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)