Heat Stability, Solubility, and Functionality of Whey Protein:

Heat Stability, Solubility, and
Functionality of Whey Protein:
Tonya C. Schoenfuss, University
of Minnesota
Department of Food Science and
Nutrition
Milk Proteins
Caseins
Random Coil
Poor in sulfur AA's
Whey Proteins
Ordered protein structure
Higher in sulfur AA's
Present in a colloidal suspension
Generally stable to heat
Generally stable to heat
Soluble in milk
Easily denatured by heat
Easily denatured by heat
Non‐crystalline
Can crystallize (to see structure)
Distinct hydrophobic and hydrophilic regions
Balanced distribution of hydrophobic/hydrophilic regions
Insoluble at pH 4.6 (when warm)
Soluble at 4.6, and at their isoelectric Soluble
at 4 6 and at their isoelectric
points in native state
Whey Proteins
Brown. 1988. Fundamentals of Dairy Chemistry
What Functional Properties
Do We Desire in Whey
Proteins?
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Solubility
y
Gel formation (or not)
Emulsification
Water binding
β-lactoglobulin
•
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50% of the protein in the serum phase
Present as dimers (associates with itself) – pH dependant
– Monomer <3, >8
– Dimer 5.1
1–8
– Octomer 3 to 5.1
Easily denatured by heat starting at 73°C (163°F)
– reversible dissociation of oligomers to monomers
– can form aggregates
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Two disulphide bonds & 1 free sulfhydryl group
With skim milk UHT treatment (148°C (298°F), 2.5 seconds),
half of the β-lac complexes w/ Κ-casein
Two main g
genetic variants ((A&B),
) that vary
y in their heat sensitivity
y
and tendency to form octomers – vary at two amino acids.
Cows with the BB-allele have less β-lactoglobulin and increased
casein
Probably the most allergenic of the milk proteins (not present in
human milk)
B-lactoglobulin B, red box indicates where A & B differ (Asp64 & Val118)
Farrell et al. 2004. JDS 87:1641-1674
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Sulfhydryl
groups buried
until heating
Beta-barrel fold
that internally
binds Vit. A, fatty
acids and other
lipophilic
compounds
J.J. Adams et al. 2006. Journal of Structural Biology 154:246–254
β-lac Dimer Association
Hydrogen bonds between
two β-lac monomers
Brownlow et al. 1997. Structure 5:481-495
Another View
Edwards et al. 2009. Chpt. 6 in Milk Proteins: from Expression to Food. Academic Press
α lactalbumin
α-lactalbumin
• High
g in cysteine
y
– 4 disulfide bonds, no
free sulfhydryl group
• Rich in tryptophan
• 49% of the aa sequence identical to
chicken eggwhite lysozyme
• Interacts with β-1,4galactos ltransferase to modif
galactosyltransferase
modify its
structure to synthesize lactose
α-lactalbumin
Ca2+ binding
site
Edwards et al. 2009. Chpt. 6 in Milk Proteins: from Expression to Food. Academic Press
α-lactalbumin
α
lactalbumin B
Farrell et al. 2004. JDS 87:1641-1674
Effect of Heat on Whey
Proteins
• Wh
Whey proteins
t i denature
d
t
– complex with casein (K-casein)
• Issue for most cheese makes – reduces gel strength and
lengthens coagulation time
– Increase viscosity and water holding
• Can precipitate at their isoelectric point if heat
denatured (way ricotta is made)
Interaction w/ Caseins How We
Classify Powder Treatments
Temp/Time
Whey Protein Nitrogen
Low Heat
75C/15 s
Not less than 6.0 mg/g
Medium Heat
85C/20 s
High Heat
High Heat
124C/30s
1.51‐5.99 mg/g
Not more than 1.5 mg/g
Powder Class
As you heat, whey protein interacts with casein, and
you have less in the whey fraction by this test
Effect of pH of Heating
on Solubility
• U
Used
d WPI
• Varied pH and temp
and measured
solubility
• Least soluble if
heated at around 4.5
Pelegrine & Gasparetto. 2005.
Lebensm.Wiss. u-Technol. 38:77–80
Effect of Ionic Strength on
Whey Proteins
• Eff
Effects
t S
Solubility
l bilit
• Small amounts of
NaCl can salt-in
protein and
increase solubility
Effect of Ionic Strength on Whey Protein
y at pH
p 4.6
Solubility
• WPI from ion exchange min
solubility at pH 5.2, membrane
derived at 4.6 (mainly
denaturation effect). WPC’s 4.6
–5
• Authors recommend ionic
strength of 0.1 when testing
solubility
l bilit att pH
H4
4.6
6
• A&B genetic variants β-lac will
behave differently because of
charge density
de Wit and van Kessel. Food
Hydrocolloids 1996, 10: 143-149
Genetic Variants Can Affect
Interaction w/ K
K-casein
casein O’Sullivan et al. Lait (1999)
79, 229-244
AA had higher sulfhydryl content than BB indicating
l
less
d
denaturation
t ti
Thermal Stability Measured by
DSC ffor G
Genetic
ti V
Variants
i t
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Imifidon at al. 1991J Dairy Sci 74:1791-1802
Study also measured the in:teractions with K-casein genetic variants
Concluded the substitution of Gly64 and Ala118 for Asp and Val in β-LG AA
rendered the structural integrity of β-LG BB more resistant to heat in the
presence of CaCl
Solubility and gel strength at failure for 20%
(w/w) WPC gels in 0.6 M NaCI, 0.1 M Na
phosphate buffer
buffer, pH 7
7.
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The protein most
denatured (least
soluble)
l bl ) was ββ
lactoglobulin
As denaturation
i
increased:
d
– Red. solubility
– Red. gel strength
– Emulsification
index increased
w/ denaturation to
41% soluble than
decreased
Beuschel et al. 1992 J Food Sci. 605-609, 634.
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Made microparticulates
from WPI (BiPro)
Blended
microparticulates w/
unparticulated WPI &
heated at 90 °C for 30
min
Above 11% WPI w/o
particles, saw gelling
The particulates
reduced the elastic
behavior of the gels i.e.
–softer gels
Found particulates to be
an inactive filler gel
Modification of Whey Gels
g Whey
y Particles
Using
Saglam et al. 2014. Food Hydrocolloids 38:163-171
State diagram of whey protein in
continous phase vs whey particles
Total protein in system
Filled
squares are
gelled
samples
Relationship Between Whey
Protein Denaturation on Acid
Gel Firmness and Syneresis
• L
Looked
k d att diff
differentt
heating regimes
– Heated only before
powder manufacture
– Heated
H t d only
l after
ft
reconstitution
– Heated both before
and after constitution
McKenna and Anema. 1993. IDF Special Issue 9303, pp. 307-1 6
Sedimentation/Aggregation of Protein in
g Protein Beverages
g
High
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Villumsen et al. 2015.
LWT - Food Science and
Technology
gy 64 164-170
Compared heat treatment
of WPI and storage
temps for 6 months for
storage induced
aggregation
70g protein/L at pH 3.0
Suggested the
aggregated spot
contained
glycomacropeptide that
self-assembled or
proteolytic fragments of
β LG or α-LA
β-LG
LA
Take-aways
y
• β-lactoglobulin the most heat sensitive protein &
responsible for much of the functionality effects (good
and bad) observed
• Changes in functional properties due to:
– denaturation
– ionic strength
– pH
• Just measuring degree of denaturation doesn’t always
correlate with functional properties
• Selective
S l ti denaturation
d
t ti can improve
i
functionality
f
ti
lit when
h
reduction in gel strength is desired
• Other proteins in the whey might be affecting stability