 1-hou
du a ion p oduced changes in p o ein lique ying
e iciency a 30 and 50 oC; by con as , no ma ked
di e ences we e eco ded be ween pa icula
enzymes. The g ea es lique ying e iciency a e 1-
hou hyd olysis a 30 oC was eco ded wi h enzyme
Alcalase (app ox. 23%), he lowes wi h enzyme
Pola zyme (app ox. 21 %), as shown in Fig. 1-A.
A e hyd olysis o same du a ion, bu a 50 oC,
g ea es lique ying e iciency was achie ed when
enzyme Sa inase was employed (app ox. 25.5 %),
he lowes hen wi h E e lase (app ox. 23 %), as
shown in Fig. 1-B. A e 5-hou hyd olysis,
di e ences in p o ein lique ac ion a wo chosen
empe a u es we e signi ican ly g ea e , as well as
subs an ial di e ences eco ded be ween pa icula
enzymes. As shown in Fig. 1-C, a 30 oC, g ea es
e iciency was shown by enzyme Alcalase – app ox.
29 % lique ac ion. In con as , lowes e iciency was
eco ded wi h enzyme Pola zyme – app ox. 20 %
lique ac ion. Simila ly, a 50 oC he highes lique ying
e iciency o p o eins was displayed by enzyme
Alcalase when almos 37 % p o eins we e lique ied.
Compa ed wi h his, enzyme Pola zyme again
showed lowes lique ying e iciency – 26.5 % (Fig.
1-D).
a) hyd olysis du a ion 1 hou a 30 oC b) hyd olysis du a ion 1 hou a 50 oC
c) hyd olysis du a ion 5 hou a 30 oC d) hyd olysis du a ion 5 hou a 50 oC
Fig. 1: Pe cen age o p o ein con e sion h ough applica ion o a ious enzymes
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De e mining bond s eng h o p o ein o solid
polysaccha ide esidue
When lique ying ama an h p o ein by
hyd olysis ca alysed wi h p o eoly ic enzyme
Alcalase 2.5 L DX, a e cen i uging he eac ion
mix u e he e is a side p oduc – polysaccha ide
esidue (solid cake) con aining bound esidual
p o ein. Bond s eng h o p o ein o polysaccha ide
may be exp essed in quan i a i e manne by he
adso p ion coe icien o Langmui ’s iso he m (
K
).
The adso p ion iso he m is gene ally de ined as he
dependence o componen concen a ion (ama an h
p o ein
cA
) bound o solid phase (polysaccha ide-
s a ch) on he concen a ion o p o ein in solu ion
(c0)
unde equilib ium condi ions. Langmui ’s
iso he m is quan i a i ely exp essed by he
exp ession as ollows:
...(1)
In case concen a ions
(c
,
c0)
a e small,
p oduc
Bc
o
B
ε
c0
in he denomina o o equa ion
(1) may be neglec ed and we ob ain a linea ela ion
be ween
cA
and
C
o
c0
:
...(2)
Linea cons an
K
was subs i u ed he e o
adso p ion coe icien
A
. I holds ha :
...(3)
When p ac ically de e mining adso p ion
coe icien
K
, he p ocedu e is as ollows: A sample
o solid phase (polysaccha ide esidue) o known
mass and s a ing o al p o ein concen a ion
(cs)
is
imme sed in a known olume o sol en
(V0),
clean
wa e in ou case, and he dependency o p o ein
concen a ion on ime is s udied. On achie ing a
s eady s a e, when p o ein concen a ion in he
solu ion emains unchanged in ime, i is ega ded
as he equilib ium concen a ion. A mass balance
may hen be pe o med:
...(4)
Applying (2) and alidi y
...(5)
we may adap and a i e a :
...(6)
whe e dimensionless consump ion o
sol en (wa e ),
Na
, was in oduced, de ined by
ela ionship:
...(7)
In ex ac ion by decan ing, a ecu en
ela ionship o he
(i+l)
h s ep in concen a ing he
washed-ou componen om he solid phase can
be de i ed:
...(8)
Ex ac ion e iciency in he
i
h s ep is
quan i a i ely de e mined by washing-ou deg ee
(y)
, de ined as he a io o mass o washed-ou
componen om he solid phase o o al con en o
he same componen in solid phase (in ou case, %
decan ed esidual p o eins om polysaccha ide
esidue):
...(9)
To al e iciency is hen de e mined by he
sum o e iciencies in e e y decan ing s ep:
...(10)
Assuming he dimensionless consump ion

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Vol. 26(3), 781-788 (2010)
Table 1: Composi ion o ama an h lou
Pa ame e Value (%)
D y ma e 86.9
Ino ganic solids in d y ma e 3.6
To al Kjeldahl ni ogen in d y ma e 2.8
Coa se p o eins (ni ogen x 5.70) in d y ma e 16.1
Fa in d y ma e 9.8
S a ch in d y ma e 65.8
Fib e in d y ma e 4.9
Table 2: Decan ing ex ac ion o esidual p o eins om polysaccha ide esidue
Na n
y (%) y K
Du a ion o one washing cycle
20 min 60 min 20 min 60 min 20 min 60 min
5 1 7.14 10.29 0.0714 0.1029 131 86
4 15.73 18.45 0.1573 0.1845 227 189
10 1 10.03 15.15 0.1003 0.1515 178 111
4 19.31 21.71 0.1931 0.2171 362 316
20 1 12.37 15.26 0.1237 0.1526 282 221
4 21.39 22.13 0.2139 0.2213 644 619
100 1 31.52 34.67 0.3152 0.3467 434 376
4 39.41 40.80 0.3941 0.4080 1497 1428
Mean alue o K 469 418
Legend o Table 2: Nadimensionless consump ion o sol en
n numbe o washing cycles y (%)% decan ed esidual p o eins om polysaccha ide esidue
y washing deg ee K adso p ion coe icien
o sol en is he same in e e y
i
h s ep
(Na1 = Na2 =
Nai)
and applying (8) and (9), he inal ela ionship
de e mining o al e iciency o mul iple ex ac ions
(decan ing) may be de i ed:
...(11)
F om he e i ollows ha
K
is:
..(12)
Du ing ac ual de e mina ion o adso p ion
coe icien (
K
), we es ed 4 dimensionless sol en
consump ions
(Na)
: 5, 10, 20, 100. We cake (d y
mass 8.8 g) emaining a e enzyma ic hyd olysis
o ama an h lou p o eins was dosed. Speci ied
dosage: wi h Na=5, 44 g wa e was dosed pe we
cake; wi h Na=10 i was 88 g wa e , wi h Na=20 i
was 176 g wa e and wi h Na=100 i was 880 g dosed
wa e . One and ou washing cycles we e examined,
one washing cycle las ing 20 o 60 min. Wo k
p oceeded a labo a o y empe a u e (22±1 oC)
unde s i ing wi h a sha s i e a 2,000 pm in
glass beake s o olumes as ollows: 150 ml (Na=5),
300 ml (Na=10), 600 ml (Na=20) and 3,000 ml
(Na=100). A e a de e mined ime, he mix u e was
cen i uged (10 min) a 6,000 pm. The liquid phase
(decan ed wa e -soluble p o eins) and emaining
787Mok ejs
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Vol. 26(3), 781-788 (2010)
solid ac ion (polysaccha ide basis wi h esidual
bound p o ein) was analysed o d y ma e con en
and o al Kjeldahl ni ogen. Resul s o decan ing
ex ac ion o p o eins om he polysaccha ide
esidue emaining a e enzyma ic hyd olysis o
ama an h lou p o eins a e indica ed in Table 2.
De e mined alues o adso p ion
coe icien
(K)
exhibi ela i ely la ge sca e , hei
mean alues a e qui e high: K=469 wi h a 20-min
washing cycle and K=418 wi h a 60-min washing
cycle. High le els o adso p ion coe icien p o e he
conside able s eng h bonding p o ein o
polysaccha ide basis; hence, decan ing ex ac ion
is o low e iciency and economically dispu able.
CONCLUSIONS
Ou aim was o de elop a echnology o
isola ing ama an h p o ein. The selec ed me hod was
a biochemical p ocedu e consis ing in employing
enzymes ac ing speci ically on p o ein deg ada ion.
Fi e enzyma ic p epa a ions (p o einases) we e
selec ed o his pu pose. Wa e -soluble low-
molecula p o ein hyd olysa e was sepa a ed om
solid polysaccha ide ac ion (s a ch) by
cen i uga ion. F om esul s i is ob ious ha he
highes e iciency in lique ying ama an h lou
p o eins was exhibi ed by enzyme Alcalase. Fi e-
hou hyd olysis a 50 oC p oduced 37 % p o ein
lique ac ion. The adso p ion coe icien (
K
)
exp essing he s eng h bonding p o ein o
polysaccha ide emnan is qui e high, which
demons a es conside able s eng h o his bond.
Decan ing ex ac ion o p o eins is o low e iciency
and economically doub ul. Composi ion, p ope ies,
pas , p esen and u u e po en ial applica ions o
pa icula ama an h componen s demons a es he
ood po en ial o his c op. Ne e heless, p oblems
conce ning comme cialisa ion o ama an h s ill las ,
pa icula ly due o insu icien expe imen al da a.
In ag icul u e, o example, i is necessa y o s udy
mo e closely speci ic soil equi emen s o ama an h
nu ien s, how o inc ease ha es s, ama an h
composi ion in a ious phases o g ow h, e c, as
well as he choice o ama an h a ian s o he mild
clima e. In he ood indus y, esea ch and
de elopmen wo ks should ocus on s o age
p ope ies, unc ionali y o ce eal ama an h and
ama an h o p o ein concen a es. Fu he mo e, a
quali y de e ing comme cialisa ion is he small size
o ama an h g ains. Fo his eason, scien i ic eams
a e conce ned wi h he sea ch o a ian s yielding
bigge g ains o , on he o he hand, adap ing
handling echnologies o small g ains. The chie
challenge o esea ch and de elopmen comp ises
inco po a ing ama an h in s anding oods u
ecipes, modi ying hei unc ional and nu i i e
quali y, c ea ing comple ely new p oduc s om co n
and om ama an h. I u he comp ises e alua ing
quali y o ama an h-based oods u s and e ec s
o pa icula componen s on he heal h o man,
e alua ing and de e mining he mos sui able a ian
as eed o li es ock. No las , elabo a ing
p ocedu es o isola e pa icula componen s o
ama an h h ough ac iona ion, and o iden i y hei
physiological ac i i y. And o use as ood addi i es,
p oducing modi ied ama an h p o ein isola es wi h
imp o ed unc ional p ope ies.
ACKNOWLEDGEMENTS
The au ho s would like o hank o Minis y
o Educa ion o The Czech Republic o inancial
suppo o his wo k execu ed unde MSM G an
No. 7088352102.
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