Compositional Characterization of Acidic Whey Dispersions in Relation to

Development of Visible Changes during Storage

PhD Defence Nanna Stengaard Villumsen

Food Science, Aarhus University

Background – whey

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Acid Whey

Sweet/Rennet WheyContaining caseinomacropeptide (CMP)

Background

Whey Protein IsolateProteinsBeta-lactoglobulin ~ 50 %Alpha-lactalbumin ~ 20 %Caseinomacropeptide ~ 20 %

Other proteins - including enzymes ~10 %

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WPI

• Re-dispersions (7 %) and acidification (pH 3) • Heat treatment (conventionally below 100 ˚C)

Storage induced aggregation (SIA)

Cheese DairyDairy Farm

Whey proccessing plant

BackgroundInitial 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

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Low HighIntermediate

Effect of Heat Treatment and Storage Temperature

MineralsContent (g/100 g) Batch Low Batch High

Calcium 0.0169 0.0018 Magnesium 0.0019 0.0009 Phosphorus 1.31 1.44 Chloride 0.12 0.11 Sodium 0.002 0.002 Potassium 0.0006 0.0039

Control of Heat Treatment and Storage Temperature Prevents the Formation of Visible Aggregates in Acidic Whey Dispersions Over a 6-month Storage Period

Nanna Stengaard Villumsen1, Marianne Hammershøj1, Line Ravn Nielsen2, Kristian Raaby Poulsen2, John Sørensen3, Lotte Bach Larsen1

Accepted for publication in LWT – Food science and Technology

Paper 1

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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…….

Detectives curse: When the answer is right under your nose, but your to busy to notice.

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True detectives

• 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

Proteolysis

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N-terminal detection

Days of storageDays of storage

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Days of storage

Free

am

inot

erm

inal

s

low

Days of storage

HighIntermediate

Fluorescamin

Peptidomic Profiling In order to reveal protease cleavage sites and thereby identify proteases

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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

AspXxx XxxXxx Xxx

Weak acid hydrolysis at aspartic acid

No apparent trend in proteolytic activity

Whey Protein Aggregation

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Initial assumption: SIA and clear liquid had identical protein compositions

SIA Clear Liquid

α-LA og β-LGSIA

Clear liquid

Reversed Phase - HPLC

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Caseinomacropeptide

β-LG

α-LA

CMP

• 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

Protein Content in WPI

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Hits in web of science

SIA Clear liquid

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2D-GE

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

gCMP

aCMPHydrophobic domain

Acidic glycoside

Ca2+

H+ Hydronium ion-

-++ ++

+

+

- - -- --

+

-

+

+ +

+

+

+

--

--

--

-

+

H+

H+

H+

H+

H+

H+

+H

Neutral glycoside

++ +

+

+

+ +Effect of increased acidity Effect of modified glycosylation

Model for CMP self assemblyA

Effect of calciumB C D

Mechanisms

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-

+

++

+

+

+

--

--

- --

+

++

+

+

+ +

• Addition of calcium • Increased acidity• Enzymatic de-sialidation

Minor Storage Experiment

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Heated at 95 ˚C /180 s, stored at 25 ˚C for 40 dTurbidity: 500 nm

Calcium pH 2.5 Sialidase

Paper 2

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Heat induced de-sialidation

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--+

+ ++

+

+

- - -- --

+

Paper 3In preparation

Conclusions

SIA can be prevented by controlling

• Heating procedure• Storage temperature• pH• Sialidase treatment• calcium

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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Prevent CMP selfassembly

Further Studies2D - 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?

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Achnowledgements

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Department of Food Science&

Graduate School of Science and Technology

Arla Foods Ingredients

The Danish Dairy Board

Supervisors Lotte Bach Larsen & Marianne Hammershøj

Arla Foods IngredientsLine Ravn, Søren Bang, Kristian Raaby, John Sørensen and Jacob Holm

TechniciansHanne Søndergaard Møller

ColleaguesHanne Bak Jensen, Thao Le, Mette Marie Løkke, Jean Møller, Sandra Beyer, Jesper Schmidt,

Valentin RauhAalborg UniversityHenrik Kjeldal, Rasmus Hansen, Florian Herbst

Rune Thorbjørn

My family and friends

Simon

Supervisors Lotte Bach Larsen & Marianne Hammershøj

Arla Foods IngredientsLine Ravn, Søren Bang, Kristian Raaby, John Sørensen and Jacob Holm

TechniciansHanne Søndergaard Møller

ColleaguesHanne Bak Jensen, Thao Le, Mette Marie Løkke, Jean Møller, Sandra Beyer, Jesper Schmidt,

Valentin RauhAalborg UniversityHenrik Kjeldal, Rasmus Hansen, Florian Herbst

Rune Thorbjørn Nordvang

My family and friends

Simon

Thank you for your attention

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