The effect of a combined pressure/temperature treatment on the structure of -lactoglobulin with
or without dextran sulfate
Amar Aouzelleg, Laura-Anne Bull
High Pressure
• Advantages– Increased shelf life– Fresh-like organoleptic qualities– Nutrients and vitamins unaffected– Opportunity for new products and
functionalities– Energy efficiency, no pressure gradient
Disadvantages
– Bacterial spore resistance– High cost of equipment
• Use of moderate pressure in combination with temperature
Pressure effect on -lactoglobulin
• Bovine -lactoglobulin
• Pressure induces aggregation of -lactoglobulin at high concentration
• At low concentration the secondary and tertiary structure are quite resistant even at 800-900 MPa
• Effect of the presence of Dextran Sulfate
Experiments• Conditions: Tris-HCl pH 7, 0.5-5 mg/ml
• Parameters:– Pressure: 136-294 MPa
– Temperature: 38-62°C
– Time: 10-30 min.
• Circular dichroism analysis– near-UV 260-320 nm, far-UV 190-260 nm
• Differential Scanning Calorimetry
• Surface Hydrophobicity
Experimental designExperiment Pressure
x1/P (MPa)
Temperature
x2/T (°C)
Time
x3/t (min.)
1 -1/150 -1/40 -1/10
2 -1/150 -1/40 1/30
3 -1/150 1/60 -1/10
4 -1/150 1/60 1/30
5 1/280 -1/40 -1/10
6 1/280 -1/40 1/30
7 1/280 1/60 -1/10
8 1/280 1/60 1/30
9 1.215/294 0/50 0/20
10 -1.215/136 0/50 0/20
11 0/215 -1.215/38 0/20
12 0/215 1.215/62 0/20
13 0/215 0/50 0/20
14 0/215 0/50 1.215/32
15 0/215 0/50 -1.215/8
Parameter -1.215 -1 0 1 1.215
P (MPa) 136 150 215 280 294
T (°C) 38 40 50 60 62
t (min) 8 10 20 30 32
Near-UV results-tertiary structure
260 270 280 290 300 310 320
-80
-70
-60
-50
-40
-30
-20
-10
0
10 Native 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
[](
deg.
cm2 .d
mol
-1)
Wavelength (nm)
• Characteristic tyrosine and tryptophan trough
• Progressive loss of dichroism due to the tryptophan residue (@293 nm)
• Loss of tertiary structure
Far-UV results - secondary structure
180 190 200 210 220 230 240 250 260 270
-10000
-5000
0
5000
10000
15000 Native 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
[](d
eg.c
m2 .d
mol
-1)
Wavelength (nm)
• Characteristic of -sheet protein
• The amplitude of the spectra is increased
• The minimum shifts from 216 nm to 205 nm
• Increase in the -helical content of the protein
Near-UV Circular Dichroism
260 270 280 290 300 310 320
-80
-60
-40
-20
0 Native 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
[](de
g.cm
2 .dmol
-1)
Wavelength (nm)
260 270 280 290 300 310 320
-80
-70
-60
-50
-40
-30
-20
-10
0
10 Native 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
[](
deg.
cm2 .d
mol
-1)
Wavelength (nm)
With Dextran SulfateWithout Dextran Sulfate
Differential Scanning Calorimetry
20 40 60 80 100
0
2
4
6
8
10
Native 1 2 3 4 5 6 7 8
Cp
/ kca
l mol
e -1
K -1
Temperature / oC
10 20 30 40 50 60 70 80 90 100 110-2
0
2
4
6
8
10
Native 1 2 3 4 5 6 7 8
Cp
/ kca
l mol
e -1
K -1
Temperature / oC
Without Dextran Sulfate With Dextran Sulfate
Surface HydrophobicityCondition Without
dextran sulfate
With dextran sulfate
native 355 (57) 333 (33) 1 610 (42) 474 (44) 2 781 (67) 630 (27) 3 812 (37) 542 (76) 4 943 (1) 951 (15) 5 936 (18) 819 (64) 6 1076 (60) 1006 (53) 7 1143 (57) 1092 (33) 8 1134 (55) 1058 (67)
• P/T effect on -lactoglobulin
• Molten globule state– Intermediate in protein unfolding/folding– Increased functionality– Opportunity for industry
• Effect of Dextran Sulfate– Potential for increased functionality
Discussion
Conclusion and Future work
• Validity of combined treatment• Different molecular structures obtained• Possible control of structures obtained• Sensitising effect of dextran sulfate• Future work should
– Characterise further these species surface (Infra Red)
– Test if functional benefits have been obtained (e.g. foaming)