4
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
Conformation and Functionality of M i l k Proteins C. V. MORR Department of Food Science, Clemson University, Clemson, SC 29631
We are indebted to numerous researchers who have contributed sufficient basic knowledge to enable us to unravel the complexities of the milk protein system, e.g., the caseins and whey proteins and their subfractions. These findings have been collected, evaluated and organized into a comprehensive report by the "Milk Protein Nomenclature Committee" (1). This latter report contains excellent details on the primary structure and physicochemical properties of most of the casein monomer subunits and the whey proteins, as well as for most of their genetic polymorphs. Milk proteins are widely recognized for their superior nutritional, organoleptic, and functional properties, as components of milk and in milk-containing food formulations. Although the caseinates have superior functional properties, especially for stabilizing emulsions and foams, there is a need to improve the functionality of the whey protein concentrates. For example, the whey protein concentrates generally exhibit poor functional properties in food applications requiring high solubility. This paper draws heavily upon the "Nomenclature Committee Report" (1) as well as several recent comprehensive reports that have considered the primary structure and conformation of the casein monomer subunits and how they are assembled into submicellar aggregates and casein micelles (2, 3) . These basic relationships were utilized to develop additional projections relating to the conformation and functional properties of the major milk proteins, e.g., commercial caseinates and whey protein concentrates in food applications. Fractionation and Distribution of Major Milk Proteins. The fractionation scheme and distribution data for the major milk proteins and their subfractions are given in Figure 1 (1). Although the caseins are easily separated from whey proteins by adjusting milk to pH 4.6-5.0, further separation and purification of the individual caseins is extremely difficult, due to their strong interaction, and requires the most sophisticated protein 0-8412-0478-0/79/47-092-065$05.00/0 © 1979 American Chemical Society
66
F U N C T I O N A L I T Y A N D PROTEIN STRUCTURE
f r a c t i o n a t i o n techniques a v a i l a b l e . S i m i l a r l y , the i n d i v i d u a l whey p r o t e i n components a r e s e p a r a t e d a n d p u r i f i e d o n l y b y a p p l i cation of elaborate fractionation techniques.
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
Nomenclature
and P h y s i c o - c h e m i c a l P r o p e r t i e s o f the C a s e i n s .
The f o u r m a j o r c a s e i n f r a c t i o n s c o n t a i n s u b f r a c t i o n s a n d g e n e t i c polymorphs (Table 1 ) , each of which p o s s e s s e s a unique p r i mary s t r u c t u r e a n d a s s o c i a t e d p h y s i c o - c h e m i c a l p r o p e r t i e s (1). F o r e x a m p l e , α - c a s e i n c o n t a i n s one m a j o r s u b f r a c t i o n (α - - C n ) p l u s s e v e r a l minor components. The f o u r g e n e t i c v a r i a n t s o f a i ~ c a s e i n have s i m i l a r i s o i o n i c p o i n t s , which would be e x p e c t e d from t h e i r s i m i l a r primary s t r u c t u r e s . The β - c a s e i n s , κ - c a s e i n s , a n d "whole α - c a s e i n s " have a l s o been f r a c t i o n a t e d and t h e i r s u b f r a c tions c h a r a c t e r i z e d (Table 1 ) . The i s o i o n i c p o i n t s f o r a l l o f these c a s e i n s u b u n i t s range from about 4.9 to 6.0 and t h e i r mole c u l a r w e i g h t s range from about 11,500 t o 2 4 , 0 0 0 . s
P r i m a r y S t r u c t u r e o f t h e M a j o r C a s e i n Monomer
s
Subunits.
The p r i m a r y s t r u c t u r e s ( F i g u r e s 2, 3 and 4) a r e g i v e n f o r a - g ^ e a s e i n , β - c a s e i n , a n d κ - c a s e i n (I). The most s i g n i f i c a n t a s pects o f these primary s t r u c t u r e s are t h e i r d i s p r o p o r t i o n a t e d i s tribution o f a c i d i c amino a c i d s , serine phosphate g r o u p s a n d h y d r o p h o b i c amino a c i d s a l o n g t h e p o l y p e p t i d e c h a i n . As d e m o n s t r a t e d b y B l o o m f i e l d a n d Mead ( 2 ) , t h e u n e v e n d i s t r i b u t i o n o f amino a c i d s f o r t h e m a j o r c a s e i n monomer s u b u n i t s l e a d s t o h i g h l y charged ( n e g a t i v e ) r e g i o n s , which a r e s e p a r a t e d from the s t r o n g l y hydrophobic r e g i o n s , a l o n g the p o l y p e p t i d e c h a i n s . These amphophilic molecules are h i g h l y susceptible to intermolecular i n t e r a c t i o n and p o l y m e r i z a t i o n through the formation o f hydrophobic a n d i o n i c , e s p e c i a l l y Ca b o n d s . S l a t t e r y (3) u s e d t h i s a n d o t h e r r e l e v a n t i n f o r m a t i o n t o d e v e l o p c o n f o r m a t i o n a l models f o r each o f t h e m a j o r c a s e i n monomer s u b u n i t s . I n h e r e n t i n t h e s e models i s the c o n s i d e r a t i o n t h a t the u n i f o r m d i s t r i b u t i o n o f p r o l i n e a l o n g t h e p o l y p e p t i d e c h a i n r e s u l t s i n a random c o i l , w i t h l i t t l e h e l i cal structure. The m o d e l f o r a - c a s e i n t h a t b e s t f i t s t h e d a t a i s a c o m p a c t , p r o l a t e e l l i p s o i d c o n t a i n i n g most o f t h e h y d r o p h o b i c amino a c i d r e s i d u e s , p l u s a 40 amino a c i d r e s i d u e c h a i n c o n t a i n i n g most o f t h e a c i d i c amino a c i d s a n d s e r i n e p h o s p h a t e s that e x t e n d i n t o t h e aqueous p h a s e as a l o o p ( 3 ) . The compact h y d r o p h o b i c r e g i o n i s s t a b i l i z e d by i n t r a m o l e c u l a r hydrophobic b o n d i n g , whereas the e x p o s e d , a c i d i c loop i s r e a d i l y a c c e s s i b l e f o r i n t e r a c t i o n w i t h adjacent c a s e i n molecules through formation o f i o n i c , mainly Ca, bonds. The m o d e l d e v e l o p e d b y S l a t t e r y (3) f o r β - c a s e i n a l s o c o n t a i n s a compact h y d r o p h o b i c r e g i o n , w i t h a b o u t t h e same d i m e n s i o n s a s f o r a - c a s e i n , a n d w i t h a s t r o n g l y a c i d i c p o l y p e p t i d e c h a i n , e . g . , amino a c i d r e s i d u e s 1 - 2 5 , e x p o s e d t o t h e aqueous m e d i a . The m o d e l f o r κ - c a s e i n ( 3 ) a l s o c o n t a i n s a compact h y d r o p h o b i c r e g i o n , as a b o v e , p l u s a f r e e l y e x p o s e d p o l y p e p t i d e c h a i n c o n t a i n i n g a h i g h p o r t i o n o f a c i d i c amino a c i d s as w e l l a s s
s
23-35 3-7
(S-Caseins
γ-Caseins
Adapted from Whitney et a l . , 1976
""Calculated from primary structure where
possible
^ T o t a l of genetic v a r i a n t s and minor components
a
4
2-6
Proteose peptones
4
1.9-3.3
Immunoglobulins
2
1
2-5
.7-1.3
4
9
3.30-3.7
5.50-8.3
4.20-4.5
5.13
5.35-5.41
5.80-6.0
5.20-5.85
4,100-40,800
150,000-1 m i l l i o n
14,146-14,174
66,500-69,000
18,275-18,362
11,556-20,629
23,939-24,089
19,005-19,037
5.37
2 7
22,068-22,723
0
Molecular weight
4.92-5.35
Isoionic point
3
9
Number of components**
0. -Lactalbumins
Bovine serum albumin
3-Lactogiobulins
Whey Proteins 7-12
8-15
K-Caseins
s
45-55
Approx % of skimmilk protein
Amount and Selected P r o p e r t i e s of M i l k Proteins and Their Components
a -Caseins
Caseins
Protein
Table I .
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
FUNCTIONALITY
A N D PROTEIN
STRUCTURE
SKIMMILK pH 4.6 @ 20 C ι
1
PRECIPITATE
SUPERNATANT
I
I
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
CASEINS (75-85 %)
WHEY PROTEINS (15-22 %)
a -
(45-55)
BSA
(.7-1.3)
β-
(25-35)
£-Lg
(7-12)
K-
(8-15)
a-La
(2-5)
y-
(3-7)
Ig (1.9-3.3)
g
PP (2-6) Figure 1.
Fractionation and distribution of the major milk proteins
10 20 H.Arg-Pro- L y s - H i s - P r o - I le - Lys-His- G l n - G l y - L e u - P r o - G l n - | G l u - V a l - L e u - A s n - G l u - A s n - L e u Absent in variant A 30 40 Leu-Arg-Phe-Phe-Val-Ala|-Pro-Phe-Pro-Gln-Val- Phe-Gly-Lys-Glu-Lys-Val -Asn-Glu-Leu50 60 Ser—Lys-Asp-I le - G l y - S e r - G l u - S e r - T h r - G l u - A s p - G i n 4-Ala Met-Glu-Asp-lle - Lys-Glu-MetP Ρ ThrP (variant D) 70 80 G l u - A l a - G l u - S e r - l l e - S e r - S e r - S e r - G l u - G l u - l l e - Val-Pro-Asn-Ser —Val—Glu - G i n - L y s - H i s Ρ Ρ Ρ Ρ Ρ 90 100 Ile - G l n - L y s - G l u - A s p - V a l - P r o - S e r - G l u - A r g - T y r - L e u - G l y - T y r - L e u - G l u - G l n - L e u - L e u - A r g L
110 120 Leu -Lys -Lys -Ty r-Lys -Val - Pro - G i n - L e u -Glu-Ile - Val -Pro -Asn -Ser - Ala - G l u - G l u - A r g - L e u P 130 140 His-Ser-Met-Lys-GIn-Gly-lle - His-Ala-GIn-GIn-Lys-Glu-Pro-Met-Ile - Gly-Val-Asn-GIn150 160 Glu-Leu^Ala-Tyr-Phe-Tyr-Pro-Glu-Leu-Phe-Arg-GIn-Phe-Tyr-GIn-Leu-Asp-Ala-Tyr-Pro170 180 Ser-Gly-Ala-Trp-Tyr-Tyr-Val-Pro-Leu-Gly-Thr-GIn-Tyr-Thr-Asp-Ala-Pro-Ser-Phe-Ser190 199 A s p - l l e - Pro- Asn-Pro-Me - Gly-Ser -Glu-Asn-Ser-|Glu|- Lys -Thr - T h r - M e t - P r o - Leu-Trp.OH Gly (variant Cî
Journal of Dairy Science Figure 2.
Primary structure of bovine a -Cn-B s
(1)
MORR
Milk
Proteins
10 20 H.Arg-Glu-Leu-Glu-Glu-Leu^sn-Val-Pro-Gly-Glu-lle - Val-Glu-Ser-Leu-Ser-Ser -Ser-Glu Ρ Ρ Ρ Ρ 7, -caseins 30 40 ^,μ-^, - .... - ™ - . , - u . - He - G l u - L y s - Phe - GI n- Ser-fcluV|Glul-Gln-Gln-GIn(absent in variant C) Ρ Lys Lys (variante) (variant E) 50 60 Thr-Giu-Asp-Glu-Leu-GIn-Asp-Lys-lIe - His-Pro-Phe-Ala-Gln-Thr-GIn-Ser -Leu-Val - T y r -
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
y
c
L y 3
C y a
70 80 P r o - P h e - P r o - G l y - P r o - I l e -| Prof- A s n - S e r - L e u - P r o - G I n - A s n - l l e - P r o - P r o - L e u - T h r - G i n - T h r (variants C, A , and B) His 90 100 Pro-Val - V a l - V a l - P r o - P r o - P h e - L e u - G I n - P r o - G l u - V a l - M e t - G l y - V a l - S e r - L y s - V a l - L y s - G l u 1
! •γ3-caseins (R-, TS-B) 110 120 A la - Met - A la - Pro - LvsrfHTi - Lvs - GI u - Met -Pro - Phe - P r o - Lys - T y r - P r o - V a l - G l n - P r o - P h e - T h r Gln (variant A ) I -caseins (S-, T S - A ) 130 140 Glu-jSerf-GIn-Ser - Leu - T h r - Leu -Thr - A s p - V a l - Glu - Asn - L e u - H i s - L e u - P r o - P r o - L e u - L e u - L e u Arg (variant B) 150 160 Gin- Ser - T r p -Met- His - G i n - Pro - H i s - G l n - P r o - Leu-Pro - P r o - T h r - V a l - Met- Phe-Pro - P r o - G i n 3
2
170 180 Ser-Val—Leu-Ser- Leu-Ser-GIn-Ser-Lys-Val-Leu-Pro - V a l - P r o - G l u - L y s - A l a - V a l - P r o - T y r -
P r o - G I n - A r g - A s p - M e t - P r o -Ile -
Val-Arg-Gly-Pro-Phe-Pro-Ile
190 200 Gln-Ala-Phe-Leu-Leu-Tyr-GIn-GIn-Pro-Val-Leu-Gly-Pro-
- Ile
209 -Val.OH
Journal of Dairy Science Figure 3.
Primary structure of bovine β-Cn-A
2
PyroGlu-Glu-GIn-Asn-GIn-Glu-GIn-Pro (Gln) (Glu) (Glu)
(1)
10 20 -Ile - A r g - C y s - G l u - L y s - A s p - G l u - A r g - P h e - P h e - S e r - A s p -
30 40 Lys -Ile - A l a - L y s - T y r - I l e - P r o - H e - G l n - T y r - V a l - L e u - S e r - A r g - T y r - P r o - S e r - T y r - G l y - L e u 50 60 A s n - T y r - T y r - G l n - G l n - L y s -Pro - V a l - A l a - L e u - l l e - A s n - A s n - G I n - P h e - L e u - P r o - T y r - P r o - T y r 70 80 Tyr-Ala - L y s - P r o - A l a - A l a - V a l - A r g - S e r - P r o - A l a - G I n - l l e - Leu-GIn-Trp-GIn-Val- Leu-Ser90 100 Asp -Thr - V a l - P r o - A l a - L y s - S e r - C y s - G l n - A l a - G I n - P r o - T h r - T h r - M e t - A l a - A r g - H i s - P r o - H i s (Asn) (Pro) 1051106 110 120 Pro - H i s - L e u - S e r - P h e - M e t - A l a - I le - P r o - P r o - L y s - L y s - A s n - G I n - A s p - L y s - T h r - G l u - l l e - P r o mis) 130 136 140 Thr-lle - Asn-Thr-lle - A l a - S e r - G l y - G t u - P r o -Thr-Ser -Thr-Pro-Thr-ffuj- G l u - A l a - V a l - G l u I Thr (variant A) NeuNAc(2-»3(6)) Gal(l->-3(6))GalNAc (carbohydrate-containing κ-caseins) 148 1 50 1 60 Ser - T h r - V a l - A l a - T h r - L e u - G l u - p U â r - S e r - P r o - G l u - V a l - l l e - G l u - S e r - P r o - P r o - G l u - I l e - A s n (variant A) Asp Ρ 169 Thr-Val-Gin-Val-Thr-Ser-Thr-Ala-Val.OH
Journal of Dairy Science Figure 4.
Primary structure of bovine κ-Cn-B (l)
70
F U N C T I O N A L I T Y A N D PROTEIN STRUCTURE
t h e n e g a t i v e l y c h a r g e d g l y c o m a e r o p e p t i d e (GMP) g r o u p . It is be l i e v e d t h a t the exposed, GMP-containing p o l y p e p t i d e c h a i n i s r e s p o n s i b l e f o r κ - c a s e i n s pronounced s t a b i l i z i n g a c t i o n f o r the m i l k c a s e i n m i c e l l e s y s t e m , s i n c e c l e a v a g e o f t h i s e n t i t y by e n z y m a t i c r e n n e t m o d i f i c a t i o n c o m p l e t e l y removes i t s s t a b i l i z i n g ability. C o m p a r i s o n o f t h e d i s t r i b u t i o n o f t h e number o f i m p o r t a n t ami n o a c i d t y p e s f o r e a c h o f t h e c a s e i n s ( T a b l e 2) p r o v i d e s an i n d i c a t i o n o f t h e i r o v e r a l l c a p a c i t y f o r a g g r e g a t i o n a n d i n t e r a c t i o n v i a h y d r o p h o b i c , i o n i c and d i s u l f i d e bond f o r m a t i o n . I t w i l l be n o t e d t h a t κ - c a s e i n i s t h e o n l y monomer s u b u n i t t h a t c o n t a i n s a d i s u l f i d e group, and t h i s i s undoubtedly r e s p o n s i b l e f o r i t s a b i l i t y t o s e l f a g g r e g a t e as w e l l as t o i n t e r a c t w i t h β l a c t o g l o b u l i n during heat p r o c e s s i n g of m i l k .
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
1
Association of
C a s e i n Monomer
Subunits.
A l l t h r e e o f t h e c a s e i n monomer s u b u n i t s e x h i b i t a s t r o n g c a p a c i t y to i n t e r a c t at n e u t r a l pH. A d d i t i o n o f Ca i o n s s t r o n g l y promotes t h e i r tendency to a g g r e g a t e , presumably by c r o s s - l i n k i n g c a r b o x y l and phosphate e s t e r groups l o c a t e d i n t h e i r exposed, a c i d i c peptide c h a i n s . A d d i t i o n o f CA p r o b a b l y a l s o p r o m o t e s a g g r e g a t i o n o f t h e c a s e i n monomer s u b u n i t s by r e d u c i n g t h e i r molecular charge. I t i s p o s s i b l e t h a t , due t o s t e r i c h i n d r a n c e , κ - c a s e i n ' s n e g a t i v e l y c h a r g e d GMP group may be u n a b l e t o p a r t i c i p a t e i n i n t e r m o l e c u l a r Ca c r o s s - l i n k s w i t h a d j a c e n t c a s e i n monomers a n d a g g r e g a t e s . T h i s may a c c o u n t f o r t h e m i c e l l e s t a b i l i z i n g f u n c t i o n d i s c u s s e d above f o r κ - c a s e i n . These c a s e i n - c a s e i n i n t e r a c t i o n s , e s p e c i a l l y those i n v o l v i n g β - c a s e i n , are s t r o n g l y t e m p e r a t u r e - d e p e n d e n t ; a phenomenon t h a t i s i m p o r t a n t f o r a s s o c i a t i o n / d i s s o c i a t i o n o f t h e s e monomer s u b u n i t s w i t h and f r o m t h e milk c a s e i n m i c e l l e system (4). κ - c a s e i n a l s o c o n t a i n s two h Cys r e s i d u e s p e r monomer s u b u n i t a n d i s t h u s c a p a b l e o f i n t e r a c t i n g w i t h t h e whey p r o t e i n s , e.g., m a i n l y β - l a c t o g l o b u l i n , v i a the d i s u l f i d e i n t e r c h a n g e mechanism a t t e m p e r a t u r e s a t o r above 6 5 ° C . T h i s l a t t e r phenomenon i s b e l i e v e d t o be i m p o r t a n t i n p r o v i d i n g c o l l o i d a l s t a b i l i t y t o the m i l k c a s e i n m i c e l l e s y s t e m , as w e l l a s t o t h e whey p r o t e i n s , i n h i g h temperature processed m i l k p r o d u c t s . I t has a l s o been p o s t u l a t e d t h a t t h i s l a t t e r i n t e r a c t i o n w i t h 3 - l a c t o g l o b u l i n may a l t e r t h e a v a i l a b i l i t y o f κ - c a s e i n i n the m i c e l l e , a n d t h u s h a s a d e t r i m e n t a l e f f e c t upon t h e c h e e s e m a k i n g p r o p e r t i e s o f m i l k (4). The C a s e i n M i c e l l e
System.
The m a j o r c a s e i n s e x i s t i n m i l k as h i g h l y s t r u c t u r e d , s p h e r i c a l a g g r e g a t e s , c o n s i s t i n g o f 450 t o 1 0 , 0 0 0 s u b u n i t s (3), commonly r e f e r r e d t o as m i c e l l e s . The i m p o r t a n t p h y s i c o - c h e m i c a l p r o p e r t i e s o f t h e m i c e l l e s a r e s u m m a r i z e d i n T a b l e 3. Casein m i c e l l e s a r e s y n t h e s i z e d i n v i v o by b i o c h e m i c a l l y c o n t r o l l e d p r o c e s s e s , w h i c h have not been t o t a l l y c h a r a c t e r i z e d (5). Even
si
A r g and H i s
1 2
20
17
1
25
169
Lys,
0
0
35
20
5
25
209
0
STÂLIC ACID
0
h CYS
PROLINE
17
2
25
BASIC
8
SERINE Ρ
Monomers
42
1
Casein
the Major
199
G l u and T y r
2
-Β
ACIDIC
Amino A c i d C o m p o s i t i o n o f
TOTAL
II.
Asp,
K-B
3-A
α
CASEIN
Table
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
III.
nm
2
Q
w
Adapted
from M o r r ,
25 C
1975
cc/g micelle 3.5-6
2-3
Q
S
1.6-2
dry micelle
ο
2-18 χ 10
8-22 χ 10
C
Voluminosity,
a
S
Daltons
g water/g
Weight,
Constant,
100-250
25 C
0-5
Solvation,
Molecular
Sedimentation
Diameter,
95-98 %
in Milk
25 C
of C a s e i n M i c e l l e s
75-80 %
micelle/total
Physico-Chemical Properties
C
0-5
Casein,
Table a
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S
°
>
3
g
π
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
4.
MORR
Milk
73
Proteins
though they a r e remarkably s t a b l e i n m i l k under normal c o n d i t i o n s , t r e a t m e n t s s u c h as c o o l i n g a n d w a r m i n g ( b e t w e e n 5 a n d 37 C ) , a d j u s t i n g t h e pH b y c h e m i c a l o r m i c r o - b i o l o g i c a l m e a n s , adjusting Ca a n d o t h e r i o n c o n c e n t r a t i o n s , h e a t i n g , e v a p o r a t i o n a n d d r y i n g , e n z y m a t i c m o d i f i c a t i o n (as w i t h r e n n e t ) a l l e f f e c t t h e s t a b i l i t y o f the m i c e l l e s ( 4 ) . T h e r e h a v e b e e n a number o f excellent p a p e r s p u b l i s h e d t h a t p r o v i d e an i n s i g h t i n t o t h e c o m p l e x i t y o f t h e m i l k c a s e i n m i c e l l e s t r u c t u r e as w e l l as a n u n d e r s t a n d i n g o f the f a c t o r s t h a t s t a b i l i z e i t . S c i e n t i s t s who h a v e made s p e c i a l c o n t r i b u t i o n s i n t h i s r e g a r d a r e : Waugh, e t a l . ( 6 ) , P a y e n s (7), G a m i e r a n d R i b a d e a u - D u m a s ( 8 ) , M o r r ( 9 ) , Rose (10) a n d B l o o m f i e l d and Mead ( 2 ) . S l a t t e r y (3) r e c e n t l y p r o p o s e d a m o d e l f o r t h e m i c e l l e s a n d t h e i r m e c h a n i s m o f f o r m a t i o n , b a s e d upon t h e a m p h o p h i l i c n a t u r e o f t h e i n d i v i d u a l c a s e i n monomer s u b u n i t s a n d their submicellar aggregates. A l t h o u g h S l a t t e r y s model c o n s i ders the r o l e o f the s u b m i c e l l a r a g g r e g a t e s o f the t h r e e major caseins ( a - , β - , and κ - c a s e i n ) and t h e i r assembly mechanism, it i g n o r e s the importance and i r r e v e r s i b l e s t r u c t u r e o f " c o l l o i d a l p h o s p h a t e " (11) i n t h e m i c e l l e . 1
g
Commercial
Caseinates.
C a s e i n i s i s o l a t e d f r o m m i l k a n d p r o d u c e d as N a , K , a n d Ca c a s e i n a t e s by the g e n e r a l s t e p s o u t l i n e d i n F i g u r e 5. Both the a c i d i f i c a t i o n and n e u t r a l i z a t i o n s t e p s p r o f o u n d l y a f f e c t the s t r u c t u r e and assembly o f r e s u l t i n g c a s e i n a t e p a r t i c l e s . These d e r i v e d c a s e i n a t e aggregate p a r t i c l e s have l i t t l e s i m i l a r i t y to milk casein m i c e l l e s . A c i d i f i c a t i o n to i s o e l e c t r i c c o n d i t i o n s , e . g . , pH 4 . 5 - 5 . 0 , c o m p l e t e l y d i s s i p a t e s t h e c o l l o i d a l p h o s p h a t e s t r u c t u r e a n d f r e e s t h e c a s e i n p r e c i p i t a t e o f Ca a n d o t h e r i n o r ganic i o n s . N e u t r a l i z a t i o n o f the c a s e i n p r e c i p i t a t e r e s o l u b i l i zes the c a s e i n , p r e s u m a b l y by a l t e r i n g i t s charge s u f f i c i e n t l y t o overcome i n t e r m o l e c u l a r h y d r o p h o b i c b o n d i n g . The r e s u l t i n g c a s e i n a t e s y s t e m c o n t a i n s a g g r e g a t e s o f t h e monomer s u b u n i t s i n a range o f s i z e s , depending upon temperature and e l e c t r o l y t e composition (12). The o r i e n t a t i o n o f t h e i n d i v i d u a l c a s e i n mono mer s u b u n i t s w i t h i n t h e s e a g g r e g a t e s i s unknown, b u t i t may b e assumed t h a t , due t o t h e r a p i d i t y o f t h e a c i d i f i c a t i o n / r e s o l u b i l i z a t i o n r e a c t i o n s , they are randomly a s s o c i a t e d . B a s e d upon t h e s t r o n g s e n s i t i v i t y o f t h e s e a g g r e g a t e s t o pH a n d i o n i c a d j u s t ments, as w e l l as t o e n z y m a t i c m o d i f i c a t i o n , i t i s l i k e l y t h a t t h e s u b u n i t s a r e a r r a n g e d w i t h an o r i e n t a t i o n t h a t p r o v i d e s t h e i r h y d r o p h i l i c , a c i d i c - p o l y p e p t i d e - c h a i n s e q u e n c e maximum c o n t a c t w i t h t h e a q u e o u s medium. C o m m e r c i a l c a s e i n a t e s , p r e p a r e d as N a a n d Κ c a s e i n a t e s e x h i b i t i m p r o v e d s o l u b i l i t y and f u n c t i o n a l i t y compared t o Ca c a s e i n a t e . T h i s l a t t e r phenomenon i s p r o b a b l y due t o l a r g e r s i z e d a n d more s t r o n g l y i n t e r a c t i n g Ca c a s e i n a t e a g g r e g a t e s due t o c r o s s - l i n k i n g b y t h e d i v a l e n t c a t i o n s . Comparison o f the above c o m m e r c i a l c a s e i n a t e s w i t h m i l k c a s e i n m i c e l l e s i n d i c a t e s t h a t , under normal food p r o c e s s i n g c o n d i -
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
74
FUNCTIONALITY AND PROTEIN STRUCTURE
t i o n s , e . g . , pH 6 t o 7, c a s e i n a t e a g g r e g a t e s a r e s m a l l e r a n d more s e n s i t i v e t o pH a n d i o n i c f l u c t u a t i o n t h a n a r e t h e c o l l o i d a l phosphate-containing milk micelles. F o r example, the m i l k m i c e l l e s y s t e m s t a b i l i z e s a h i g h e r l e v e l o f Ca i o n s t h a n c a n t h e commer c i a l c a s e i n a t e s , due t o i t s i n c o r p o r a t i o n i n t o t h e r e l a t i v e l y n o n r e a c t i v e c o l l o i d a l phosphate polymer s t r u c t u r e (10). Although t h e r e h a s b e e n much c o n j e c t u r e on t h e s u b j e c t , i t i s c o n s i d e r e d h i g h l y d o u b t f u l that m i c e l l e s resembling those i n milk i n every d e t a i l , can be r e f o r m e d by c o m b i n i n g t h e i r i n d i v i d u a l components. I f t h i s l a t t e r f u n c t i o n c o u l d be a c c o m p l i s h e d , i t w o u l d p r o b a b l y p e r m i t t h e d e v e l o p m e n t o f new c a s e i n a t e forms w i t h m o d i f i e d f u n c tional properties. Functional Properties
of
Commercial
Caseinates.
Although commercial c a s e i n a t e s are used i n a wide v a r i e t y o f a p p l i c a t i o n s w i t h i n the f o o d i n d u s t r y (13, 14), they are e s p e c i a l l y u s e f u l i n those that u t i l i z e t h e i r e x c e l l e n t s u r f a c t a n t p r o perties. The p r o b a b l e e x p l a n a t i o n f o r t h e e x c e l l e n t surfactant p r o p e r t i e s o f the c a s e i n a t e s l i e s i n t h e i r unique a m p h i p h i l i c c o n f o r m a t i o n as w e l l a s t h e i r o r d e r e d a g g r e g a t e s t r u c t u r e s , as d e s c r i b e d above. I t has been r e p o r t e d t h a t c a s e i n m i c e l l e s r a p i d l y a s s o c i a t e on t h e s u r f a c e o f f r e s h l y - f o r m e d f a t g l o b u l e s (15) to s t a b i l i z e them a g a i n s t c o a l e s c e n c e a n d s e p a r a t i o n . T h u s , i t may be p o s t u l a t e d t h a t c a s e i n a t e , e i t h e r as monomer ^ u n i t s o r t h e i r a g g r e g a t e s , a l s o r a p i d l y m i g r a t e t o a n d a s s o c i a t e on f r e s h l y f o r m e d a i r / w a t e r o r o i l / w a t e r i n t e r f a c e s o f foams o r e m u l s i o n s t o s t a b i l i z e them a g a i n s t c o l l a p s e o r c o a l e s c e n c e . It i s further p r o p o s e d t h a t s u c h e m u l s i o n s a n d f o a m s , due t o t h e h i g h a v a i l a b i l i t y o f c a s e i n a t e h y d r o p h i l i c , a c i d i c p e p t i d e c h a i n s to the a q u e o u s p h a s e , w o u l d e x h i b i t a s t r o n g s u s c e p t i b i l i t y t o pH a n d Ca i o n fluctuations. A l s o , a d d i t i o n a l e m u l s i f i e r s , foaming agents and f o o d i n g r e d i e n t s t h a t a l t e r t h e a v a i l a b i l i t y and r e a c t i v i t y o f t h e exposed c a s e i n a c i d i c c h a i n s would l i k e l y have a p r o f o u n d i n f l u e n c e upon t h e f u n c t i o n a l p r o p e r t i e s o f t h e c a s e i n a t e s i n foam a n d e m u l sion applications. s u
Enzymatic M o d i f i c a t i o n of
Casein.
The a c t i o n o f r e n n e t u p o n c a s e i n m i c e l l e s i n m i l k i l l u s t r a t e s t h e t r e m e n d o u s i m p o r t a n c e o f e n z y m a t i c m o d i f i c a t i o n upon t h e c o n f o r m a t i o n , p h y s i c o - c h e m i c a l and f u n c t i o n a l p r o p e r t i e s o f c a s e i n ates. R e n n e t s p e c i f i c a l l y h y d r o l y z e s t h e 105-106 l i n k a g e o f t h e κ - c a s e i n p o l y p e p t i d e c h a i n w h i c h , although l o c a t e d w i t h i n the m i c e l l e , i s h i g h l y a c c e s s i b l e t o the enzyme. The e f f e c t o f t h i s r e a c t i o n i s t o r e l e a s e t h e s t r o n g l y a c i d i c GMP f r o m κ - c a s e i n monomer s u b u n i t s , t h e r e b y r e d u c i n g t h e m a g n i t u d e o f t h e n e g a t i v e c h a r g e o n t h e m i c e l l e s u f f i c i e n t l y t o p e r m i t them t o associate into a gel structure (4).
4.
MORR
Milk
Proteins
75
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
P r o p e r t i e s o f t h e Whey P r o t e i n s
and T h e i r
Subfractlons.
As w i t h t h e c a s e i n s , whey p r o t e i n s h a v e b e e n f u r t h e r f r a c t i o n a t e d a n d t h e s e s u b f r a c t i o n s h a v e b e e n c h a r a c t e r i z e d (I) . The p r i m a r y s t r u c t u r e s o f two o f t h e s e p r o t e i n s , 3 - l a c t o g l o b u l i n a n d α - l a c t a l b u m i n a r e g i v e n i n F i g u r e s 6 a n d 7. Comparison o f these primary s t r u c t u r e s w i t h those o f the caseins i n d i c a t e s s e v e r a l ma j o r differences which account f o r t h e i r d i s t i n c t p h y s i c o - c h e m i c a l and f u n c t i o n a l p r o p e r t i e s . I n c o n t r a s t t o t h e c a s e i n s , whey p r o t e i n s e x h i b i t a r a t h e r u n i f o r m d i s t r i b u t i o n o f a c i d i c / b a s i c and h y d r o p h o b i c / h y d r o p h i l i c amino a c i d s a l o n g t h e i r p o l y p e p t i d e chains. T h u s , t h e y l a c k t h e a m p h i p h i l i c n a t u r e o f t h e c a s e i n mo nomer s u b u n i t s , b u t a r e r a t h e r p r e s e n t i n a c o m p a c t , g l o b u l a r conformation (16), The s u b s t a n t i a l l y l o w e r p r o l i n e c o n t e n t i n t h e whey p r o t e i n m o l e c u l e s a l s o p e r m i t s a g l o b u l a r c o n f o r m a t i o n w i t h a s u b s t a n t i a l h e l i c a l c o n t e n t , which e x p l a i n s t h e i r s t r o n g suscep t i b i l i t y to d e n a t u r a t i o n by h e a t and s i m i l a r treatments ( 4 ) . Iso i o n i c p o i n t s o f t h e m a j o r whey p r o t e i n s r a n g e f r o m 4 . 2 t o 5 . 4 (Table 1 ) , which a r e comparable to those o f the c a s e i n s . D e n a t u r a t i o n o f whey p r o t e i n s h a s b e e n shown t o u n f o l d t h e i r p o l y p e p t i d e chains and c o n v e r t t h e i r conformation from a g l o b u l a r to an extended form t h a t f a c i l i t a t e s t h e i r s e l f i n t e r a c t i o n ( a g g r e g a t i o n ) and i n t e r a c t i o n w i t h κ - c a s e i n by d i s u l f i d e i n t e r c h a n g e , i o n i c and hydrophobic bonding ( 4 ) . A l t h o u g h β - l a c t o g l o b u l i n e x i s t s as a monomer w i t h a m o l e c u l a r w e i g h t o f 18,000 a t p H ' s below 3 . 5 , i t a s s o c i a t e s t o form a n octamer w i t h m o l e c u l a r w e i g h t o f 144,000 a t pH*s i n the 3.7 to 5.1 range a n d e x i s t s as a dimer w i t h a m o l e c u l a r w e i g h t o f 36,000 a t pH 5 . 1 (16) . T h e s e p H - d e p e n d e n t i n t e r a c t i o n s a r e due t o c h a n ges i n i o n i z a t i o n o f a c i d i c a n d b a s i c amino a c i d s w h i c h h a v e a n e f f e c t upon t h e f o r m a t i o n a n d d i s s i p a t i o n o f h y d r o p h o b i c a n d d i s u l f i d e bonds. A l t h o u g h 3 - l a c t o g l o b u l i n and α - l a c t a l b u m i n a r e c a p a b l e o f the above a s s o c i a t i o n / d i s s o c i a t i o n r e a c t i o n s , t h e y p r o b a b l y h a v e l i t t l e importance i n determining f u n c t i o n a l p r o p e r t i e s i n food a p p l i c a t i o n s i n t h e pH 6 t o 7 r a n g e . H o w e v e r , p r o t e i n c o n c e n t r a t i o n , pH a n d o t h e r r e l a t e d f a c t o r s do a f f e c t t h e i r s u s c e p t i b i l i t y t o h e a t denaturation (17). P r e p a r a t i o n o f Whey P r o t e i n
Concentrates.
I n c o n t r a s t t o t h e c a s e i n s , whey p r o t e i n s r e t a i n t h e i r s o l u b i l i t y i n t h e pH 4 . 5 - 5 . 0 r a n g e , p r o v i d e d t h e y h a v e n o t b e e n denatured. I t i s therefore r e l a t i v e l y d i f f i c u l t t o recover and p u r i f y undenatured p r o t e i n c o n c e n t r a t e s on a commercial s c a l e . P r o c e s s e s t h a t s e p a r a t e t h e whey p r o t e i n s f r o m t h e l o w m o l e c u l a r w e i g h t , n o n p r o t e i n components o f whey h a v e b e e n u s e d w i t h o n l y moderate success t o date ( 1 8 ) . Such p r o c e s s e s u t i l i z e u l t r a f i l t r a t i o n / r e v e r s e o s m o s i s membrane t e c h n o l o g y , g e l f i l t r a t i o n b y t h e basket c e n t r i f u g e t e c h n i q u e , p o l y v a l e n t i o n p r e c i p i t a t i n g agents
76
FUNCTIONALITY
A N D PROTEIN STRUCTURE
SKIMMILK pH 4.6-5.0 CENTRIFUGE
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
CASEIN
—1
CURD
WHEY
WASH pH 8.5-9.0 (Na,K,Ca) Figure 5.
Procedure for preparation of commercial caseinate
10 H.Leu-lle - V a l - T h r - G l n - T h r - M e t - L y s - G l y - L e u - A s p - l l e
SPRAY
DRY
(A/Dr 20 - Gin-Lys 1 V a l - A l a - G i y - T h r - T r p - T y r -
30 401 S e r - L e u - A l a - M e t - A l a - A l a - S e r - Asp-lle - Ser- L e u - L e u - A s p - A f a - G l n - S e r - A l a - P r o - L e u - Arg)50 60 V a l - T y r - V a l - G l u - G l u - L e u - L y s - P r o - T h r - P r o - G l u - G l y - A s p - L e u - G l u - l l e - Leu-Leu-JGIn-j-Lys(variant C) His ! 70 80 T r p - G l u - A s n fAsp[-G I u - Cys - A la - G In - Lys - Lys -1 le - I l e - A l a - G l u - L y s - T h r - Lys-Ile - P r o - A l a - | Gly (variant B and C) 90 100 V a l - P h e - L y s - L e u - A s p - A l a -Ile - A s n - G l u - A s n - L y s - V a l - L e u - V a l - L e u - A s p - T h r - A s p - T y r -LysI 110 fSHli 120 L y s - T y r - L e u - L e u - P h e - C y s - M e t - G l u - A s n - S e r - A l a - G l u - P r o - G l u - G l n - S e r - L e u f V a l f Cys'-GIn ] (variant B and C) Ala !S~H> " 130 140 "C'fs - Leu -Val - A r g - T h r - P r o - G l u - Val - A s p - A s p - G l u - Ala - L e u - G l u - L y s - Phe - Asp - L y s - Ala - L e u - ^ 150 Lys- Ala - Leu -Pro -Met -His -1 le - Arg - L e u -Ser - Phe - A s n -Pro - T h r - Leu-Gin - G l u - Glu - G i n 162 His-lle.OH
160 Cys -
Journal of Dairy Science Figure 6.
Primary structure of bovine β-Lg-A
(1)
20
I 10 H . G I u - G l n - L e u - T h r - L y s - C y s - G l u - V a l - P h e -{Argj-Gtu-Leu - L y s - A s p - L e u - L y s - G l y - T y r - G l y - G l y (variant A) Gin 30 40 V a l - S e r - Leu-Pro - G l u - T r p - V a l - C y s - T h r - T h r - P h e - H i s - T h r - S e r - G l y - T y r - A s p - T h r - G l u - A l a -
I 50 60 lie - V a l - G l u - A s n - A s n - G l n - S e r - T h r - A s p - T y r - G l y - L e u - P h e - G l n - l l e - A s n - A s n - L y s - l l e - T r p I 70 I 80 C y s - L y s - A s n - A s p - G l n - A s p - P r o - H i s - S e r - S e r - Asn-lle - C y s - Asn-Ile - S e r - C y s - A s p - L y s - P h e 90 I 100 L,eu-Asn-Asn-Asp-Leu-Thr-Asn- Asn-lle - M e t - C y s - V a l - L y s - L y s - l i e - Leu-Asp-Lys - V a l - G l y 110 J 120 l i e - A s n - T y r - T r p - L e u - A l a -His - L y s - A l a - L e u - C y s - S e r - Glu - L y s - L e u - A s p - G i n - T r p - Leu - C y s 123 Glu-Lys-Leu.OH
Journal of Dairy Science Figure 7.
Primary structure of a-La-B (1)
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
4.
MORR
Milk
Proteins
77
(carbοxymethyl c e l l u l o s e , hexametaphosphate, acrylic acid, etc.), e l e c t r o d i a l y s i s , and i o n - e x c h a n g e r e s i n treatments to fractionate and r e c o v e r t h e whey p r o t e i n c o n c e n t r a t e s w h i c h commonly c o n t a i n o n l y 35 t o 50% p r o t e i n o n a d r y w e i g h t b a s i s . Inherent i n each o f t h e s e p r o c e s s e s i s t h e n e c e s s i t y t o c o n c e n t r a t e t h e d i l u t e whey p r o t e i n s o l u t i o n under c o n d i t i o n s t h a t minimize d e n a t u r a t i o n o f t h e h e a t - s e n s i t i v e whey p r o t e i n s . B o t h o f t h e commonly u s e d c o n c e n t r a t i o n p r o c e s s e s , e . g . , vacuum e v a p o r a t i o n and s p r a y d r y i n g , i n t r o d u c e a s m a l l b u t d e f i n i t e amount o f p r o t e i n d e n a t u r a t i o n i n t o the o v e r a l l p r o c e s s (19). Whey p r o t e i n c o n c e n t r a t e p r e p a r a t i o n p r o c e s s e s b a s e d upon h e a t d e n a t u r a t i o n t o i n s o l u b i l i z e a n d p r e c i p i t a t e them f r o m whey have been d e v e l o p e d (20). S u c h whey p r o t e i n c o n c e n t r a t e p r o d u c t s a r e s u i t a b l e f o r a p p l i c a t i o n s i n p a s t a p r o d u c t s (21) due t o t h e i r i n s o l u b i l i t y and l a c k o f s t i c k i n e s s . Functional Properties
of
Whey P r o t e i n
Concentrates.
A l t h o u g h whey p r o t e i n c o n c e n t r a t e s p o s s e s s e x c e l l e n t n u t r i t i o n a l and o r g a n o l e p t i c p r o p e r t i e s , t h e y o f t e n e x h i b i t o n l y p a r t i a l s o l u b i l i t y a n d do n o t f u n c t i o n as w e l l as t h e c a s e i n a t e s for s t a b i l i z i n g a q u e o u s foams a n d e m u l s i o n s ( 1 9 ) . A number o f compo s i t i o n a l and p r o c e s s i n g f a c t o r s are i n v o l v e d which a l t e r the a b i l i t y o f whey p r o t e i n c o n c e n t r a t e s t o f u n c t i o n i n s u c h f o o d formulations. These i n c l u d e : p H , redox p o t e n t i a l , Ca c o n c e n t r a t i o n , heat d e n a t u r a t i o n , enzymatic m o d i f i c a t i o n , r e s i d u a l p o l y phosphate o r o t h e r p o l y v a l e n t i o n p r e c i p i t a t i n g a g e n t s , residual m i l k l i p i d s / p h o s p h o l i p i d s and c h e m i c a l e m u l s i f i e r s (22). I t i s l i k e l y t h a t t h e i n a b i l i t y o f whey p r o t e i n s t o f u n c t i o n as w e l l a s c a s e i n a t e i n s t a b i l i z i n g foams a n d e m u l s i o n s i s due t o c o n f o r m a t i o n a l a n d s t r u c t i o n a l d i f f e r e n c e s i n t h e two p r o t e i n s . I t i s t h e r e f o r e p o s t u l a t e d t h a t whey p r o t e i n s , w h i c h l a c k an a m p h o p h i l i c c o n f o r m a t i o n , do n o t o r i e n t s u f f i c i e n t l y w e l l a t a i r / w a t e r i n t e r f a c e s t o s t a b i l i z e foam o r e m u l s i o n s y s t e m s as effec t i v e l y as caseinates. D e n a t u r a t i o n o f t h e whey p r o t e i n m o l e c u l e , i f p r o d u c e d a t t h e proper stage of the p r o t e i n concentrate i s o l a t i o n / u t i l i z a t i o n p r o c e s s , can improve the f u n c t i o n a l i t y . The i m p r o v e m e n t i n f u n c t i o n a l i t y i s p r o b a b l y due t o a n u n f o l d i n g o f t h e m o l e c u l e t o e x p o s e h y d r o p h o b i c amino a c i d r e s i d u e s , t h u s m a k i n g t h e p r o t e i n more a m p h o p h i l i c and c a p a b l e o f o r i e n t i n g a t the a i r / w a t e r o r o i l / w a t e r interface. However, i f the p r o t e i n c o n c e n t r a t e i s d e n a t u r e d d u r i n g p r e l i m i n a r y p r e p a r a t i o n s t a g e s , e . g . , p r i o r to o r d u r i n g d r y i n g , t h e i r s o l u b i l i t y and r e l a t e d f u n c t i o n a l i t y a r e a d v e r s e l y affected. Summary. The c o n f o r m a t i o n casein
and p h y s i c o - c h e m i c a l
and whey p r o t e i n c o m p o n e n t s ,
properties
and t h e i r
of
the
subfractions,
major are
78
FUNCTIONALITY AND PROTEIN STRUCTURE
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r e l a t e d to the f u n c t i o n a l p r o p e r t i e s of commercial c a s e i n a t e and whey p r o t e i n c o n c e n t r a t e s u s e d i n t h e f o o d p r o c e s s i n g i n d u s t r y . The e f f e c t s o f p H , t e m p e r a t u r e , i o n i c e n v i r o n m e n t , h e a t d e n a t u r a t i o n , enzymatic m o d i f i c a t i o n and p r o c e s s i n g are a l s o c o n s i d e r e d i n this regard. The m a j o r c a s e i n monomer s u b u n i t s h a v e random c o i l c o n f o r m a t i o n that f a c i l i t a t e s strong p r o t e i n - p r o t e i n i n t e r a c t i o n v i a h y d r o p h o b i c and i o n i c b o n d i n g . The u n i q u e a m p h i p h i l i c s t r u c t u r e , which a r i s e s from s e p a r a t e l y c l u s t e r e d h y d r o p h o b i c and n e g a t i v e l y c h a r g e d ( a c i d i c a n d e s t e r p h o s p h a t e ) amino a c i d r e s i d u e s a l o n g t h e p o l y p e p t i d e c h a i n , makes them s u s c e p t i b l e t o pH and Ca i o n c o n centration effects. This amphiphilic nature i s probably responsi b l e f o r the e x c e l l e n t s u r f a c t a n t p r o p e r t i e s of commercial c a s e i n ate i n a v a r i e t y of food a p p l i c a t i o n s . The m a j o r whey p r o t e i n s h a v e c o m p a c t , g l o b u l a r conformations, w i t h a s u b s t a n t i a l amount o f h e l i c a l s t r u c t u r e . This conformation i s p r o b a b l y due t o t h e r a t h e r u n i f o r m d i s t r i b u t i o n o f a c i d i c / b a s i c a n d h y c r o p h i l i c / h y d r o p h o b i c amino a c i d s a l o n g t h e i r p o l y p e p t i d e chains. T h e i r s e n s i t i v i t y to h e a t and o t h e r d e n a t u r i n g a g e n t s i s due t o t h e h i g h c o n t e n t o f s u l f h y d r y l / d i s u l f i d e g r o u p s , w h i c h s t a b i l i z e t h e i r d e r i v e d , random c o i l c o n f o r m a t i o n b y t h e d i s u l f i d e bond i n t e r c h a n g e mechanism. Exposure of s u l f h y d r y l , hydrophobic a n d a c i d i c amino a c i d r e s i d u e s d u r i n g d e n a t u r a t i o n makes t h e whey p r o t e i n s s u s c e p t i b l e to s e l f p o l y m e r i z a t i o n and i n t e r a c t i o n w i t h casein, e . g . , mainly κ-casein. Although heat denaturation gene r a l l y r e d u c e s t h e s o l u b i l i t y a n d f u n c t i o n a l i t y o f whey p r o t e i n s , i t can be u t i l i z e d , i f conducted a t the p r o p e r p o i n t i n the p r o c e s s , to improve t h e i r f u n c t i o n a l i t y .
Functionality and Protein Structure Downloaded from pubs.acs.org by NANYANG TECHNOLOGICAL UNIV on 05/29/16. For personal use only.
4. MORR
Milk Proteins
79
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October 23, 1978.