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Introduction
UHT milk processing - options
Impact of plasmin activity on the shelf life
and stability of UHT milk
160
Indirect UHT
Direct steam
infusion/injection
140
120
100
80
Valentin Rauh
Arla Strategic Innovation Center
Mejeriforskningens Dag 23. April 2015
Direct steam
infusion - ultra
short holding time
60
40
20
0
0
20
40
60
80
100
120
140
Time (s)
2
Introduction
Effect of heat treatment on milk
17 April 2015
Introduction
Protein denaturation
UHT milk processing the balance
Enzymes
Residual enzymatic
acitivty
Microorganisms
(Off-) Flavour formation
Direct steam
infusion - ultra
short holding time
3
Maillard reaction
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Heat induced
changes
4
17 April 2015
Indirect UHT
Heat severity
Introduction
Project overview: Hypotheses and aims
The plasmin system in milk
Blood
Tissue-type (tPA)
Milk
Understanding factors influencing plasmin acitvity measurements is necessary for a correct
interpretation of the results
to develop a new assay or improve existing assays and investigate the effect of sample
preparation and composition on plasmin activity
2.
Residual plasmin activity causes quality defect in UHT milk during storage
to characterize plasmin mediated proteolysis and physical-chemical changes during the storage
of UHT milk
3.
Plasmin activity measurements, proteolysis and quality defects can be connected and used
to predict shelf life of UHT milk
to correlate the proteolysis and quality defects with plasmin activity
measurements
Urokinase-type (uPA)
Plasminogen activator (PA)
Plasminogen activator
inhibitor (PAI)
Plasmin inhibitor (PI)
Plasminogen (PLG)
Plasmin (PL)
Proteolysis
Ratio:
10 : 1
5
1.
Adapted from Ismail & Nielsen, 2010
17 April 2015
Determination of plasmin activity
6
17 April 2015
Determination of plasmin activity
How to measure plasmin?
Summary
Plasmin
Skimming of homogenized milk results in a underestimation of plasmin activity
Concentration
Activity assays
Proteolysis
ELISA/Western blotting
Synthetic substrates
Absorbance/Fluorescence
Chromatography/
Electrophoresis
Recovery rates give information on the inhibitory and interfering effects in a sample
matrix
S-2251
7
17 April 2015
Dissociation of plasmin and caseins affects interaction and inhibition by caseins and
inhibitors
8
17 April 2015
Plasmin activity in UHT milk
Plasmin activity in UHT milk
UHT treatment
Feed pump
Heat treatment evaluation
Pre-heat section
Feed
160
140
Holding tube
120
Flash cooling
Pre-heat treatment
Direct
Steam
Infusion
72°C/180 s
Cooling section
60
Residual enzyme activity (%)
-lg
-la
Plasmin
Plasminogen
37
13
31
14
-
95°C/180 s
94
46
-
Indirect 140 °C/4 s
94
70
-
100
80
Whey protein denaturation (%)
Homogenizer
40
20
Packaging
0
0
20
40
60
80
100
120
140
Time (s)
9
10
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Plasmin activity in UHT milk
Plasmin activity in UHT milk
Milk development
Start
Bitterness
17 April 2015
Plasmin activity
Gelation
Gelation
1000
800
600
Plasmin
Plasminogen
400
200
0
-200
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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4
6
8
10
Storage time (wk)
Age in weeks
11
2
12
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12
14
Plasmin activity in UHT milk
Plasmin activity in UHT milk
Proteolysis
Activation of
plasminogen
Proteolysis
Constant rate
of proteolysis
Substrate
depletion/gelation
5e+5
100
4e+5
80
3e+5
60
S1-casein
-casein
40
2e+5
Bitterness
20
Gelation
1e+5
0
0
0
2
4
6
8
10
12
14
16
0
2
4
Storage time (wk)
13
14
17 April 2015
Affinity of plasmin & peptide development
Interaction regions
Ionic
79 identified peptides
12 wk
33 from
S2-casein
29 from
S1-casein
17 from -casein
4000
0 from -casein
8 wk
11 by plasmin and cathepsins
4 wk
7 by cathepsins and other proteases
0 wk
Based on hydrophobicity:
10
17 April 2015
14
S1 casein
Phosphoserine
Phospho- Hydrophobic
serine/ionic
20
30
40
1,0
*
*
Ser-P cluster
Major
Minor
Not hydrolysed
Hydrophobic
0,5
* *
0,0
-0,5
Hydrophilic
-1,0
23 potentially bitter peptides
0
50
100
Residue
Retention time (min)
15
12
61 by plasmin
14 wk
0
10
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Identified peptides after 14 weeks:
6000
2000
8
Affinity of plasmin & peptide development
Overview
8000
6
Storage time (wk)
16
17 April 2015
150
200
Conclusions
1.
2.
3.
17
Perspectives
Understanding of plasmin-casein interaction can be used to improve plasmin inactivation
Application of knowledge for different products
Residual plasmin activity causes quality defect in UHT milk during storage
Plasmin can cause bitter off flavours and is involved in the age gelation of UHT milk
Extensive proteolysis is necessary for these quality defects to occur
Infant formulas with higher nutritional value
Better functionality of ingredients
More natural products
Ambient distribution of products
Plasmin activity measurements, proteolysis and quality defects can be connected and used
to predict shelf life of UHT milk
Results indicate that a correlation is possible
18
17 April 2015
Acknowledgements
Marianne Hammershøj
Lotte Bach Larsen
Richard Ipsen
Marie Paulsson
Mette Bakman
Anja Sundgren
Carola Edsman
Jakob Holm Nielsen
Grith Mortensen
Lene Johansen
19
Correlation of plasmin activity, defects and shelf life seems possible
Understanding the factors influencing plasmin activity measurements is necessary for a
correct interpretation of the results
Plasmin activity is very dependent on the assay conditions
Inhibition and interference need to be taken into account
17 April 2015
17 April 2015