and Hyperfiltration During Production of ... - ACS Publications

10 Application of Ultra- and Hyperfiltration During Production of Enzymatically Modified Proteins

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 2, 2015 | http://pubs.acs.org Publication Date: May 27, 1981 | doi: 10.1021/bk-1981-0154.ch010

H. SEJR OLSEN and J. ADLER-NISSEN Enzyme Applications Research and Development, Novo Industri A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark A p p l i c a t i o n of membrane processes during production of purified food p r o t e i n s i s a m i l d treatment which ensures that the f u n c t i o n a l p r o p e r t i e s of the n a t i v e p r o t e i n s are r e t a i n e d . (1) These p r o p e r t i e s are mostly found t o be s u p e r i o r t o those of denatured p r o t e i n s . However, not all p o s s i b l e needs o f the modern food i n d u s t r y are fulfilled by using n a t i v e p r o t e i n s i n s t e a d of denatured ones. Therefore, enzymatic m o d i f i c a t i o n of p r o t e i n s has been demonstrated as a p o s s i b l e means o f meeting the needs of the food i n d u s t r y f o r h i g h - q u a l i t y p r o t e i n i n g r e d i e n t s ( 2 ) , (13), (14). Membrane processes have a potential a p p l i c a t i o n w i t h i n many areas of industrial enzymatic h y d r o l y s i s of p r o t e i n s . Table I shows how membrane processes can be a p p l i e d i n the d i f f e r e n t types of enzymatic m o d i f i c a t i o n of p r o t e i n . Thus membrane processes may be used f o r pre-treatment of p r o t e i n s , f o r the r e a c t i o n step and as an essential p a r t of the purification o r post -treatment step. In the f o l l o w i n g , r e s u l t s from our work w i t h these processes i n Novo's pilot p l a n t f o r Enzyme A p p l i c a t i o n will be presented. The r e s u l t s demonstrate t h a t the f u n c t i o n a l p r o p e r t i e s of some of the p r o t e i n products obtained were improved to such an extent that the membrane processes may become very important in the modern p r o t e i n technology. Owing to the i n t e r e s t i n g p r e l i m i n a r y r e s u l t s obtained regarding f u n c t i o n a l p r o p e r t i e s , l e s s a t t e n t i o n has been paid to a thorough i n v e s t i g a t i o n of the u n i t operations as such. General C h a r a c t e r i s t i c s of Enzymatic H y d r o l y s i s . As e a r l i e r reported (2_) , a l i m i t e d h y d r o l y s i s of a p r o t e i n product may improve c e r t a i n f u n c t i o n a l p r o p e r t i e s such as whipping and emulsifying capacity. These improvements are dependent on the enzyme and on the p r i n c i p l e by which the r e a c t i o n i s c o n t r o l l e d . The p r e f e r a b l e way of c o n t r o l l i n g the m o d i f i c a t i o n i s by a p p l i c a t i o n of the pH-stattechnique, by which the base consumption used f o r maintaining pH 0097-6156/81/0154-0133$09.25/0 © 1981 American Chemical Society

In Synthetic Membranes: Volume II; Turbak, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


134

SYNTHETIC

MEMBRANES:

H F AND U F

USES

during the p r o t e o l y t i c r e a c t i o n i s d i r e c t l y converted to degree of h y d r o l y s i s 02). Degree of h y d r o l y s i s , DH, i s the p r o p o r t i o n between the number of peptide bonds cleaved and the t o t a l number of peptide bonds i n the i n t a c t p r o t e i n ( 3 ) . P r o t e o l y t i c a l l y modified p r o t e i n s which have been thoroughly enzyme digested are low molecular p r o t e i n h y d r o l y s a t e s . Such products have o f t e n l e s s pronounced foaming o r e m u l s i f y i n g propert i e s than p r o t e i n s which have been only s l i g h t l y hydrolyzed (2) . However, a need f o r t h i s k i n d of p r o t e i n products appears i n the beverage i n d u s t r y f o r enrichment o f s o f t d r i n k s w i t h p r o t e i n and i n the meat i n d u s t r y f o r pumping of whole meat cuts w i t h low cost p r o t e i n s . Important p r o p e r t i e s of low molecular p r o t e i n h y d r o l y sates are a bland t a s t e and a complete s o l u b i l i t y over the wide pH-range used i n foods. The production method f o r low molecular p r o t e i n hydrolysates has been described e a r l i e r (40 . A c o n t r o l l e d batch h y d r o l y s i s using the pH-stat i s performed, and the p r o t e i n hydrolysate i s then recovered by e.g. s o l i d s s e p a r a t i o n . H y p e r f i l t r a t i o n may be used f o r c o n c e n t r a t i o n and/or d e s a l i n a t i o n . Instead of using the c o n t r o l l e d batch h y d r o l y s i s and s o l i d s s e p a r a t i o n processes, the s e p a r a t i o n of peptides may be performed from an enzyme-substrate r e a c t i o n mixture under continuous u l t r a f i l t r a t i o n i n a s o - c a l l e d membrane r e a c t o r . H i g h l y F u n c t i o n a l Soy P r o t e i n s Native soy p r o t e i n i s o l a t e may be produced by u l t r a f i l t r a t i o n of an aqueous e x t r a c t of d e f a t t e d soy bean meal, (1), ( 5 ) . The process layout i s shown i n F i g . 1. A c a r e f u l s e l e c t i o n of membrane parameters such as flow v e l o c i t y , pressure drop, temper a t u r e , and of the type of membrane and modules i s important i n order to o b t a i n a bean p r o t e i n i s o l a t e by a d i r e c t u l t r a f i l t r a t i o n of the c l a r i f i e d e x t r a c t ( 5 ) . The p r o t e i n i s o l a t e has a prot e i n - d r y matter r a t i o higher than 90% (N x 6.25), when using t h i s process. H i t h e r t o , enzymatic m o d i f i c a t i o n of u l t r a f i l t e r e d soy prot e i n s has not been described. The present i n v e s t i g a t i o n shows that p r o t e i n products w i t h b e t t e r p r o p e r t i e s than e n z y m a t i c a l l y modified a c i d p r e c i p i t a t e d p r o t e i n s can be produced by a s u i t a b l e combination of the i n v o l v e d u n i t o p e r a t i o n s . M o d i f i c a t i o n of U l t r a f i l t e r e d versus A c i d P r e c i p i t a t e d Soy P r o t e i n . When the r e t e n t a t e obtained from the u l t r a f i l t r a t i o n of soybean e x t r a c t i s subjected to an enzymatic h y d r o l y s i s as described e a r l i e r (2) f o r a c i d p r e c i p i t a t e d p r o t e i n , a h y d r o l y s i s curve (DH versus time) may be drawn. A comparison of such h y d r o l y s i s curves i s shown i n F i g . 2 f o r a c i d p r e c i p i t a t e d soy p r o t e i n i s o l a t e and u l t r a f i l t e r e d soy p r o t e i n i s o l a t e . The curves a r e drawn on the b a s i s of the same h y d r o l y s i s parameters. The enzyme used i s the m i c r o b i a l a l k a l i n e protease s u b t i l i s i n C a r l s b e r g (ALCALASE® ).


O L S E N A N D ADLER—NISSEN

Table I.

TYPES

OF

MATIC

Enzymatically Modified Proteins

1

Application of Membrane Processes During Enzymatic Modification of Proteins

ENZY-

ENZYMATIC

MODIFIED

PRETREATMENTS

REACTION

STEP

POSTTREATMENTS


PROTEINS

HIGHLY

PRODUCTION

FUNCTIONAL

NATIVE

PROTEINS

ISOLATE

OF

MOLECULAR

-

PROTEIN

SEPARATION

BY UF

BY

UF

CONCENTRATION

LOW

-

AND/OR

MOLECULAR

PROTEIN

MEMBRANE

-

REACTOR

CONCENTRATION BY

AND/OR

UF

UF

~

ULTRAFILTRATION

HF

~

HYPERFILTRATION

Soy

BY HF

HYDRO-

LYZATES

meal

50% (Nx6.25)

DESA-

LINATION

DESA-

LINATION

BY HF

Water

T\ Extraction

(Liq./solid-ratio:

Extract

4% ( N x 6 . 2 5 )

10:1)

_j

C oonncceei n t r a t e 2 2 - 2 5 %

(Nx6.25)

Centrifuge

Remanence (sludge) 8% ( N x 6 . 2 5 )

filtration (batch)

meate 0.15% (Nx6.25) Spray drying

Figure 1.

Protein Q

Q

%

isolate

( N x 6 > 2

5)

Production of soy protein isolate by UF in pilot plant



136

SYNTHETIC

MEMBRANES:

HF

AND

UF

USES

I t appears from F i g . 2 t h a t the u l t r a f i l t e r e d soy p r o t e i n i s o l a t e i s hydrolyzed c o n s i d e r a b l y more slowly than the a c i d prec i p i t a t e d p r o t e i n . This i s due to the compact molecular s t r u c t u r e of the u l t r a f i l t e r e d p r o t e i n , which i s s t i l l i n the n a t i v e s t a t e . That the degree of denaturation of a p r o t e i n s u b s t r a t e has a profound i n f l u e n c e on the k i n e t i c s of the p r o t e o l y s i s has been known f o r long, see Christensen ( 6 ) . I t should be noted that s u b t i l i s i n C a r l s b e r g i s not i n h i b i t e d by the protease i n h i b i t o r s present i n n a t i v e bean p r o t e i n ( 7 ) . Using the u l t r a f i l t e r e d soy p r o t e i n i s o l a t e described prev i o u s l y ( F i g . 1 ) , a s e r i e s of hydrolysates covering a range of DH-values (DH = 0 to DH = 6%) was made i n the l a b o r a t o r y using the method o u t l i n e d i n F i g . 3. In a l l cases the h y d r o l y s i s was terminated by a d d i t i o n of HC1 to pH = 4.2 to i n a c t i v a t e the enzyme. A f t e r 30 minutes, pH was readjusted to pH = 7.0 by using NaOH. NaCl was added u n t i l the f i n a l c o n c e n t r a t i o n i n a 10% prot e i n (N x 6.25) s o l u t i o n was 0.25 M NaCl ( 2 ) . The whipping expansion was determined by 4 minutes whip of a s o l u t i o n having 3% p r o t e i n ( 2 ) . F i g u r e 4 shows whipping expans i o n versus DH f o r u l t r a f i l t e r e d and a c i d - p r e c i p i t a t e d soy prot e i n s modified by A l c a l a s e . S i m i l a r r e s u l t s have a l s o been found f o r u l t r a f i l t e r e d and a c i d - p r e c i p i t a t e d p r o t e i n s from faba beans ( V i c i a faba) ( S e j r Olsen, unpublished r e s u l t s ) . I t appears from F i g . 4 that i n the case of the a c i d - p r e c i p i t a t e d p r o t e i n , higher DH-values cause a d i s t i n c t r e d u c t i o n of the whipping expansion. The i n c r e a s i n g content of small peptides r e s u l t i n g from a more pronounced degradation of the p r o t e i n s at the high DH values i s assumed to be r e s p o n s i b l e f o r t h i s r e d u c t i o n of foaming a b i l i t y . Removal of the small peptides during the proc e s s i n g of the u n r e f i n e d soy meal to the f i n a l whipping agent might t h e r e f o r e have a p o s i t i v e e f f e c t on the foaming a b i l i t y . Experimental D e t a i l s . In order to examine the above hypot h e s i s , the enzymatic h y d r o l y s i s was c a r r i e d out at d i f f e r e n t stages during the soy i s o l a t e process. Four process combinations examined i n our p i l o t p l a n t are o u t l i n e d s c h e m a t i c a l l y i n F i g . 5. In a l l cases the f o l l o w i n g procedure was used: D e f a t t e d , dehulled white soy meal from Aarhus O l i e f a b r i k A/S was e x t r a c t e d w i t h water at pH = 8.0 using a l i q u i d : s o l i d r a t i o of 10 : 1. A l l c e n t r i f u g a t i o n s were c a r r i e d out i n a W e s t f a l i a solids ejecting centrifuge.

SB-7

A l l u l t r a f i l t r a t i o n s were made on a DDS-modul type 35 at 50°C using polysulphone membranes type GR6-P at 3 kp/cm . The module had 2.25 m2 of membrane area. 2

Hydrolyses were made by use of ALCALASE 0.6 i n pH-stat.

L at pH =


8.0

OLSEN A N D ADLER—NISSEN

Emymatically Modified Proteins

137

% DH'

pH -

12


Soy

Cone, of substrate: _ S= 8% N x 6 . 2 5

16

protein

Purin?.

isolate

500 E ^

8.0

50°C

Cone, o f enzyme: E/S = 2 . 0 % A l c a l a s e (E = 0 . 1 6 % )

0.6L

-

8

Soy p r o t e i n i s o l a t e ultrafi1tered

4

r

i 30

i

1

60

120

i

_

i

180

240 min

Figure 2.

Hydrolysis curves for soy protein isolates

> ^ ^ D i l u t e d (Soy

UF-retentate

V^_8%

Nx6.25

ALCALASE

pH-STAT

4N

at

6N

NaOH

HC1

pH

j y

hydrolysis

=

8 . 0 ,

50°C

\

Enzyme

i n a c t i v a t i o n

pH

50°C,

4 . 2 ,

30

min.

• 4N

NaOH

»•

Adjustment pH

to

of

7.0

• Sol i d

NaCl

Adjustment 14.6g

Freeze

Figure 3.

to

NaCl/lOOg

Nx6.25

drying

Laboratory method for proteolytic modification of ultraflltered soy protein isolate


138

SYNTHETIC

i

i

i

% Whipping

i

r— i

T

MEMBRANES:

1

H F A N D U F USES

•

expansion

1600 Ultrafiltered

-


1200

800

- i

/

/ /

/ /

/ /

400

A*

Acid

/ T 1

i

i 2

3

i

i

i

i

i

4

5

6

7

8

precipitated

% DH

Figure 4.

Whipping expansions vs. DH for soy protein hydrolysates

EXTRACTION

EXTRACTION

1 HYDROLYSIS

CENTRIFUGATION

1 INACTIVATION

CENTRIFUGATION

1

1

1

ULTRAPILTRATIOR"

•OLTRAPIETRATION"

1

1

' H Y D R O L Y S I S '

' H ? G R 0 C ? 5 I 5 '

1 CENTRIFUGATION

1 DRYING

CENTRIFUGATION

1 INACTIVATION

1

"OCTRAFILTRATIUR"

EXTRACTION

1 HYDROLYSIS

1 CENTRIFUGATION

EXTRACTION

!

INACTIVATION 1

1

1 "OCrariCTRATTOR"

1

1 INACTIVATION

DRYING

CENTRIFUGATION

I

I

DRYING

1 III

'OCTRAPICTRATIOflJ l

II DRYING

IY Figure 5.

Process combinations investigated for production of highly functional enzymatically modified soy proteins


10.



139

In a l l cases h y d r o l y s i s to DH = 3% and DH = 6% was made and i n a c t i v a t i o n was c a r r i e d out a t pH = 4.0 (50°C) f o r 30 minutes. The general h y d r o l y s i s parameters were:


Substrate c o n c e n t r a t i o n s : Enzyme/substrate r a t i o : Temperature: pH:

S = 3 to 8% (N x 6.25) E/S = 2% ALCALASE 0.6 L T = 50°C pH = 8.0

Some of the d i f f e r e n t u l t r a f i l t r a t i o n processes w i t h i n the four process combinations i n c l u d e d i a f i l t r a t i o n as w e l l . When d i a f i l t r a t i o n was included at a stage, the sequence, u l t r a f i l t r a t i o n - d i a f i l t r a t i o n - u l t r a f i l t r a t i o n , was used i n order to obt a i n a high s e p a r a t i o n e f f i c i e n c y and membrane c a p a c i t y ( 8 ) . The f i n a l p r o t e i n products were analysed and evaluated f o r t h e i r whipping expansion, foam s t a b i l i t y and i n the case of the DH-6%-products f o r baking performance i n a meringue b a t t e r as w e l l . The a n a l y t i c a l procedures are d e s c r i b e d below. Methods of A n a l y s i s Whipping Expansion.

C a r r i e d out as d e s c r i b e d p r e v i o u s l y

(2). Foam S t a b i l i t y . A p l a s t i c c y l i n d e r (diameter 7 cm, h e i g h t 9 cm) having a w i r e net w i t h a mesh s i z e of 1 mm x 1 mm i s f i l led w i t h foam and the amount of foam i s found by weighing (A gram) . The c y l i n d e r i s then placed on a funnel on top of a g l a s s c y l i n d e r of 100 ml. A f t e r 30 minutes the weight (B) of drained l i q u i d i n the g l a s s c y l i n d e r i s determined. The foam s t a b i l i t y FS i s d e f i n e d by the equation: A

FS =

B

~ A

x 100%

Baking Performance of Meringue B a t t e r . To 100 ml of a 12% w/w (N x 6.25) s o l u t i o n a t pH = 7.0 of the whipping agent, 150 g of saccharose i s added. The saccharose i s completely d i s s o l v e d by gentle s t i r r i n g at room temperature. The s o l u t i o n i s then whipped a t speed I I I (260 rpm) f o r 10 minutes i n a Hobart Mixer (model N - 50) u s i n g a w i r e whisk. Immediately a f t e r w a r d s , t e n samples of 10 ml are t r a n s f e r r e d to an aluminium t r a y a t separate p o s i t i o n s by means of a s y r i n g e . Baking i s then performed a t 130°C f o r 1 hour. A f t e r c o o l i n g t o ambient temperature, the weight, the height (h) and the diameter (d) are determined. The volume i s c a l c u l a t e d assuming that the meringue i s a s p h e r i c a l segment having the volume: 1 2 ^ 2 V=-g-xTrx(h +|- x ) Z

z

d

The apparent d e n s i t y i s then c a l c u l a t e d , and the s m a l l e r the dens i t y , the b e t t e r the baking performance, provided that the s u r face i s s t i l l smooth and the shape i s maintained.


140

SYNTHETIC

Amino-acid Analyses. K o l d i n g , Denmark.

MEMBRANES:

HF

AND

UF

USES

C a r r i e d out by B i o t e k n i s k I n s t i t u t ,

TCA-soluble N i t r o g e n . Measured i n 0.8 N t r i c h l o r o a c e t i c a c i d (TCA) u s i n g the method of Becker et a l . ( 9 ) . Nitrogen S o l u b i l i t y .

C a r r i e d out as d e s c r i b e d p r e v i o u s l y

(2).


Free Alpha-amino Groups.

Measured by the TNBS-method (10).

Crossed Immunoelectrophoresis. Weeke (11) .

The method i s d e s c r i b e d by

R e s u l t s and D i s c u s s i o n Data from the membrane p r o c e s s i n g are given i n Table I l a and l i b . Average permeate f l u x e s of the same order of magnitude were seen i n a l l combinations, whether the u l t r a f i l t r a t i o n s were performed on enzyme t r e a t e d p r o t e i n s or on raw bean e x t r a c t . This i n d i c a t e s that p r o t e i n molecules capable of forming a g e l on the membrane surface are s t i l l present a f t e r the enzymatic m o d i f i c a t i o n . The s i z e of the permeate f l u x e s obtained i s i n the i n t e r v a l of about 20-40 l/h/m2, which i s i n the economically a t t r a c t i v e range of the process ( 5 ) . The p r o t e i n y i e l d s shown i n Table I l i a and I l l b are based on 100% recovery of phases. The reason f o r the r a t h e r low y i e l d s are low n i t r o g e n s o l u b i l i t y of the soy meal used, v i z . about 60% at pH = 8. As about 90% of p r o t e i n may be water e x t r a c t e d from a l e s s denatured soy meal (12), the o v e r a l l y i e l d s would be about 50% higher i f such a raw m a t e r i a l i s used. I f the combinations I , I I and IV are compared w i t h respect to the y i e l d s and f u n c t i o n a l p r o p e r t i e s , i t appears that both whipping expansion and foam s t a b i l i t y are h i g h e s t at the h i g h DHv a l u e . However, due to h i g h e r content of low molecular peptides at the high DH-value, the o v e r a l l p r o t e i n (N x 6.25) y i e l d s are lower. The processes have to be evaluated more thoroughly i n o r der to f i n d a compromise between the p r o t e i n y i e l d s and the funct i o n a l i t y wanted. When comparing the f u n c t i o n a l i t y s t u d i e s w i t h the chemical p r o p e r t i e s of the DH = 3%-products g i v e n i n Table I l i a , no s i g n i f i c a n t c o r r e l a t i o n i s found between T C A - s o l u b i l i t y , p s i ( p r o t e i n s o l u b i l i t y index) at pH = 4.5 or the p s i at pH = 7.0. Omitting the I I I combination which i s made by a method which r e t a i n s some denatured p r o t e i n (see ( 2 ) ) , only the content of leu-NH^ equival e n t s s i g n i f i c a n t l y c o r r e l a t e s w i t h the whipping expansion and the foam s t a b i l i t y . I n c l u d i n g the r e s u l t s from Table I l l b , the curves shown i n F i g . 6 and F i g . 7 c l e a r l y show that the higher the content of f r e e NTJ^-groups, the h i g h e r the whipping expansion and foam s t a b i l i t y of the i s o l a t e d p r o t e i n s , although the process combinations were d i f f e r e n t . This confirms the s i g n i f i c a n c e of


10.



Table H a . Ultrafiltration/Diafiltration Processing Data ( D H =

Retentates


Process combination

I

UF or DF

%(Nx6.25)

%(Nx6.25)

UF DF UF

2.9 9.3 9.3

9.3 9.3 18.0

Final

Before

permeate

Prot.re-

flux

tention %

7

1/h/nT

70.7

30.1 26.7 20.0

2.3

10.9

75.5

35.6

DF

10.9

10.1

92.9

34.7

10.1

ap20.0

93.1

UF

2.8

18.3

93.1

36.0

IV 1 s t

UF DF

3.2

9.8

92.2

41.9

9.8

15.7

98.2

26.7

IV 2 n d

UF

3.3

10.4

84.4

III

UF

3%)

Average Average

UF II

no

data

Table l i b . Ultrafiltration/Diafiltration Processing Data ( D H =

Retentates Process combination

UF Before or DF % ( N x 6 . 2 5 )

Final %(Nx6.25)

Average Prot.retention

UF DF

3.1 7.4

6.7

55.8 83.2

II

UF

1.8

12.4

69.1

III

UF

I

IV 1 s t IV 2 n d

UF

See

7.4

table

2a

141

See

%

Average permeate flux 7 1/h/ni 38.5 no d a t a

31.1

6 - 1061a

2.8 9.9

9.9 12.5

95.4

43.4

DF

98.5

21.7

UF

3.3

6.3

74.2

39.1


6%)

142

SYNTHETIC

MEMBRANES:

H F A N D U F USES

Table Ilia. Protein Yields and Some Properties of the D H 3 % Products Made by Different Process Combinations

Process combination

I

II

III

IV

% Protein y i e l d (based on soy meal)

39.5

30.9

55.3

28.6

96.0

93.1

77.9

95.5


Coniposi t i o n : % PY/HY Functionality: % Whipping exp. ) 7o Foam s t a b i 1 i t y

Chemical prop.: % psi i n TCA 10.0 % psi at pH - 4.5 42.7 % psi a t pH = 7.0 99.9 leu-NH^, mol/kg prot. 0.34 ~ DH= -0.2 Immuno precip. 6 archs l

1566 no data

733 12

667 9

17.7 13.2 42.6 47.8 59.9 94.7 0.60 ~ 0.41 ~ DH = 0.8 DH = 3.1 6 archs 6 archs

1650 42

13.4 49.7 97.0 0.49 ~ DH = 1.8 6 archs

) at pH 7.0

Table Illb. Protein Yields and Some Properties of the D H Products Made by Different Process Combinations

6%

Process combination

I

II

III

IV

7o P r o t e i n y i e l d (based on soy meal)

34.3

22.3

55.3

23.8

Composition: % PY/HY Methionin (g/16gN) C y s t i n (g/16gN)

90.1 1.06 1.76

91.1 1.10 1.93

77.6 1 .36 1.44

91.2 0.95 1.75

Functionali ty: % Whipping exp. * % Foam s t a b i 1 i t y Density o f mering. g/m3 **

833 20 0.11 ± O.Ol

1317 50 0.096 ± 0.004

1570 no data 0.21 ± 0.01

2484 69 0.17 ± 0.02

Chemical prop.: % psi i n TCA % psi at pH = 4.5 % psi a t pH = 7.0 leu-NH^,mol/kg protein

20.0 51.0 92.0 0.42 ~ DH = 0.9

19.0 54.6 100.0 0.49 ~ DH = 1.7

32.0 53.8 68.9 0.87 ~ DH = 6.4

30.1 71.7 99.5 0.67 ~ DH = 4.0

3 archs

5 archs

4 archs

2 archs

Immuno p r e c i p . * a t pH 7.0

** w i t h

eggwhites:

0.12 g/cm

3


10.

OLSEN AND ADLER—NISSEN


%

0.3


WHIPPING

0.4

143

EXPANSION

0.5

0.6 mol

0.7

NH2/kg

(Nx6.25)

Figure 6.

Whipping expansion vs. the number of free NH groups for highly functional soy protein ultraflltered after hydrolysis

Figure 7.

Foam stability vs. the number of free NH groups for highly functional soy protein ultraflltered after hydrolysis

2

2



144

SYNTHETIC

MEMBRANES:

HF

AND

UF

USES

using DH as the c o n t r o l l i n g parameter during p r o t e i n h y d r o l y s i s . The values of p s i i n TCA and p s i a t pH = 4.5 give a rough measure of the content of low molecular p r o t e i n s . The presence of arches determined by crossed Immunoelectrophoresis demonstrates that some high molecular p r o t e i n s are r e t a i n e d i n the modified products. For example IV-DH 6%, which has the best f u n c t i o n a l i t y , c o n s i s t s of only two s i n g l e high molecular p r o t e i n f r a c t i o n s compared to the IV-DH 3% which has s i x f r a c t i o n s . Further studies are r e q u i r e d to e l u c i d a t e the s t r u c t u r a l composition of these high molecular p r o t e i n f r a c t i o n s i n r e l a t i o n to the f u n c t i o n a l i t y . The presence of c y s t i n e i s important f o r i r r e v e r s i b l e g e l formation, but as the content of c y s t i n e i s p r a c t i c a l l y the same i n the hydrolysate made by I , I I and IV as shown i n Table I l l b , i t i s concluded that the c y s t i n e content i s not r e s p o n s i b l e f o r the d i f f e r e n c e s seen i n whipping p r o p e r t i e s . A p r e l i m i n a r y o r g a n o l e p t i c e v a l u a t i o n of the products d i d not show any b i t t e r n e s s , i n accordance w i t h the observation that the b i t t e r n e s s of soy p r o t e i n hydrolyzed w i t h A l c a l a s e only becomes pronounced a t DH-values of 7% and above ( 2 ) . Therefore, the present products may be used as n u t r i t i o u s i n g r e d i e n t s and h i g h l y f u n c t i o n a l p r o t e i n s as w e l l . I n many food formulations they may serve as s u b s t i t u t e s f o r egg-white. This was f o r example demons t r a t e d i n meringue b a t t e r s (see Table I l l b ) . I s o e l e c t r i c Soluble P r o t e i n Hydrolysates An i n d u s t r i a l process has been developed f o r production of i s o e l e c t r i c s o l u b l e soy p r o t e i n hydrolysate w i t h no b i t t e r n e s s and a bland t a s t e (13). The raw m a t e r i a l may be a c i d washed soy white f l a k e s , soy p r o t e i n concentrate o r soy p r o t e i n i s o l a t e The raw m a t e r i a l i s hydrolyzed by the a l k a l i n e protease ALCALASE® to a s p e c i f i e d degree o f h y d r o l y s i s using the pH-stat a t pH = 8.0 (4). Extensive p r o t e o l y s i s of a p r o t e i n o f t e n r e s u l t s i n the f o r mation of b i t t e r peptides ( 2 ) . Therefore, a compromise between high p r o t e i n y i e l d and low b i t t e r n e s s has to be found when choosing the DH-value a t which the h y d r o l y s i s r e a c t i o n should be terminated. For the present process a DH-value of about 10% seems to be a reasonable value. The t e r m i n a t i o n i s performed by a c i d i n a c t i v a t i o n of the enzyme and the a c i d used should be chosen i n accordance w i t h the d e s i r e d o r g a n o l e p t i c c h a r a c t e r i s t i c s of the f i n a l h y d r o l y s a t e . A t o t a l l y n o n - b i t t e r product can be produced by use of an organic a c i d l i k e m a l i c or c i t r i c a c i d . Due to the masking e f f e c t s o f such a c i d s , a b s o l u t e l y no b i t t e r n e s s can be detected even when the t a s t e e v a l u a t i o n i s performed a t n e u t r a l pH. Such products are found most s u i t a b l e f o r s o f t d r i n k s . However, when i n o r g a n i c a c i d s , e.g. h y d r o c h l o r i c or phosphoric acids are used, a s l i g h t b i t t e r n e s s may be detected i n the pure h y d r o l y sate. However, when evaluated i n f o r instance a meat product, no b i t t e r n e s s a t a l l can be t a s t e d even when the hydrolysate i s added up to a p r o p o r t i o n of 1 : 3 of hydrolyzed p r o t e i n to meat p r o t e i n . v



10.



145

A flow sheet of ISSPH-production i s given i n F i g . 8. The carbon treatment removes the l a s t t r a c e s of soy o f f - f l a v o u r s . Using the recommended process parameters, the f i l t e r e d hyd r o l y s a t e c o n t a i n s about 3% p r o t e i n . H y p e r f i l t r a t i o n i s t h e r e f o r e an a t t r a c t i v e process to use f o r c o n c e n t r a t i o n before d r y i n g . In p i l o t p l a n t experiments we have used a 7 m^ DDS-module type 40 w i t h t i g h t c e l l u l o s e acetate membranes type DDS-990. Conc e n t r a t i o n has been performed at pH = 4.0-4.5 i n a batch system at ambient temperature using 30 kp/m^ d e l i v e r e d by a Rannie p i s ton pump. In Table IV the composition of r e t e n t a t e s and average permeate f l u x e s are shown f o r d i f f e r e n t types of ISSPH. Volumes^of 700-900 l i t r e s of c l e a r h y d r o l y s a t e s were t r e a t e d on the 7 m DDSmodule, except f o r one experiment i n which o n l y 60 l i t r e s were t r e a t e d on a 0.36 m DDS-LAB-module. The p r o t e i n l o s s i n the permeate was below 3% i n a l l experiments. F i g u r e 9 shows a t y p i c a l f l u x and dry matter curve versus the per cent of water removed as permeate. U n f o r t u n a t e l y the f l u x decreases r a t h e r much d u r i n g the process. Both increase i n osmotic pressure and c o n c e n t r a t i o n pol a r i z a t i o n are r e s p o n s i b l e f o r t h i s dependence. L a t e r s t u d i e s have shown that the f l u x r a t e can be improved by i n c r e a s i n g the flow v e l o c i t y over the membrane s u r f a c e . D e s a l i n a t i o n of ISSPH. S p e c i f i c a p p l i c a t i o n s of ISSPH may r e q u i r e a reduced content of s a l t s , mainly NaCl. The membrane DDS-865, a c e l l u l o s e membrane, has been used f o r both d i r e c t hyp e r f i l t r a t i o n and f o r d i a f i l t r a t i o n , and i t appears that i t has a h i g h r e t e n t i o n of hydrolyzed p r o t e i n and a low r e t e n t i o n of s a l t . In Table V r e s u l t s are shown from an experiment i n which a h y d r o l y s a t e of soy p r o t e i n i s o l a t e c o n t a i n i n g NaCl-HCl i s d e s a l i nated. From a mass balance on n i t r o g e n (N) as w e l l as on n o n - n i t r o gen m a t e r i a l (NNM) the f o l l o w i n g has been found: direct hyperfiltration: hyperfiltration and d i a f i l t r a t i o n :

11% l o s s of N, 74%removing of NNM 23% l o s s of N, 93% removing of NNM.

For most a p p l i c a t i o n s the product which may be obtained by the d i r e c t h y p e r f i l t r a t i o n i s s u f f i c i e n t l y d e s a l i n a t e d and the p r o t e i n l o s s of about 11% may be accepted. P r e l i m i n a r y r e s u l t s a l s o seem to i n d i c a t e a s l i g h t r e d u c t i o n i n b i t t e r n e s s and soy o f f - f l a v o u r due to removal of very small b i t t e r peptides and other f l a v o u r compounds i n the permeate when t h i s d e s a l i n a t i o n membrane i s used. Process f o r D e c o l o r a t i o n of Slaughterhouse Blood. A novel p r o t e i n i n g r e d i e n t can be manufactured by a c o n t r o l l e d enzymatic


146

SYNTHETIC


MIXING

MEMBRANES: HF

H20, S5°C Protei n (Soy concentrate

or

UF

USES

isolate)

HYDROLYSIS IN S T I R R E D TANK

NaOH (pH-stat)

S = 8% protein E/S = 2.0% A l c a l a s e 0.6 50-55°C, pH 8.0

ENZYME INACTIVATION

Acid

DH = 10% - 3 h organic acid, pH 4.0-4.2

1.

AND

Soli ds-ejecting c e n t r i fuge

CENTRIFUGATION H20

FURTHER

CENTR.

FILTRATION

~1 SIudge (unconverted protein and other insoluble material)

CARBON

50°C, 0.1%

TREATMENT

30

min.

w/v

FILTRATION

Further

treatment

Figure 8. Flow sheet: production of a nonbitter, soluble soy protein hydrolysate suitable for incorporation into soft drinks and other low pH foods.

__1 20

1 40

1

1

60

80

1 X%

2

Figure 9. Hyperfiltration of HCl-containing ISSPH: flux in 1/h/m ; X, percentage of water removed; DM, percentage of dry matter (rejractometer).


OLSEN

A N D ADLER—NISSEN

Table I V .


Processing Data Regarding Hyperfiltration of ISSPH


Average Av. p r e s - permeate flux « sure Bar l/h/nf

Retentate

Type o f ISSPH

Vol. cone. ratio

Final

Before

DH(%)

A c i d used for inact.

10

ma 1 i c

2.7

4.5

14.3

22.3

5.1

20

3.80

10

HC1

3.5

4.3

21.1

26.2

6.1

34

4.46

10

HC1

2.0

2.8

14.2

17.6

6.7

34

4.67

15

mal i c

1.8

3.2

13.0

23.3

7.3

29

4.80

10

ma 1 i c

3.9

5.6

12.4

19.8

3.4

32

4.03

15

mal i c

3.5

5.4

16.8

27.5

4.9

32

7.08

10

HC1

30.2*

7.1

30

7.48

10

mal i c

-

26.3

5

32

10

malic

3.9

19.7

3.6

43

* by r e f r a c t o m e t e r

Table V .

Permeate flux o 1/h/nT

X /iwater removed

%x6.25

5.8*

Y %water added

83.3 87.5

8.7

87.5

0

7.8

33.0

33.0

16.7

38.3

41.7

25.3

66.6

14.0

%DM

5.96 32.83**

t r e a t e d on a D D S - L A B - m o d u l e

Desalination of ISSPH by Hyperfiltration on a DDS-865 C A Membrane

7.0

45.0

5.4

**60 l i t r e s

13.0

0

-

3.9*

-

51.7

%Nx6.25

%DM

8.3

133.0

133.0

8.0

233.3

233.3

8.7

300.0

300.0

Retentate

Permeate

0.25

1.25

-

3.44

/o

4.20

6.00

0.80

2.52

(10.44) ( Z l . 0 8 )

-

0/

%Nx6.25

%Nx6.25

-

Nx6.25 DM 81.9

6.89

87.1

-

-

-

18.31

19.90

92.0

-

-

-

1.38

2.23

20.38

21.27

95.8

0.69

0.77

18.50

18.23

101.5

(10.94) (11.28) 2

DDS-LAB-module, average pressure: 29 kp/cm , 20-30°C


148

SYNTHETIC MEMBRANES:

HF

AND

UF

USES


h y d r o l y s i s and subsequent d e c o l o r a t i o n of the red blood c e l l f r a c t i o n a r i s i n g as a by-product i n plasma recovery (14). A flow sheet of t h i s process i s shown i n F i g . 10. The process i s much s i m i l a r to t h a t of ISSPH-production. H y p e r f i l t r a t i o n serves the purpose of c o n c e n t r a t i o n of both plasma and h y d r o l y s a t e s e p a r a t e l y . F l u x data are very s i m i l a r to those obtained on soy p r o t e i n h y d r o l y s a t e s , and a l s o the t o t a l economy of such process seems a t t r a c t i v e . The main reason i s that slaughterhouse blood i n most cases i s regarded as a waste product having no v a l u e , or even a negative v a l u e . D i s c u s s i o n . In the above-mentioned examples membrane processes are found u s e f u l f o r both c o n c e n t r a t i o n and d e s a l i n a t i o n . One reason f o r recommending h y p e r f i l t r a t i o n i n s t e a d of evaporat i o n i n t h i s area i s the economical f a c t o r s . M u l t i - s t e p - e v a p o r a t o r s are s t i l l more economic than reverse osmosis i n very b i g p l a n t s , but the production of p r o t e i n hydrolysates w i l l i n a l l p r o b a b i l i t y be d i s t r i b u t e d between a number of m i d d l e - s i z e d p l a n t s r e q u i r i n g new investments. At a time w i t h i n c r e a s i n g costs of energy, h y p e r f i l t r a t i o n i s recommendable i n such p l a n t s . A l s o , the freedom of choosing membranes which may improve the q u a l i t y of the p r o t e i n s , f o r example by removing of o f f - f l a v o u r s and s a l t , speaks f o r h y p e r f i l t r a t i o n . Continuous P r o t e i n H y d r o l y s i s i n a Membrane Reactor The membrane r e a c t o r i s an u l t r a f i l t r a t i o n system, i n which a high c o n c e n t r a t i o n of h y d r o l y t i c enzyme i s confined. High mol e c u l a r weight s u b s t r a t e i s fed continuously to the r e a c t o r , and the low molecular weight products are removed simultaneously as permeate. I d e a l l y , a steady s t a t e i s reached, i n which the degrad a t i o n of the s u b s t r a t e i s c a r r i e d out i n d e f i n i t e l y w i t h high e f f i c i e n c y and n e g l i g i b l e l o s s of enzyme. The membrane r e a c t o r concept was demonstrated i n l a b o r a t o r y scale a decade ago by Butterworth et a l . (15) and by Ghose and K o s t i c k (16) i n s t u d i e s on the h y d r o l y s i s of s t a r c h and c e l l u l o se, r e s p e c t i v e l y . L a t e r on s e v e r a l p u b l i c a t i o n s have appeared d e s c r i b i n g the analogous, continuous conversion of v a r i o u s prot e i n s i n t o peptides intended f o r human n u t r i t i o n (17-22). Among these works only that of Iaccobucci et a l . (18) presents a quant i t a t i v e model of the membrane r e a c t o r i n continuous p r o t e i n hyd r o l y s i s , and i t i s a l s o the only demonstration of the p r a c t i c a l f e a s i b i l i t y of the concept i n p i l o t p l a n t s c a l e . Iaccobucci et a l . (18) a p p l i e d an a c i d , thermostable fungal protease from P e n i c i l l i u m duponti i n t h e i r work. The choice of t h i s enzyme had two advantages: The h y d r o l y s i s c o n d i t i o n s ensured v i r t u a l s t e r i l i t y (pH = 3.7, 60°C) and the peptides were q u i t e p a l a t a b l e (23). A major disadvantage of working i n the a c i d range i s that soy p r o t e i n i s o l a t e , which was used as s u b s t r a t e , i s i n s o l u b l e . In p r a c t i c e t h i s causes mechanical problems i f the subs t r a t e c o n c e n t r a t i o n i s not kept s u f f i c i e n t l y low (18).



10.



149

As described p r e v i o u s l y i n the present p u b l i c a t i o n , we have developed a batch process f o r producing i s o e l e c t r i c s o l u b l e soy p r o t e i n hydrolysate (ISSPH) w i t h a bland t a s t e . From s t u d i e s o f the k i n e t i c s of the h y d r o l y s i s r e a c t i o n , which takes place i n t h i s process, we have come to the c o n c l u s i o n that the r e a c t i o n i s adequately c o n t r o l l e d by keeping pH constant and monitoring DH. Termination of the r e a c t i o n at a preset value of DH ensures a r e p r o d u c i b l e , optimal o r g a n o l e p t i c q u a l i t y of the product. In the f o l l o w i n g the p o s s i b i l i t i e s are discussed of produc i n g ISSPH w i t h a f i x e d DH-value during a continuous h y d r o l y s i s r e a c t i o n i n a membrane r e a c t o r using s i m i l a r h y d r o l y s i s c o n d i t i o n s as i n the batch process. The s l i g h t l y a l k a l i n e c o n d i t i o n s are advantageous from a mechanical p o i n t of view, because the substrate i s d i s p e r s i b l e / s o l u b l e , but may a l s o imply a greater r i s k of i n f e c t i o n . The change from an i n s o l u b l e to a s o l u b l e subs t r a t e , and i n p a r t i c u l a r the a p p l i c a t i o n of the DH concent, immediately l e d to the c o n c l u s i o n that the q u a n t i t a t i v e model described by Iaccobucci e t a l . (18) would i n our case have to be s u b s t i t u t e d by an independently derived and more complete model, which only on c e r t a i n p o i n t s i s i n s p i r e d by the former. The f u l l d e r i v a t i o n of the model i s described i n the Appendix of the present p u b l i c a t i o n . Based on the k i n e t i c s of the batch h y d r o l y s i s i t i s demonstrated i n t h i s model that i t i s p o s s i b l e to run the membrane r e a c t o r i n steady s t a t e , i . e . DH can be kept constant i n the r e a c t o r . The steady s t a t e i s i n t r i n s i c a l l y s t a b l e and can be achieved immediately by c a r r y i n g out the h y d r o l y s i s as a batch r e a c t i o n w i t h zero membrane f l u x u n t i l the d e s i r e d DH-value i s reached, c f . F i g . 11. A t t h i s p o i n t , the f l u x i s increased to a preset v a l u e , and i f the various parameters i n the system have been chosen c o r r e c t l y , DH w i l l be maintained constant. A few experiments have been c a r r i e d out i n the l a b o r a t o r y s c a l e w i t h a one l i t r e h y d r o l y s i s v e s s e l , connected to a small i m p e l l e r pump and a S a r t o r i u s l a b o r a t o r y module f i t t e d w i t h DDS GR6-P membranes (0.2 m^). However, the flow r e s i s t a n c e i n t h i s module was too l a r g e , and i t was soon concluded that a resonably constant f l u x was u n a t t a i n a b l e . Despite these d i f f i c u l t i e s , the q u a l i t a t i v e behaviour of the r e a c t o r v a r i a b l e s could be p r e d i c t e d from the model and v e r i f i e d e x p e r i m e n t a l l y . For example, w i t h dec r e a s i n g f l u x DH i n c r e a s e d , but the r a t e of the base consumption decreased, w h i l e the p r o t e i n c o n c e n t r a t i o n i n the permeate r e mained q u i t e s t a b l e as p r e d i c t e d . The hydrolysate was evaluated and found comparable i n q u a l i t y t o ISSPH produced i n the batch process. These r e s u l t s have encouraged us to continue the work i n p i l o t p l a n t w i t h the DDS-35 module, where we can expect cons i d e r a b l y more favourable flow c o n d i t i o n s . The f i r s t experiments c a r r i e d o^t so f a r i n d i c a t e ^ t h a t a reasonable f l u x i n the order of 50 1/m /h (approx. 1 1/m /min.) can be a t t a i n e d but that foaming problems n e c e s s i t a t e the c o n s t r u c t i o n of p r e s s u r i z e d a i r f r e e r e a c t o r . Future s t u d i e s w i l l t h e r e f o r e be needed t o produce a complete experimental v e r i f i c a t i o n of the derived model.


150

SYNTHETIC

MEMBRANES:

Bl ood CENTRIFU6ATI0NL__^

Cell 40

H F A N D U F USES

Plasma fraction 60 % v / v

fraction

% v/v

HEMOLYSIS Acid


HYDROLYSIS

IN

STIRRED

S = 8 % protein E/S = 4 % A l c a l a s e 0 . 6 T = 550C, pH = 8.5

ENZYME

TANK

L

K

SEPARATION

FILTRATION

^Sludge

H20

INACTIVATION

DH = 1 8 % pH 4 . 0 HCL o r o r g a n i c acid

Supernatant

HYPER SEPARATION

II

FILTRATION

OR

FILTRATION

EVAPORATION

1 Sludge^ Dark

coloured

CARBON

TREATMENT

-

1

SPRAY

DRYING

insolubles Decoloured

Figure 10.

Figure 11.

product

Enzymatic decoloration of blood

Base Consumption—ideal steady-state protein concentration at t = Po


0


10.



151

An overview of the v a r i a b l e s i n the membrane r e a c t o r process i s given i n F i g . 12, and those equations, which are most r e l e v a n t from an engineering p o i n t of view, are summarized i n Box 1. The s i g n i f i c a n c e of most of the v a r i a b l e s should appear from F i g . 13 and Box 1 immediately, - f o r a f u l l e x p l a n a t i o n , the reader i s r e f e r r e d to the appendix. The main r a t i o n a l e behind the membrane r e a c t o r g e n e r a l l y appears to be savings of enzyme and the high conversion y i e l d , compared w i t h a batch h y d r o l y s i s process, I t should perhaps be ment i o n e d that the emphasis on enzyme c o s t s i s not p a r t i c u l a r l y r e levant i n the present case, as the major cost f a c t o r s f o r the exi s t i n g batch process are the raw m a t e r i a l s and the c a p i t a l costs (13). In any case the r a t i o n a l e i s based on the assumption that the r e a c t i o n can be c a r r i e d on f o r many c y c l e s w i t h no or only a s l i g h t purging. However, i f a s u b s t a n t i a l f r a c t i o n of the subs t r a t e i s non-degradable, i n e r t m a t e r i a l w i l l r a p i d l y b u i l d up i n the r e a c t o r causing mechanical problems. A c o n s i d e r a b l e purge i s necessary i f the c o n c e n t r a t i o n of t h i s m a t e r i a l s h a l l be kept at a reasonably low l e v e l . This has a d r a s t i c , negative i n f l u e n c e on the instantaneous y i e l d , as demonstrated i n F i g . 14 and Table VI. A l s o the enzyme l o s s d u r i n g purging w i l l be c o n s i d e r a b l e unl e s s the f r a c t i o n (y) of degradable p r o t e i n i n the s u b s t r a t e i s c l o s e to 100%. For soy p r o t e i n i s o l a t e Iaccobucci et a l . (18) found t h a t 6.2% of the p r o t e i n i n the s u b s t r a t e accumulated as i n e r t m a t e r i a l - i n other words, when comparing the membrane process w i t h the batch process, i t seems most r e l e v a n t to use y = 94%. I f a short c y c l e time i s chosen (e.g. 10 min.) Table V I shows that the enzyme consumption w i l l be much higher than i n the batch process, as soon as purging s t a r t s (1.8 hours from s t a r t ) . The enzyme consumption can be decreased by e n l a r g i n g the r e a c t o r s i z e , as enzyme c o n c e n t r a t i o n and r e a c t o r s i z e are i n v e r s e l y prop o r t i o n a l (eq. I , Box 1 ) , but i t w i l l s t i l l be of the same order of magnitude as i n the batch process. The i n c r e a s e i n r e a c t o r s i z e has, however, the disadvantage that more p r o t e i n s u b s t r a t e i s confined and l o s t i n the end (Table V I ) . The concomitant l o s s of confined enzyme i s found to be the same i n both cases, which i s obvious from eq. I . I f we look at Table V I I , the f i g u r e s f o r a t o t a l run of twelve hours are g i v e n , and i t appears that the short c y c l e time w i l l give a 1% higher p r o t e i n y i e l d than the long c y c l e time, but at the expense of a much higher enzyme consumption. A y i e l d above 80% appears at the f i r s t glance favourable compared w i t h the batch process where the h y d r o l y s i s process y i e l d s a l i t t l e above 60% (13). However, the y i e l d s must be compared w i t h respect t o the o r i g i n a l raw m a t e r i a l : soy white f l a k e s . I t i s , of course, necessary to feed the r e a c t o r w i t h soy i s o l a t e and t h i s i s produced from white f l a k e s i n a y i e l d of 60-65%. Based on white f l a k e s the p r o t e i n y i e l d of the membrane process as w e l l as the batch process w i l l be approximately 50%.


152

SYNTHETIC

MEMBRANES:

H F A N D U F USES

INPUT: SUBSTRATE NaOH

FEED FROM

pH-STAT

(ENZYME)

(COMPENSATED FLOWS)

FOR

PR

= PROTEIN

CONCENTRATION

SR

= A C C E S S I B L E PROTEIN

FEED

VELOCITY

NaOH AND

= &

ENZYME

CONC.

x(l+&)

REACTOR:


MEMBRANE REACTOR pH

8.0

= PROTEIN

= A C C E S S I B L E PROTEIN SUBSTRATE CONC.

OS

= SOLUBILIZED, PERMEABLE PROTEIN CONC.

50°C

i—r

PERMEATE FLUX = *

P S

PURGE = 3 x

*

CONCENTRATION

E

= ENZYME

M

= CONFINED

=

CONC. MASS

(CONSTANT)

OUTPUT: PY

= PROTEIN (-OS)

3

= PURGE

CONC.

IN

PERMEATE

COEFFICIENT

Figure 12.

Variables in reactor model

Figure 13.

SHC for soy isolate & alcalase





Table V I . Some Key Figures for the Production of ISSPH on the Membrane Reactor

M/ $

3

y

V

b

n

a

%

AU/kg

Substrate processed

M/*

% 10 mi n .

40 mi n .

hours

%

at

98

2.7

6

94. 1

9.9

96

5.6

2.8

8 8 . 3

19.9

&

94

8.7

1 . 8

82.7

30.0

kg

5.0

AU

27.8

2.7

24

94.1

2.5

11.3

88.3

5.0

150 &

8.7

7.3

82.7

7.5

11.9

5.3

77.3

10.0

90

15.4

4.0

72.0

12.5

a)

Enzyme c o n s u m p t i o n

b)

Enzyme a n d s u b s t r a t e

c)

Unrealistic

Table VII.

y

10

98

mi n .

96

present

in practice

n

t

at start

because

n

o

10.2

in

reactor

of microbial

prm2

E

AU/1 36 72

17

15

11 36

98 94

13.4

kg

deterioration

Total-Yield Calculations on the Membrane Reactor

94

96

18.2

2.4

used

%

C

per kg substrate

cycle time

7.4

5 8 . 2

Subst.

40 min.

0.6

5.6

92

, kg

15.0

98 94

Q

AU

150

96

c

n

18

17 11

3.75

kg

membrane E n z . / subst. Enz. ratio used AU/kg AU

Total yield

%

29.8

296

9.9

94.0

30.6

612

20.0

88.0

31 . 5

946

30.0

82.3

31 . 2

150

4.8

31 . 3 32.2

158 241

5.0

87.2

7.5

81.3

89.7


154

SYNTHETIC

I:

E ~

MEMBRANES:

HF

AND

UF

USES

1

i x PR x (E/S) „ x M ' 'SHC k(DH) cu

v


PR

III:

e

IV:

n

p = y x |\ - -2. x ( i - y ) ] m

n

= (t-t ) x ~

VI:

[PR X (1-y)

(number of c y c l e s )

Q

Z m n = b PR x (1-y)

P o PR

Substrate used:

M x [p^ + PR x ( n + 3 x

(n -n ))]

Enzyme used:

MxEx

(n -n )]

t

n Total y i e l d :

Box 1.

Q

[ l + n

t

t

x C + $ x

t

Q

Q

x y + ( n - n ) x T] - P /PR t

Q

Q

^

Equations used i n engineering c a l c u l a t i o n s of the membrane r e a c t o r

The d e r i v a t i o n of the equations i s given i n the appendix. I

i s used f o r c a l c u l a t i n g the enzyme c o n c e n t r a t i o n from the k i n e t i c data of the batch h y d r o l y s i s .

II

gives P

III

gives 3, the purge c o e f f i c i e n t from acceptable l e v e l of i n e r t matter = Z

q

( p r o t e i n cone, a t s t a r t i n r e a c t o r )

m IV

gives n, the instantaneous

yield

V

gives the number of c y c l e s f o r a given p e r i o d

VI

gives the number of c y c l e s before purging must be s t a r t e d .



10.



155

I f we b r i e f l y consider the main investments i n the two processes f o r a production o f 1000 tons of ISSPH per year and i n clude i n the membrane process equipment f o r producing soy i s o l a t e , the membrane process appears to r e q u i r e s l i g h t l y higher t o t a l investments. The r e s u l t s given above i n d i c a t e that there i s no obvious advantage o f s u b s t i t u t i n g the e x i s t i n g batch process f o r product i o n of ISSPH by a membrane r e a c t o r process. However, t h i s does not i n general mean that continuous p r o t e i n h y d r o l y s i s i n a membrane r e a c t o r w i l l be uneconomical. For example i f the subs t r a t e i s more completely degradable than soy p r o t e i n ( c a s e i n might be such a s u b s t r a t e ) , i t i s expected that i n a small s c a l e p l a n t (where the c a p i t a l costs would favour the membrane r e a c t o r ) the membrane r e a c t o r process could be very a t t r a c t i v e . The prod u c t i o n of p r o t e i n hydrolysates f o r d i e t e t i c and medical use, could w e l l be considered i n t h i s context. APPENDIX Development of a K i n e t i c Model f o r P r o t e i n H y d r o l y s i s i n a Membrane Reactor General c o n s i d e r a t i o n s Basis f o r the k i n e t i c model i s a standard batch h y d r o l y s i s experiment (2,). F i g . 14 shows the standard h y d r o l y s i s curve f o r soy p r o t e i n i s o l a t e - A l c a l a s e . The r e a c t i o n constant (pseudo f i r s t order r a t e constant) i s c a l c u l a t e d from the standard curve by f i t t i n g the inverse curve i n a small DH-range (ADH ^1.3%) to a second order Newton-Gregory polynomium (24), and f i n d i n g v(DH) by d i f f e r e n t i a t i o n . This procedure has i n our experience proved t o be the simplest and most r e l i a b l e way o f o b t a i n i n g values o f the r e a c t i o n r a t e . k(DH) i s shown i n f i g . 15. - i t v a r i e s s t r o n g l y w i t h DH. As demonstrated p r e v i o u s l y (25) there i s substrate s a t u r a t ion throughout the r e a c t i o n which means that f o r a constant E/S v(DH) and t h e r e f o r e k(DH) i s independent o f S. A l s o , E/S and v(DH) are p r o p o r t i o n a l to each other as usual ( i b i d ) . F i g . 12 gives an overview of the v a r i a b l e s i n the r e a c t o r model. In accordance w i t h what was demonstrated by Iacobucci e t a l . (18) i t i s assumed that the c o n c e n t r a t i o n of s o l u b i l i z e d , permeable p r o t e i n i s equal on both sides of the membrane. This assumption i s s u b s t a n t i a t e d by the f a c t that the p r o t e i n h y d r o l y zate c o n s i s t s mainly o f s m a l l e r , s o l u b l e peptides and unconverted p r o t e i n (2). The c o n c e n t r a t i o n of a c c e s s i b l e p r o t e i n i n the feed stream, SR, w i l l be smaller than PR, as i t i s l i k e l y that a s m a l l , constant percentage of the p r o t e i n i s undegradable, i n accordance w i t h what was found by Iacobucci e t a l . (18). This f r a c t i o n counts as p r o t e i n i n a K j e l d a h l a n a l y s i s , but i s otherwise c o n s i -


156

SYNTHETIC

p =6%, PR=4%, 1=3% o m

x

MEMBRANES: HF

(50% o f

AND

UF

USES

PJ o'


100 80 \ 60 40

Number o f c y c l e s \

b e f o r e purging becomes n

necessary

20 1 ,

Figure 14.

Figure 15.

Relationship between certain important variables in the membrane reactor

The reaction constant, k(DH), from the standard hydrolysis curve



10.

OLSEN A N D ADLER—NissEN

157


dered i n e r t , i . e . i t i s assumed that i t s accumulation i n the r e tentate does not i n f l u e n c e the h y d r o l y s i s k i n e t i c s a p p r e c i a b l y . This assumption w i l l be s u b s t a n t i a t e d l a t e r . I t has been found i n batch h y d r o l y s i s experiments that the p r o p o r t i o n of s o l u b l e n i t r o g e n to t o t a l n i t r o g e n i s g r a d u a l l y i n creasing w i t h DH (2). For a constant DH t h i s p r o p o r t i o n i s independent of S (26) i n accordance w i t h the f a c t that v(DH) i s independent of S. I t thus seems reasonable to assume that OS/S w i l l be a monotonously i n c r e a s i n g f u n c t i o n of DH and independent of S. The r e l a t i v e increase of OS/S w i l l always be equal to or smaller than the corresponding r e l a t i v e increase i n DH - t h i s i s a mathem a t i c a l consequence of the f a c t that the average peptide chain l e n g t h i n the s o l u b l e f r a c t i o n of a hydrolyzate w i l l be constant or decreasing w i t h DH (26). In batch h y d r o l y s i s experiments we have g e n e r a l l y not d i s t i n g u i s h e d between S and P, but have assumed S = P f o r the standard h y d r o l y s i s curve. However, the d i s t i n c t i o n between S and P i s c r u c i a l i n the present case where i n e r t p r o t e i n (N*6.25) accumulates. P denotes the p r o t e i n c o n c e n t r a t i o n i n the beginning of the experiment. F i g . 11 shows the p r i n c i p l e s i n an i d e a l , steady s t a t e experiment. A t t=0 the h y d r o l y s i s i s s t a r t e d as a batch h y d r o l y s i s ( = 0). When the d e s i r e d DH-value has been reached (DH=DH a t t=t ) the membrane r e a c t o r i s s t a r t e d , i . e . peptides are permeat i n g through the membrane w i t h the volume f l u x , $, and f r e s h subs t r a t e i s added continuously to r e p l a c e the degraded p r o t e i n . In the f o l l o w i n g i t w i l l be proved that i f the values of the i n dependent v a r i a b l e s ( i . e . PR, P , E , M, $ and 3) have been chosen c o r r e c t l y , the r e a c t o r w i l l immediately be i n steady s t a t e , most g e n e r a l l y defined as DH remaining c o n s t a n t l y equal to D H . The equations which describe the r e l a t i o n s h i p between the v a r i a b l e s w i l l be derived i n the f o l l o w i n g s e c t i o n s . The above general c o n s i d e r a t i o n s are summarized i n Box 2. q

Q

q

The steady-state

equations

The most general d e f i n i t i o n of steady s t a t e was given p r e v i o u s l y namely that DH should remain constant. However, t h i s d e f i n i t i o n i s too general to be of p r a c t i c a l use, and i t i s t h e r e f o r e necessary i n the f o l l o w i n g to assume that the f o l l o w i n g parameters a l so remain constant throughout the experiment: PR, M, E, $ (and 3)

a l l constant

(9)

Convenience d i c t a t e s that PR and M should remain constant. E i s kept constant by r e p l a c i n g the l o s s of enzyme through i n a c t i v a t i o n , permeation and purging. $ can be regulated by the pressure drop and can be maintained reasonable constant i n a w e l l b u i l t system i n which high flow rates are obtained. Q u a s i - s t a t i o n a r y c o n d i t i o n s , i . e . slow changes i n the above parameters w i l l be d e a l t w i t h l a t e r .


158

SYNTHETIC

MEMBRANES:

HF

Mass balance c o n s i d e r a t i o n s i n the time p e r i o d t lead to the f o l l o w i n g :

AND

Q

UF

USES

to ( t +dt) °

E/P v(DH) =

(E/S) 'SHC

x v(DH) _, which can be w r i t t e n as CIJ

c

d(DH) dt

_E P

=

(E/S)


c

x DH x k(DH)

PY = OS

y

(2)

SR S P R P ~ o

=

-

, . ( y

=

Accumulated Z= OS

i

o

1

The r i s e i n OS means that P Y w i l l r i s e , because OS = P Y , eq. (11):


From

10.



| | = | x [SRx(l+3) - PY - B*S]

163

(11)

we can then conclude t h a t because both PY and S are l a r g e r than p r e v i o u s l y , dS/dt (which i s zero i n steady s t a t e ) must be n e g a t i ve .


A s i m i l a r l i n e of arguments leads to the c o n c l u s i o n t h a t i f AS < 0, dS/dt w i l l be p o s i t i v e . There i s t h e r e f o r e negative feed-back i n the system w i t h r e s p e c t to S. The demonstration of negative feed-back f o r the two dependent v a r i a b l e s , DH and S, i s proof of the i n t r i n s i c s t a b i l i t y of the steady s t a t e . Quasi-stationary conditions I f the changes i n the independent parameters are slow, we have q u a s i - s t a t i o n a r y c o n d i t i o n s and the steady s t a t e equations w i l l h o l d . By slow changes i s meant that the r a t e of change i n DH caused thereby i s slow compared to the r e a c t i o n r a t e , d(DH)/dt, as given by eq. (1). ADH At

«

jT- x -^t|j

o

x DH x k(DH)

(30)

SHC

The r e l e v a n t steady s t a t e equations are: E

=| x R x ( l S

+

e

)

x ( /S) E

S H C

x- L ^

PY - SR x (1 + 3) - 3 x s f(DH) =

x (1+3)

-

(17a)

(19a)

3

(20a)

From f i g . 15 i s obtained t h a t : DH =

8 =>k(DH)

DH =

9 =>

:= 160 min

do.

= 230 min

DH = 10 =>

do.

= 310 min

DH = 11

do.

= 420 min

Consequently, even r a t h e r l a r g e changes i n the independent v a r i a b l e s w i l l o n l y lead to comparatively small changes i n DH. For example, a 20% r e l a t i v e change i n E, $, M, SR og (1+B) w i l l only change DH approximately 0.6 u n i t s (e.g. from 10 to 10.6%).


164

SYNTHETIC

MEMBRANES:

HF

AND

UF

USES

The corresponding r e l a t i v e change i n f(DH) i s l e s s than 6% and t h i s leads again to a change i n S of l e s s than 6%, eq.(20a), prov i d e d 3 1 - This i s of c o n s i d e r a b l e importance i n p r a c t i c e , as i t i s d i f f i c u l t to avoid some decrease i n the values of E and $ d u r i n g continuous o p e r a t i o n of the membrane r e a c t o r due to f o u l ing and enzyme l o s s e s . F o r t u n a t e l y , according to eq. (17a) E and $ w i l l p a r t i a l l y counteract each other i n t h e i r e f f e c t on DH. < < :


The above has the p r a c t i c a l consequence t h a t because y x (1+3)c*l i n most cases, E i s adequately given by the s i m p l i f i e d equation: E

i x PR x

(E/S) SHC

X

k(DH)

(17b)

At any time the DH-value can be c a l c u l a t e d q u i t e simply from the base consumption i n the p e r i o d , At (13) (31) As the r e l a t i v e change i n DH i s l e s s than the r e l a t i v e decrease i n i t appears from eq. (31) that AB/At w i l l decrease w i t h decreasing $, although DH a c t u a l l y i n c r e a s e s ! The change i n PY as a r e s u l t of changes i n the independent parameters i s n e g l i g i b l e . This i s evident from eq. (19a) because S changes only s l i g h t l y and 3 0). The boundary c o n d i t i o n i s t h a t Z = P x (1-y) f o r t = t , independently of 3 . The s o l u t i o n to eq. (35) i s then: q

-3 x e

For

t =» oc

Q

x ^x( -t ) M o t

z

1 lm

(36)

= PR x

( 1

3 )

+ p

x (1-y)

(37)

In case 3 = 0, (35) i s immediately s o l v e d : Z = - x PR x ( t - t ) + P Q

Q

x (1-y)

(

3

8

)

As expected, Z increases l i n e a r l y w i t h time, when there i s no purge.


166

SYNTHETIC

Compensation

MEMBRANES:

HF

AND

UF

USES

f o r Enzyme Losses

Enzyme i s l o s t through p u r g i n g , i n a c t i v a t i o n and permeation. A l l three are p r o p o r t i o n a l t o E. The mass balance on the enzyme gives: M x d E = $ x 3 x E x d t + C x E x d t (purge) (mactiv.) 1


+ C

x x E x dt (permeation)

2

(39)

or: =

¥

[I

X

( 6 + C

2

)

+

C

l

x

d t

( 4 o )

I f the i n a c t i v a t i o n l o s s e s are s m a l l , eq. (40) can be simp l i f i e d to ~ | x (3

f-

+ C

) x dt

(41)

or on f i n i t e form: J«

(S+C) x At

(42)

Yield Calculations The instantaneous y i e l d , r| i s d e f i n e d as the p r o p o r t i o n between the s u b s t r a t e f l u x and the permeate f l u x ( p r o t e i n b a s i s ) . From the p r o t e i n mass balance (33), r| i s immediately obtained: P n — X (43) n = y x 1 ~ PR (1+3) 0

The t o t a l y i e l d i s the y i e l d based on a complete run, i n c l u d i n g the s t a r t i n g up. The p r o t e i n present i n the r e a c t o r at t = t cannot be recovered i n the end, because i f the s u b s t r a t e o . feed i s i n t e r r u p t e d (PR = 0 ) , DH cannot be maintained constant, but w i l l increase r a p i d l y . The most economical way of running the r e a c t o r i s to keep 3 = 0 u n t i l Z reaches a p r e s e t l i m i t , Z . The number of c y c l e s , m i . e . : ri = ( t - t ) x ^, which corresponds to Z i s found by solving (38): =

n

o

Z 2 PR x (1-y)

P - _° PR

(44) ™ K

F o l l o w i n g the p e r i o d g i v e n by n , purging i s e s t a b l i s h e d and i s found s o l v i n g ( 3 7 ) :


J


10.


167

z

r

2

(45)

1

-y)

(l-y

LPR x

Combining eq. ( 4 5 ) w i t h eq. ( 4 3 ) g i v e s :

n = y x [ l - _° x ( l - y ) l ^m •*

(46)


L

In one c y c l e the mass of substrate f e d i s PR x M x ( 1 + 3 ) . In n c y c l e s (n >n ) the t o t a l y i e l d ( i n c l u d i n g the l o s s of prot e i n confined a t tfte end), can be c a l c u l a t e d from the instaneous yields: Product formed: n

Q

x y x PR x M + (n - n ) x n x PR x M x

Substrate Mxp

(1+3)

used: o

+ n x PR x M + (n -n ) x PR x M x o t o y

The l a s t can be s i m p l i f i e d

(1+3)

slightly to:

M x [P + PR x (n + 3(n -n ))] Q

s.

t

t

m i • u n x + (n -n ) x Total y i e l d = o t o P p| + n + 3(n -n ) J

y

t

t

o

(1+3)

x n (47)

Q

In most p r a c t i c a l cases, where 3 i s small and the number of cycl e s i s 1 0 - 1 0 0 , eq. ( 4 7 ) can be s i m p l i f i e d t o : n Total y i e l d « —

x y + (n -n ) x n - P /PR — t

(48)

n

The amount of enzyme consumed i s M x E x (3+G) per c y c l e . T o t a l amount of enzyme i n n c y c l e s i s then ( i n c l u d i n g i n i t a l enzyme) : fc

Enzyme used:

(n^-n ) x (3+C) x M x E t o

M xE +n x C x M x E + o

(49)

or: Enzyme used:

MxEx

[ l + n

t

x C

+3

X

(n ~n ) ] t


(50)

168

SYNTHETIC

MEMBRANES:

HF

AND

UF

USES

Acknowledgement


We wish to thank the s t a f f a t Enzyme A p p l i c a t i o n Technology P i l o t P l a n t and at Enzyme A p p l i c a t i o n Technology I f o r t h e i r s k i l l f u l a s s i s t a n c e i n a l l the p r a c t i c a l work c a r r i e d out i n connection w i t h t h i s paper. A l s o we want to thank Mr. Peer N. J0rgensen a t Pharmacological Laboratory - Novo I n d u s t r i A/S, Bagsvaerd f o r the immunochemical examinations. F i n a l l y we thank our c o l l e a g u e , Mr. F i n n Jacobsen f o r drawing our a t t e n t i o n to the p o s s i b i l i t i e s of u s i n g h y p e r f i l t r a t i o n f o r d e s a l i n a t i o n of ISSPH. Literature cited 1.

Manak, L . J . , Lawhon, J.T., and Lusas, E.W., (1980) 45, 236-245.

2.

A d l e r - N i s s e n , J., and S e j r Olsen, H., ACS Symp. Ser. (1979) 92, 125-146.

3.

A d l e r - N i s s e n , J., J . A g r i c . Food Chem.

4.

A d l e r - N i s s e n , J., Process Biochem.

5.

Sejr Olsen, H., Lebensm.-Wiss. u.-Technol., (1978) 11,57-64.

6.

C h r i s t e n s e n , L.K., Compt.-rend. Lab C a r l s b e r g , Sér. chim., (1952) 28 ( 1 ) , 39-169.

7.

Novo I n d u s t r i A/S, " P r o t e o l y t i c Enzymes f o r the M o d i f i c a t i o n of Food P r o t e i n s " , IB 163, Bagsvaerd 1978.

8.

S e j r Olsen, H., Isolation of Bean P r o t e i n by Ultrafiltration In: Proceedings of the I n t e r n a t i o n a l symp. on Sep. proc. by Membranes, Ion-exchange and Freeze-concentration i n Food I n d u s t r y . IUF0ST and FEEC, P a r i s (1975), p. A 6-1, A6-21.

9.

Becker, H.C., M i l n e r , R.T., and Nagel, R.H., Cereal Chem. (1940) 17, 447-457.

10.

J . Food Sci.

(1976) 24, 1090-93

(1977) 12 ( 6 ) , 18-23,32.

A d l e r - N i s s e n , J . , J. A g r i c . Food Chem., (1979) 27, 1256-62.

11.

Weeke, B., 3. Crossed Immunoelectrophoresis, p. 47-56. I n : N.H. Axelsen, J . Krøll and B. Weeke: A Manual of Q u a n t i t a t i v e Immunoelectrophoresis - Methods and A p p l i c a t i o n s . Universitetsforlaget, Oslo, 1973. 12.

Smith, A.K., and 1414-1418.

Circle,

S.J., Ind. Eng. Chem. (1938) 30,



10.

OLSEN AND ADLER-NISSEN


169

13.

Sejr Olsen, H., A d l e r - N i s s e n , J . , Process Biochem. (1979) 14 (7) 6-8, 10-11.

14.

Novo I n d u s t r i A/S, " D e c o l o r a t i o n o f Slaughter House Blood by Enzymatic M o d i f i c a t i o n " . IB 225, Bagsvaerd 1980.

15.

Butterworth, T.A., Wang, D.I.C., Sinskey, A . J . , B i o t e c h n o l . Bioengin. (1970), 12, 615-631.

16.

Ghose, T.K., K o s t i c k , J.A., B i o t e c h n o l . B i o e n g i n . (1970), 12, 921-946.

17.

Roozen, J.P,

18.

I a c o b u c c i , G.A., Myers, M.J., Emi, S., Myers, D.V., Proc. IV I n t . Cong. Food S c i . Technol., Madrid 1974, .5, 83-95.

19.

Bhumiratana, S., Hill Jr., C.G., Amundson, C.H., J . Food Sci. (1978), 42, 1016-1021.

20.

Payne, R.E., Hill Jr., C.G., Amundson, C.H., J . Food Sci. (1978), 43, 385-389.

21.

Cunningham, S.D., Cater, CM., (1978), 43, 1477-1480.

22.

Roozen, J.P., Pilnik, W., Enzyme Microb. Technol. (1979), J., 122-124.

23.

Myers, D.V., R i c k s , E., Myers, M.J., W i l k i n s o n , M., IacobucG.A., Proc. IV I n t . Congr. Food Sci. Technol., Madrid 1974, 5, 96-102.

24.

Bennet, A.A., M i l n e , W.E., Bateman, H.,"Numerical I n t e g r a t i o n of Differential Equations", Dover P u b l . , I n c . , N.Y. 1956, p. 27.

25.

A d l e r - N i s s e n , J., Ann. Nutr. A l i m . (1978), 32, 205-216

26.

A d l e r - N i s s e n , J., Unpublished

ci,

Pilnik,

W., Process Biochem. (1973), 8, 24-25

Mattil,

K.F.,J.Food Sci.

experiments.

R E C E I V E D December 4, 1980.


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