1

The effect of Lupinus albus protein on the oxidation of

sunflower oil-in-water emulsions
Isabel Sousa, Paula Lourenço, Anabela Raymundo e José Empis
DAIAT, Sec. de Ciência e Tecnologia dos Alimentos, Inst. Sup. Agronomia/Univ. Técnica de Lisboa
Tapada da Ajuda, 1349-017 Lisboa, Portugal

ABSTRACT
Four sunflower o/w emulsions (commercial, lupin, lupin + xanthan gum and home-made
mayonnaises) stabilised by two different types of proteins (egg yolk and lupin isolate) were studied in
terms of oxidation development (peroxide and p-Anisidine values), textural parameters (firmness and
adhesiveness), and rheological characteristics (mechanical spectra and plateau modulus -G0N ), to
observe if the Lupinus albus protein has a protective role against oxidation of food emulsions like
mayonnaise.
The emulsions were left at room temperature and incubated at 50  2ºC. Once a week the emulsions
were stirred with an inox spoon to introduce some metal contact and incorporate some oxygen into
the emulsions, to accelerate the oxidation phenomena.
A pro-oxidative effect of the lupin material, due to residual oil on the protein isolate, was observed
and one can say that lupin protein isolates will perform better if they are fat free. In terms of
structure, lupin emulsions kept their emulsion texture, in spite of the severe thermal conditions, while
egg-yolk emulsions were drastically destabilised.
1. INTRODUCTION
Lipid oxidation is one of the most common causes of quality deterioration in food emulsions.
Oxidation of lipids results in chemical changes affecting the sensory characteristics, nutritional
quality, and safety of food emulsions, creating economic losses in the food industry.
The design of food emulsions with improved quality depends on a better understanding in the
physicochemical mechanisms of lipid oxidation in these systems. The oxidation of emulsified lipids
differs from that of bulk lipids, because of the presence of the droplet membrane, the interactions
between the ingredients, and the partitioning of ingredients between the oil, aqueous and intefacial
regions 1.
Food manufacturers must develop methods of preventing, or at least retarding, lipid oxidation in
foods. To do this effectively, it is necessary to have a thorough understanding of the mechanisms of
lipid oxidation, and how these are affected by the physicochemical environment of the lipids. The
lipid oxidation has been extensively studied in bulk fats and oils, and there is a fairly good
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understanding of the factors that affect oxidation in such sistems2, but there are substantial
differences between the lipid oxidation in bulk oils and that in emulsified oils 1. Understanding of the
factors that affect lipid oxidation in systems in which the oil is dispersed as emulsion droplets is still
an active and important area of research2.
Emulsions are thermodynamically unstable systems. To form kinetically stable emulsions for a
reasonable period of time, chemical substances known as emulsifiers must be added before
homogenisation. Emulsifiers, are surface-active molecules that adsorb to the surface of droplets,
forming a protective membrane that prevents coalescence 2. Many proteins, by virtue of their
amphiphatic structure and unfolding capabilities can act as emulsifiers, forming coherent, cohesive
interfacial films that surround the oil droplets with charged layers, thereby retarding coalescence 3.
Food emulsions, such as mayonnaise, are usually stabilised by egg yolk. The use of plant proteins as
functional additives to improve specific properties of “new foods” is a subject of growing interest.
The plant protein, such as soy and lupin, will replace the traditional animal protein with dietetic
(cholesterol free), technological (salmonella free), and ethical (animal free) advantages, and will
increase the offer for “green products”4,5.
Various factors, such as solubility of the emulsifier, pH, salts, emulsifier concentration, temperature,
and the properties of the interfacial film itself, all affect the coalescence stability of emulsions 6,7,8
Temperature, affects physical stability, and influences also the chemical instability of food emulsions,
such as lipid oxidation (heat induced hydroperoxides degradation). Many of the physicochemical
properties of emulsions, essentially their high dynamic nature, may also have a significant influence
on lipid oxidation in colloidal systems1.
Unsaturated oils are oxidised by free radical autoxidation, a chain reaction process catalysed by the
products of the reaction. Oxidation of oils may also be initiated by lipoxygenase or photosensitizers.
The lipid hydroperoxides formed during the oxidation process are colourless, tasteless and odourless.
Undesirable flavours associated with lipid oxidation arise from the decomposition of the labile
hydroperoxides. The volatile secondary products of oxidation formed include hydrocarbons,
aldehydes, ketones, alcohol, esters, furans and lactones1.
In order to retard lipid oxidation in food emulsions, one can control the droplet membranes and
droplet composition, use antioxidants, control the partitioning of reactants/products and ingredient
interactions1. In the present study, the potential antioxidant effect of Lupin isolate in sunflower oil-
water emulsions was investigated.
2. MATERIAL AND METHODS
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All mayonnaises were sunflower oil (Helianthus annus Linnaeus) - water emulsions, containing 2%
(w/w) of vinegar, 1%(w/w) salt and 0,7%(w/w) citric acid. With exception to the commercial
mayonnaise, all the rest had 0,1% (w/w) sodium azide to prevent fungi growth.
All mayonnaises were prepared at ambient temperature using a high-pressure homogenisator
-ULTRA TURRAX T-25, during 5 minutes. The samples (commercial, lupin, lupin + xanthan and
egg-yolk) were submitted to two different temperatures - ambient temperature and 50ºC. Both
chemical and physical changes were observed through six months.
The lupin stabilised emulsions were prepared using Lupinus albus isolate L 9000, obtained from
Mittex, Weingarten (Germany), with a protein content of 90% and 8% oil. The lupin and lupin light
mayonnaises contained, respectively, 5 and 7%(w/w) of protein, and the latter also had 0,4%(w/w) of
xanthan gum to replace the oil effect9. The commercial and traditional home made mayonnaises
contained 0,6%(w/w) and 3%(w/w) of egg-yolk, respectively.
The oil extraction of the emulsions was achieved by mixing with a chloroform and hexane mixture,
following the Bligh & Dyer (1959) technique.
The primary oxidation products (hydroperoxides) were determined by the Peroxide Value, and the
secondary oxidation products (aldheydes, ketones, etc) were analysed by the p-Anisidine Value. Both
determinations were carried out following standard titrimetric and spectrophotometric procedures.
The textural measurements were carried out at 20 ± 1ºC in a texturometer TAX-T2, Stable Micro
System (U. K.) with a cylindrical probe (diameter = 37,6 mm), with a penetration of 5 mm and a
velocity of 2,0 mms-1 in emulsions on a 60 mm diameter cylindrical flask.
The rheological parameters were determined at 20 ± 0,5ºC in a stress controled rheometer Haake RS
75 (Germany), using cone-plate (C60/2º) geometry. Mechanical spectra were run in the linear
viscoelastic region of each mayonnaise. These analysis were carried out only at the beginning and
ending of the 6 month incubation time.
To test for significant evolution of the experimental parameters through the six months, Scheffé’s
ANOVA-MANOVA post hoc comparisons was used.

3. RESULTS AND DISCUSSION

The commercial mayonnaise is protected against lipid oxidation by antioxidant addition.
Nevertheless, if the exponent can be used as a measure of the rate of peroxide production, one can see
from fig. 1a, that lupin emulsion (L, T) showed slower rates: 0,106 and 0,150 against 0,180 and 0,170
for commercial (C) and traditional egg-yolk mayonnaises, respectively. An explanation to this is
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given by fig. 1b. From these data it can be seen that lupin emulsions have higher rates (0,106 and
0,05) of secondary products (S P) formation. This can be due to the presence of 8% oil on lupin
protein isolate. This oil, submitted to high temperatures during the protein isolation procedure, can
act as a starter for the oxidation of sunflower oil inside the o/w droplets, which is further supported by
the highest rate of the lupin light emulsion with higher protein content, in spite of the presence of
xanthan gum, a highly viscous polysaccharide, which will reduce diffusion phenomena within the
system.
As expected, an increase in temperature (figs. 2a, 2b), apparently resulted in a higher hydroperoxides
degradation into secondary products.

0.180x 0.170x C L
y (C)= 2.8596e y (O)= 4.4168e
2 2
O T
70 R = 0.8314 R = 0.8457
0.106x 0.150x
60 y (L)= 6.9214e y (T)= 5.1213e
2 2
50 R = 0.4366 R = 0.8209

40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (weeks)

0.031x 0.042x
y (C)= 5.4856e y (O)= 4.4059e C L
40
2 2
R = 0.4275 R = 0.5226 O T

0.106x 0.053x
30 y (L)= 2.9282e y (T)= 4.6658e
2 2
R = 0.6928 R = 0.4434

20

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time (weeks)
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Fig.1 - Peroxide (a) and p-Anisidin (b) Values curves obtained at room temperature for C-
commercial mayonnaise; L- lupin light mayonnaise; O- home made egg-yolk mayonnaise; T- lupin
mayonnaise.

y (C)= 2.2826e
0.043x
y (O)= 6.8765e
-0.0041x C
2
R = 0.1347
2
R = 0.002 L
70
0.051x 0.056x O
60 y (L)= 3.8174e y (T)= 3.997e
2 2 T
R = 0.1141 R = 0.1437
50

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time (weeks)

0.121x 0.083x C
y (C)= 3.6025e y (O)= 4.0442e
2 2 L
40 R = 0.8483 R = 0.8486
O
0.110x
y (L)= 4.2424e y (T)= 3.9597e
0.109x
T
2
30 R = 0.7823 2
R = 0.8495

20

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (weeks)

Fig. 2 - Peroxide (a) and p-Anisidin (b) Values curves obtained at 50ºC for: C- commercial
mayonnaise; L- lupin light mayonnaise; O- home made egg-yolk mayonnaise; T- lupin mayonnaise.

As it may be seen in Figs. 3a and 3b, at room temperature, all mayonnaises showed a decreasing in hardness, accompanied
by a slight decrease in adhesiveness, with exception to the commercial mayonnaise whose texture parameters showed the
highest values and did not change through the six months study. We observed that the home made egg-yolk mayonnaise
was the less hard and less adhesive mayonnaise. A small decrease (-1,425 and –1,575) in firmness was observed for the
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lupin mayonnaises (fig. 3a), confirmed by the a decrease (-1.788 and –3.467) in adhesiveness (fig 3b), being the lupin light
mayonnaise, in presence of xanthan gum, the harder and the most adhesive one but also the one with a bigger decay on
adhesiveness through time, at room temperature.

y (C)= -0.253x + 112.27 y (L)= -1.425x + 90.135 y (O)= -1.868x + 68.303 y (T)= -1.575x + 83.254
2 2 2 2
R = 0.1153 R = 0.8214 R = 0.6896 R = 0.8895
130

110

90

70

50

30 C
10 L

-10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 O
Time (weeks) T

y (C)= -0.435x + 187.5 y (O)= -1.467x + 93.548

300 2 2
R = 0.0668 R = 0.4773
250 y (L)= -1.788x + 116.75 y (T)= -3.467x + 118.13
2
R = 0.2326 2
R = 0.6737
200

150

100 C
L
50
O

0 T
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (weeks)

Fig. 3 - Textural results - hardness (a) and adhesiveness (b) - obtained at room temperature for: C-
commercial mayonnaise; L- lupin light mayonnaise; O – home made egg-yolk mayonnaise; T- lupin
mayonnaise.

The results shown in Figs. 4a and 4b, allow us to conclude that at 50ºC both the commercial and
home-made egg-yolk mayonnaises greatly decreased in hardness (-6.814,-4.606) and adhesiveness
(-14.208,-8.034). On the contrary, both lupin mayonnaises kept their texture approximately constant
(-0.149, -0.299 and –2.037, -1.752) during experimental time, pointing to a higher structural stability
of the lupin protein emulsions as it was confirmed by visual observation.
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y (C)= -6.814x + 110.25 y (O)= -4.606x + 94.02 C

2 2
R = 0.9275 R = 0.9014 L
120
O
y (L)= -0.147x + 71.011 y (T)= -0.299x + 49.813
100 2 2 T
R = 0.0191 R = 0.0665
80

60

40

20

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (weeks)

y (C)= -14.208x + 184.66 y (O)= -8.034x + 117.09 C

2 2
300 R = 0.8975 R = 0.9518 L

y (T)= -1.752x + 66.97 O

y (L)= -2.037x + 102.95
250 2 2
R = 0.2702 T
R = 0.4399

200

150

100

50

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
-50
Time (weeks)

Fig. 4 - Textural results - hardness (a) and adhesiveness (b) - obtained at 50ºC for: C- commercial
mayonnaise; L- lupin light mayonnaise; O – home made egg-yolk mayonnaise; T- lupin mayonnaise

The mechanical spectra of the emulsions at time zero and after six months, at room temperature are
shown in Figs. 5a and 5b. The egg-yolk home-made mayonnaise improved structure with ageing
which is demonstrated by loosing the critical point, were G’ and G’’ cross, observed in the first
spectra (fig. 5a). The commercial mayonnaise was always the one with the higher level of structure,
with highest values of G’ and higher distance between G’ and G’’ curves, lowest values of δ, a
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tendency to a plateau region on the G’ curve and the presence of a minimum in the G’’ curve. With
time (fig. 5b), both lupin mayonnaise G’ curve slopes slightly increased with frequency, denoting a
smooth decrease in the structural level.
With the increase in temperature (figs. 6a, 6b), an increase in the structural level was observed,
possibly dued to the protein denaturation, favouring aggregation of the protein. These observations
are confirmed by the positive variation of the “plateau” modulos (G0N) (Table 1).

G. Commercial 1
10000 G..
G. Lupin Light 1
G..
G. Home-made Egg-Yolg 1
G..
G. Lupin 1
1000 G..
G' G'' [Pa]

100

0.01 0.1 1 10 100 1000

ω [rad/s]

G. Commercial 2
G..
10000
G. Lupin Light 2
G..
G. Home-made Egg-Yolk 2
G..
G. Lupin 2
G..
1000
G' G'' [Pa]

100 The contrast

analysis of

0.01 0.1 1 10 100

ω [rad/s]
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the results showed that home made egg-yolk mayonnaise did not significantly (p>0,05) altered its G0N
value.

Fig. 5 - Frequency sweeps at room temperature, of the initial (a) and after 6 months (b)
measurements.
Table 1 – Plateau modulus variations (G0N = [G’]TAN δ minimum ) each mayonnaise between zero and ending
study times, at both temperatures.

∆ G0N
Samples Room Temperature 50º C
Commercial not change significantly 529 %
Home-made Egg Yolk -14 % 37 %
Lupin Light -33 % not change significantly
Lupin not change significantly 69 %

G.. Commercial 1 50ºC

G.
10000 G. Lupin Light 1 50ºC
G..
G. Home-made Egg-Yolk 1 50ºC
G..
G. Lupin 1 50ºC
G..
1000
G'
G''
[P
a]

100

0.01 0.1 1 10 100 1000

ω[rad/s]
 G. Com. 2 50ºC
G..
G. Light 2 50ºC
G.. 10
10000 G. Egg-Yolk 2 50ºC
G..
G. Lupin 2 50ºC
G..

G' G'' [Pa]

1000

100

0.01 0.1 1 10 100 1000

ω [rad/s]

Fig. 6 - Frequency sweeps at 50ºC, of the initial (a) and after 6 month (b) measurements.

4. CONCLUSIONS
From this introductory work about the possible protective effect of Lupin protein against oil oxidation
in emulsion systems, one can say that protein isolates will perform better if they are fat free. The
presence of residual oil can promote oxidation of the emulsified oil inside the droplets. Besides this
drawback, the lupin protein can stand severe destabilising conditions, like high temperature regimes
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and keep emulsion texture. Egg protein aggregate and emulsions phase separate with severe browning
for similar conditions.

FURTHERWORK
Test the fat free lupin protein isolate with a larger period of ageing up to 12 months, which is the
shelf life of commercial mayonnaises.

5. REFERENCES
1. Coupland, J. N. & McClements, D. J. (1996). Lipid oxidation in food emulsions. Trends in Food
Sci. & Technol., 7: 83-91.
2. Dickinson, E. (1992). An Introduction to Food Colloids, Oxford University Press.
3. Phillips L. G., Whitehead, D. & Kinsella, J. (1994) Structure-function properties of food proteins.
Academic Press, Inc. San Diego.
4. Sousa, I., Raymundo, A., Empis, J., Franco, J. & Gallegos, C.(1998). Rheology and texture of lupin
protein-stabilised emulsions: a statistical approach. Les Cahiers de Rhéologie, XVI (1): 112-116.
5. Raymundo, A., Empis, J. & Sousa, I. (1998). Optimisation of lupin protein emulsion composition.
Pol. J. Food Nutr. Sci., 7/48 (3 S): 127-134.
6. Raymundo, Franco, J., Gallegos, C. & A., Empis, J. & Sousa, I.(1998). Effect of thermal
denaturation of lupin protein on its emulsifying properties. Nahrung, 42: 220-224.
7. Franco, J.; Raymundo, A.; Sousa, I. & Gallegos, C.(1998). Influence of processing variables on the
rheological and textural properties of lupin protein-stabilised emulsions. J. Agric. Food Chem., 46:
3109-3115.
8. Kato, A. & Nakai, S. (1984). Hydrophobicity determined by fluorescence Probe method and its
correlation with surface properties of proteins. Biochim. Biophys. Acta, 624: 13-20.
9. Raymundo et al.(undated). Formulation of low oil “light” food emulsions stabilised by lupin
protein. Unpublished results.