Enzymic Hydrolysis of Ox-Blood Hemoglobin

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.


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


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



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 .


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

d) Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 1, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch017

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


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


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)


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


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



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.,


Enzymic Hydrolysis of Ox-Blood Hemoglobin

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