Transcript PhD Forsvar Nanna Villumsen

Compositional Characterization of Acidic Whey
Dispersions in Relation to Development of Visible
Changes during Storage
PhD Defence
Nanna Stengaard Villumsen
Food Science, Aarhus University
PhD defence
Background – whey
Acid Whey
Sweet/Rennet Whey
Containing caseinomacropeptide (CMP)
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Background
Proteins
Whey Protein Isolate
Beta-lactoglobulin
~ 50 %
Alpha-lactalbumin ~ 20 %
Caseinomacropeptide ~ 20 %
Other proteins - including
enzymes ~10 %
Dairy Farm
Cheese Dairy
Whey proccessing plant
WPI
• Re-dispersions (7 %) and acidification (pH 3)
• Heat treatment (conventionally below 100 ˚C)
Storage induced aggregation (SIA)
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Background
Initial work
• Identical protein compositions between
SIA and surrounding clear liquid
• Similar protein/mineral content between
batches
• Increasing heating temperature decreases
SIA formation
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Storage Experiment 1
Low
Intermediate
High
Effect of Heat Treatment and Storage Temperature
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Minerals
Content (g/100 g)
Paper 1
Batch Low
Batch High
Calcium
0.0169
0.0018
Control
of Heat Treatment and Storage Temperature
Prevents
the Formation of
Magnesium
0.0019
0.0009
VisiblePhosphorus
Aggregates in Acidic Whey Dispersions
1.31Over a 6-month
1.44Storage Period
Chloride
0.12
0.11
Sodium Villumsen1, Marianne Hammershøj1, Line
0.002
0.002 Raaby Poulsen2, John
Nanna Stengaard
Ravn Nielsen2, Kristian
Potassium
0.0006
0.0039
3
1
Sørensen , Lotte Bach Larsen
Accepted for publication in LWT – Food science and Technology
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Mechanisms ?
• Batch differences
• Effect of increased heating temperatures
• Content / mechanism (Who is the villain)
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Who is the Villain?
True mysteries requires……. True detectives
Detectives curse: When the answer is right under your nose, but
your to busy to notice.
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Proteolysis
• Hypotheses
• Proteolysis is involved in age gelation in UHT milk and may also contribute
to SIA formation in whey dispersions
• High heating temperature inhibits proteolytic activity leading to SIA
• Batch differences caused by different proteolytic activities
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N-terminal detection
Fluorescamin
Intermediate
High
Free aminoterminals
low
Days
Days of
of storage
storage
Daysofofstorage
storage
Days
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Peptidomic Profiling
In order to reveal protease cleavage sites and thereby identify proteases
Orbitrab Mass spectrometer
Comparisons of ion-intensities showed different peptide profiles between:
• Samples heated at either 95 ˚C or 120 ˚C
• Samples stored at either 4 or 30 ˚C
• Samples from either batch
Xxx Xxx
Asp
Xxx Xxx
Weak acid hydrolysis at aspartic acid
No apparent trend in proteolytic activity
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Whey Protein Aggregation
Initial assumption: SIA and clear liquid had identical protein compositions
SIA
Clear Liquid
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Reversed Phase - HPLC
SIA
Clear liquid
α-LA og β-LG
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Caseinomacropeptide
Hits in web of science
Protein Content in WPI
CMP
β-LG
α-LA
• Tendency to aggregate and migrate at 20- 25 kDa in reducing SDS –PAGE
• Stain poorly with coomassie and elute early in RP-HPLC
• pI 3.1-4.1 due to genetic variants and PTM
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2D-GE
SIA
Clear liquid
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Batch Variation
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Effect of Heat Treatment
120 °C/20 s heat treatment renders CMP spots less acidic
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Mechanism: CMP Cold Gelation
Sialic acid
• NeuAc
• Neu5Ac
• NANA
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Mechanisms
Model for CMP self assembly
A
+
+
- +-
+ -
Ca2+
Neutral glycoside
+
- -
- +-
gCMP
H+ Hydronium ion
Acidic glycoside
Hydrophobic domain
+
aCMP
Effect of calcium
B
Effect of increased acidity
+
-+
-+
+
+ +-
-
+
C
+
+
+-
+
+
--
+
+
+
-H+
H+
- +
Effect of modified glycosylation
D
+
-H+
-+
H
+
+
-
- H+
-H+
+
H
+
+
+
+
+
+
+
+
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Minor Storage Experiment
• Addition of calcium
• Increased acidity
• Enzymatic de-sialidation
Calcium
Heated at 95 ˚C /180 s, stored at 25 ˚C for 40 d
Turbidity: 500 nm
pH 2.5
Sialidase
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Paper 2
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Heat induced de-sialidation
+
+
+ -
- +-
+
- -
- +-
+
Paper 3
In preparation
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Conclusions
SIA can be prevented by controlling
•
•
•
•
•
Heating procedure
Storage temperature
pH
Sialidase treatment
calcium
Prevent CMP selfassembly
Batch differences caused by differnet mineral contents and glycosylation pattern.
Effect of high heating temperature is due to heat induced de-sialidation
Peptide hydrolyses is chemical hydrolyses – and presumably not determining for SIA
formation. Potentially different proteolytic activities between batches.
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Further Studies
2D - gels of remaining batches – CMP enrichment and effect of heat treatment
Different dyes, e.g. pass staining
MS Identification of various CMP spots on 2D-gels. Determine PTMs and genetic variants
Test effect of sialidase in all batches
Investigate the effect of divalent cations
Larger perspective: Could this knowledge be used to modulate new products?
PhD Defence
Achnowledgements
Supervisors
Supervisors
Aalborg
AalborgUniversity
University
Lotte
Lotte
Bach
BachLarsen
Larsen&&Marianne
Marianne
HenrikKjeldal,
Kjeldal,Rasmus
RasmusHansen,
Hansen,Florian
Florian
Department
of
Food ScienceHenrik
Hammershøj
Hammershøj
Herbst
Herbst
&
Graduate
School of Science and
Technology
Arla
ArlaFoods
FoodsIngredients
Ingredients
Rune
Rune
Thorbjørn
ThorbjørnNordvang
Line
LineRavn,
Ravn,Søren
SørenBang,
Bang,Kristian
KristianRaaby,
Raaby,
John
JohnSørensen
Sørensenand
andJacob
JacobHolm
Holm
Arla Foods Ingredients
Technicians
Technicians
Hanne
HanneSøndergaard
SøndergaardMøller
Møller
My
Myfamily
familyand
andfriends
friends
Simon
Simon
Colleagues
Colleagues
The
Danish
Dairy
Board
Hanne
Hanne
Bak
Bak
Jensen,
Jensen,Thao
ThaoLe,
Le,
Mette
MetteMarie
Marie
Løkke,
Løkke,Jean
JeanMøller,
Møller,Sandra
SandraBeyer,
Beyer,Jesper
Jesper
Schmidt,
Schmidt,Valentin
ValentinRauh
Rauh
PhD Defence
Thank you for your attention