17 Enzymic Hydrolysis of Ox-Blood Hemoglobin
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K. J. STACHOWICZ Institute of Fermentation Industry, Rakowiecka 36, Warsaw, Poland C. E . ERIKSSON and S. T J E L L E SIK, The Swedish Food Institute, Fack, S-40021 Göteborg, Sweden
In the s e a r c h f o r n o v e l , cheap p r o t e i n s to be used i n food e i t h e r as s u b s t i t u t e s f o r meat, f o r fortification purposes or as f u n c t i o n a l additives, the b l o o d of s l a u g h t e r e d a n i m a l s has been surprisingly little considered. Even though some whole b l o o d i s used as an i n g r e d i e n t i n a few t y p e s o f foods i n a few a r e a s i n the w o r l d , w i d e r use o f b l o o d is hampered primarily for religious and ethical r e a s o n s and by its characteristic f l a v o r and c o l o r . In a d d i t i o n to this, attempts to use b l o o d in c e r t a i n foods as a combined l p r o t e i n and i r o n fortifier and coloring agent have failed due to the enhancing o f lipid o x i d i a t i o n and off-flavor development c a t a l y z e d by the i r o n - p o r p h y r i n group p r e s e n t i n the hemoglobin (1). T h i s k i n d of catalytic activity may a l s o be i n c r e a s e d by denaturation of hemoproteins e . g . by heat (2). The total amount o f b l o o d r e p r e s e n t s a g r e a t d e a l o f p r o t e i n , which to day is used to o n l y a limited e x t e n t as an animal f e e d . O f t e n , however, it g i v e s rise to s e r i o u s pollution of water recipients and s h o u l d t h e r e f o r e be s u b j e c t to i n t e n s e s t u d i e s c o n c e r n i n g its p r o c e s s i n g into v a r i o u s p r o t e i n p r o d u c t s f o r f o o d u s e . In Sweden most blood is collected under strict h y g i e n i c c o n d i t i o n s i n immediate c o n n e c t i o n w i t h the s l a u g h t e r o f cattle and p i g s . T h i s b l o o d is then stabilized by sodium citrate, t r a n s p o r t e d c o l d to c e n t r a l plants where the plasma is s e p a r a t e d from the red cells. Plasma, e i t h e r liquid, concentrated, f r o z e n , or d r i e d is used i n the manufacture of meat p r o d u c t s , e . g . sausage m a i n l y due to its good e m u l s i fication and b i n d i n g p r o p e r t i e s . The r e d cell fraction is d r i e d and m o s t l y used i n a n i m a l f e e d i n g , a
295 Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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296
ENZYMES IN FOOD AND BEVERAGE PROCESSING
s m a l l p a r t in f o o d . In Sweden about 2000 tons of b l o o d p r o t e i n mainly hemoglobin, is available annually i n the r e d cell fraction. T h i s q u a n t i t y r e p r e s e n t s , howe v e r , l e s s than 1% of the Swedish p r o t e i n consumption to d a y . S i n c e p r o t e i n is overconsumed in our c o u n t r y , p r o d u c t i o n of b l o o d p r o t e i n f o r human nutrition is not r e l e v a n t . Hence, utilization of b l o o d p r o t e i n s s h o u l d be p r i m a r i l y based more on their p o s s i b l e technical f u n c t i o n s i n f o o d like their b i n d i n g p r o p e r t i e s , their use as f l a v o r p r e c u r s o r s , c o n s i s t e n c y r e g u l a t o r s , meat e x t e n d e r s , e t c . I t i s thus b e l i e v e d t h a t a c c e s s to a wide range of b l o o d p r o t e i n p r o d u c t s w i t h differ e n t p r o p e r t i e s s h o u l d be advantageous. The o l d e s t and still most w i d e l y employed approach is the p r e p a ration of a d e c o l o r e d p r o t e i n p r o d u c t from the r e d cell fraction by removal o f the heme group from hemog l o b i n through the a c i d - a c e t o n e method ( 3 , 4) or m o d i fications of it. In t h i s p r o c e s s apohemoglobin can be precipitated w h i l e the hematin remains in the mixed acetone-acid-water phase. Acid
acetone
Hemoglobin
> Apohemoglobin(s)+ hematin
A g r e a t d e a l o f work has been c a r r i e d out to o p t i m i z e t h i s p r o c e s s a c c o r d i n g t o p r o t e i n y i e l d , product c o l o r , f u n c t i o n a l p r o p e r t i e s , solvent r e c y c l i n g , explosion safety, etc. In A u s t r a l i a the c o s t of a f u l l s c a l e p l a n t f o r production of a decolored p r o t e i n product from whole b l o o d has been e s t i m a t e d , on the b a s i s o f extensive p i l o t plant investigations . In our l a b o r a t o r y a n o t h e r method has been a p p l i e d f o r o b t a i n i n g p r o t e i n p r o d u c t s from the r e d c e l l f r a c t i o n , i n c l u d i n g a decolored product. P r e l i m i n a r y work r e v e a l e d t h a t enzymic h y d r o l y s i s of hemoglobin f o l lowed by g e l f i l t r a t i o n or u l t r a f i l t r a t i o n y i e l d e d h y d r o l y s a t e f r a c t i o n s which c o n t a i n e d o n l y s m a l l amounts of heme. These i n v e s t i g a t i o n s w i l l be publised later. However, some l a r g e - s c a l e work was a l s o c a r r i e d out i n o r d e r to produce s i n g l e , o n e - k i l o g r a m b a t c h e s of ox b l o o d p r o t e i n h y d r o l y s a t e f o r e x p e r i mental f o o d u s e . T h i s work w i l l be p r e s e n t e d i n what follows. Pretreatment
of red
cells.
Most p r o t e o l y t i c enzymes t e i n s b e t t e r than n a t i v e ones s t e r i c u n a v a i l i b i l i t y of b o t h tive substrate. The r e d c e l l
a t t a c k denatured p r o due to the i n h e r e n t the enzyme and the n a f r a c t i o n obtained a f t e r
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
17.
plasma
separation
35-kO°/o
of
dry
by
The
hemolyzing
Denaturation by
a l k a l i
formed
treatment
and
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ration
effect
to
a
to
fram
ox
of
drawn
up
addition natant
was
content
1,
was
reported ethyl
released
with
by
water.
accomplished
by
per
a l k a l i
denaturation
alcohol
and
( j 5 , 7^)·
protein
heat
The
measurement
was
at
20°C
2,
and
were
denatu
of
in
the
the
presence
The
adding
c e l l
reached. samples
h
2k
then
by
red
and
f i r s t
diluted
k°/o.
about
performed
hemolyzed
1 Ν HC1 and further
of
them was
inorganic i s
pre-investigation
protein
p H 11
samples
of
protein
treated
of
h
2k
0.5,
The
cells
enzyme.
u n t i l
to
after
ments.
the
sample
blood
retained
soy
treatment
stirred
a
the
some
denaturation
estimated
proteolytic A l k a l i
after compare
containing
and
d i l u t i n g
dissolved
combined
was
rate
by
previously
applied
hydrolysis a
by
involving
treatment
of
to
l i q u i d
protein within
simply
the
order
viscous
protein
them, of
a
mainly
treatment,
in
methods
forms
matter
constituents.
297
Hydrolysis of Ox-Blood Hemoglobin
STACHOWICZ ET AL.
for
pH
were
with
with
water
pH 9
to
The a
factor
was
measure
to
centrifuged.
d i l u t i o n
Ν NaOH
This
k i n e t i c
adjusted
5
fraction
by
the
super
hemoglobin was
always
9. The in
a
enzyme
pH-stat
Switzerland). k°/o o f
about in
the
of
alcalase
a c t i v i t y
(Mettler Twenty
water
™
(NOVO,
consumed
being
10
min.
The
at
50
and
continuously
p H 9·
By
between
a l k a l i
and
combined
(Table in
I ) .
the
subsequent
Large-scale A l k a l i 1,
(k5-k6.5 was
grade,
owing
out
at
in
a
in
Figuve
both and
at
i t s
1 ml
for
superior
treatment
of
f i r s t
The 0.01
Ν NaOH
difference
p H 11
9
mg
/ u m o l 0H~~
with
was
one
alcohol-heat
scale
denatured, in
pH 9
to
treatment simplicity
was
preferred
operations.
1.
during feed
during
f i l t r a t e d water) and
enzyme
membrane
ture-controlled tration
to
a l k a l i
large
3.3-3-9%
proteolytic
shown
r i e d
ethyl
no
5
in
the
during
out
pH
operations.
hydro l y z e d
Denmark) as
Hence,
r e p r o d u c i b i l i t y ,
added,
method
to
Then
Denmark)
recorded
11,
containing
adjusted
was
treatment
performed
11 + D V
pH-stat.
carried
this
two and
f i r s t the
was
were
10 + D K
substrate
p H 9)
t i t r a t i o n
the
of
DK
Copenhagen, to
found h
of was
vessel
(pre-adjusted
C
ml
hemoglobin,
t i t r a t i o n
measurements
system:
by
means
time
before
volume
protein
of
(NOVO,
p r i n c i p a l l y
enzymic
certain
tank
the
C
blood
hemolyzed
a l c a l a s e ®
reactor The
a
50
ox
from
reaction i n
the
starting
reduction
red the
cells food
Copenhagen, designed was
car
tempera u l t r a f i l
period
that
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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298
E N Z Y M E S I N FOOD A N D B E V E R A G E PROCESSING
Γ
Temperature controlled feed tank (1)
Recycling of reject
Φ
(2)
(3)
)
48
1661
blood
membrane
(s)
k
start
i n
Running
(i) Protein
i n
respectively. Looking can see that both the tration increased with a small increase while the c o n c e n t r a t i o n i n -
u l t r a f i l t r a t i o n
proteins and
shown
a f a i r l y large, f i n a l y i e l d of was o b t a i n e d ; 83°/o, 73, a n d 72 in
t h e X M 5 0 , PM 1 0 , a n d PM 5 r u n s at the intermediate yields, one dry matter and the hemin c o n c e n t i m e . T h e X M 50 r u n s h o w e d o n l y in b o t h t h e PM 10 a n d PM 5 r u n s Table
are
2.9
U l t r a f i l t r a t e
0.6 0.7 0.5 0.7 0.9 0.9 0.9 1.5
1 .1 1 .2 0.9 1.4 1-5 1.5 1-5 2.9
5 5 5 5 5 5 5 5
170 174 175 184 176 177 180 188
ko ko
1424
12.0
0.8
1416
12.2
0.9
R e c o v e r y (°/o)
89
85
Membrane:
XM 5 0 .
fraction
No.
1
2 3 4 5 6 7 8 Sum,
fractions
Average
f i l t r a t e
Romicon
Cut
25 off:
50.000.
2.46
Area:
Pressure, inlet: 103 k P a ( 1 . 1 k p / c m , 15 l b / i n Pressure, outlet: 69 k P z ( 0 . 7 k p / c m , 10 l b / i n Flux: 89 l / m χ h . Enzyme: A l c a l a s e , 10.1 g.
m
). ).
Temperature: 4 8 . 5 - 51 C . T i m e : Pre-hydrolysis 107 m i n . u l t r a f i l t r a t i o n 13 m i n . T o t a l N a O H c o n s u m p t i o n : 3 · 0 m o l .
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
17.
Table
III.
H y d r o l y s i s and u l t r a f i l t r a t i o n o f ox b l o o d p r o t e i n s w i t h a l c a l a s e i n a P M 10 m e m b r a n e reactor. Composition of s t a r t i n g material and u l t r a f i l t r a t e . Running conditions. Volume
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Protein
46,5
start
Ultrafiltrate fraction No. 1
Sum,
Total dry matter
Total hemin
ω
ω
1548
2.5
0.061
0.005
1121
0.117
0.010
fractions
41 . 0 4i.0 88
Μ
1121
109 259 325 428
R e c o v e r y (°/o)
38.8
0.006 0.003 0.004 0.007
10.0 10.0 10.0 1 1 .0
f i l t r a t e
Dry matter hemin
0.007 0.007 0.014 0.033
2 3 4
Average
301
Hydrolysis of Ox-Blood Hemoglobin
STACHOWICZ ET AL.
72
0.3
Membrane: R o m i c o n PM 10. Cut o f f : 10.000. Area: 2.46 m2 ( 2 6 . 5 f t ) . Pressure, inlet: 138 k P z (l.1kp/ cm , 20 l b / i n | ) . Pressure, outlet: 103 k P a ( 1 . 1 kp/ cm , 15 l b / i n ). Flux: 12,7 l / m x h. Enzyme: Alcalase, 22.5 g . Temperature: 49.5 50.5°C T i m e : P r e - h y d r o l y s i s 21 m i n , u l t r a f i l t r a t i o n 64 m i n . T o t a l NaOH c o n s u m p t i o n : 3·0 m o l . p
2
2
c r e m e n t was m u c h l a r g e r f r o m t h e f i r s t to the last f r a c t i o n . The hemin content of the u l t r a f i l t r a t e dry m a t t e r was a l w a y s s i g n i f i c a n t l y l o w e r t h a n t h a t o f the s t a r t i n g m a t e r i a l . The average u l t r a f i l t r a t e dry matter contained 0.9, 0.01, a n d 0.02% o f h e m i n , t h u s a 3 - , 2 5 O - , a n d 1 5 0 - f o l d heme r e d u c t i o n i n t h e XM 5 0 , PM 1 0 , a n d PM 5 r u n s r e s p e k t i v e l y . It s h o u l d be n o t e d t h a t the f i g u r e s o f heme c o n t e n t , particularly in the PM 10 a n d PM 5 e x p e r i m e n t s , a r e somewhat uncertain s i n c e i t i s j u s t on the v e r g e o f d e t e c t a b i l i t y with the method used (8). The three average ultrafiltrates were freezed r i e d a f t e r h a v i n g been n e u t r a l i z e d by the a d d i t i o n of HC1, c i t r i c o r l a c t i c a c i d . The moisture and ash con t e n t were d e t e r m i n e d i n the f r e e z e - d r i e d p r o d u c t s . The results i n T a b l e V show t h a t the d r y m a t t e r contained 13-16% o f a s h . T h e a d d e d N a O H f o r p H - a d j u s t m e n t d u r i n g the r e a c t i o n and the a c i d used f o r n e u t r a l i z a t i o n be f o r e f r e e z e - d r y i n g a c c o u n t f o r a b o u t two t h i r d s o f the
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
302
E N Z Y M E S I N FOOD A N D B E V E R A G E
Table
IV.
H y d r o l y s i s and u l t r a f i l t r a t i o proteins with alcalase in a reactor. Composition of sta and u l t r a f i l t r a t e . Running Volume
Total dry matter
U)
(1)
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Protein
Ultrafiltrate fraction No.
Sum,
n o f ox b l o o d PM 5 m e m b r a n e rting material conditions.
Total hemin
Dry matter hemin
(e)
W 3.0
52.5
1767
start
PROCESSING
2 3 4 5 6 7 8
5 5 5 5 5 5 5 5
73 97 120 143 168 195 226 263
0.016 0.011 0.012 0.018 0.025 0.037 0.052 0.081
0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.03
fractions
ko
1285
0.252
0.02
ko
1307
0.292
0.02
89
73
Average
1
f i l t r a t e
R e c o v e r y (°/o)
0.6
Membrane: R o m i c o n PM 5 . Cut o f f : 7.000. A r e a : 2.18 m (23.5 p ) Pressure, inlet: 138 k P a ( 1 . 4 kp/cm? 20 l b / i n ) . Pressure, outlet: 103 k P a ( 1.1 kp/cm , 15 l b / i n ). Flux: 28 l / m χ h. Enzyme: Alcalase 22.5 g . T e m p e r a t u r e : 50 - 5 1 ° C . Time: Pre-hydrolysis 7.5 min, ultrafiltration 44.5 m i n . T o t a l NaOH c o n s u m p t i o n : 3-2 m o l . f
t
2
2
Table
V.
Some p r o p e r t i e s n e u t r a l i z e d and a t e s f r o m ox b l o was made e i t h e r c i t r i c acid.
Average
Hemin
u l t r a f i l t r a t e
of hydrolyzed, ultrafiltrated, freeze-dried protein hydrolysod red c e l l s . Neutralization with hydrochloric acid or
Moisture (°/o)
Ash
W)
50
0.9
5.8
12.9
PM
10
0.01
4.9
PM
5
0.02
6.1
XM
Color Chloride
Citrate light brown
16.3
dark brown beige
14.0
beige
cream
cream
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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17. STACHOWICZ ET AL. Hydrolysis of Ox-Blood Hemoglobin
303
ash content, while the r e s t o r i g i n a t e s from the mineral content o f the r e d c e l l f r a c t i o n . The c o l o r of the dry p r o d u c t d e p e n d e d on the a c i d u s e d f o r neutralization. H y d r o c h l o r i c a c i d (and l a c t i c a c i d ) gave brown products, whilst c i t r i c a c i d gave c r e a m - c o l o r e d ones. Particularly the product treated with lactic acid was v e r y h y g r o s c o p i c . P r o d u c t s f r o m t h e PM 10 a n d PM 5 runs were somewhat b i t t e r , no s i g n i f i c a n t difference in b i t t e r n e s s b e i n g f o u n d when e a r l y a n d l a t e f i l t r a t e fractions were compared. F u r t h e r work on the use of blood protein hydrolysates w i l l include investigations on the p o s s i b l e c o n t r o l o f the amount o f i n o r g a n i c cons t i t u e n t s and b i t t e r n e s s as w e l l as i n v e s t i g a t i o n s on the f u n c t i o n a l p r o p e r t i e s of these products.
Abstract T h i s paper p r e s e n t s evidence t h a t enzymic hydrolysis under slightly alcaline c o n d i t i o n s can be used to p r e p a r e p r o t e i n h y d r o l y s a t e s w i t h a v e r y low heme c o n t e n t from the p r o t e i n m a i n l y h e m o g l o b i n , i n the r e d cells of c a t t l e b l o o d . The r e a c t i o n s were c a r r i e d out in a h o l l o w f i b e r membrane r e a c t o r , b o t h the r e a c t i o n and the p r o p e r t i e s of the product b e i n g a f f e c t e d by the c h o i c e o f membrane. The appearance o f the p r o d u c t s c o u l d be f u r t h e r a f f e c t e d by the c h o i c e of a c i d f o r neutralization o f the h y d r o l y s a t e . The p r o d u c t s o b t a i n ed had a fairly h i g h content of ash and were somewhat bitter. Literature cited (1) Tappel, A.L., in "Autioxidation and antioxidants" L u n d b e r g , W . O . , ed., Vol. I , p . 3 2 5 , I n t e r s c i e n c e Publ., New York 1 9 6 1 . (2) Eriksson, J.
Am.
(3) Lewis, (4) (5)
C.E., Oil
U.J.,
Olsson,
Chem. S o c . J.
Biol.
P.,
and Svensson,
(1971)
Chem.
48,
(1954)
S.,
442. 206, 109.
A n s o n , M.L., and M i r s k y , A.E., J. Gen. Physiol. ( 1 9 3 0 ) 13, 469. W i l s o n , B.W. in P r o c e s s I n d u s t r i e s o f Australia Impact and growth, N a t i o n a l C h e m i c a l E n g i n e e r i n g C o n f e r e n c e , p. 431, S u r f e r s P a r a d i s e , Q u e e n s l a n d , 1974.
( 6 ) Fukushima,
D.,
C e r e a l Chem.
(1969)
46,
156.
(7)
Fukushima,
D.,
C e r e a l Chem.
(1969)
46,
405.
(8)
P a u l , K.G., Theorell, H., and Åkesson, A c t a Chem. S c a n d . ( 1 9 5 3 ) 7 , 1 2 8 4 .
Å.,
Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.