Relationship Between Hydrophobicity and Emulsifying

Properties of Some Plant Proteins

S. Nakai, L. Ho, N. Helbig, A. Kato* and M. A. Tung
Department of Food Science
University of British Columbia
Vancouver, British Columbia V6T lW5

Abstract initial slope (So) of the fluorescence intensity when a

Significant positIve correlations were observed between
bound dodecylsulfate or linoleate and dispersibility of surfactant
treated soy, sunflower and rape seed proteins, suggesting that the
bound suriactants played an important role in solubilization of
these proteins. Dispersibility was also highly correlated with
effective hydrophobicity of the proteins as measured fluorometri-
caliy. The effective hydrophobicity thus determined for soy,
sunflower and rapeseed proteins treated with surfactants and B.
subtilis proteinase for solubilization revealed good correlations
with surface tension, interfacial tension and emulsifying activity of
the ptoteins, indicating the importance of protein hydrophobicity
when used as emulsifiers.
Resume
On a observe des correlations significativement positives entre
le dodecylsulfate ou le linoleate lie et la dispersibilite des
proteines de soja, de tournesol et de colza traitees aux agents
tensio-actifs. Ceci suggere que ces derniers jouent un role
important dans la solubilisation des proteines. La dispersibilite fut
aussi en correlation etroite avec l'hydrophobicite vraie des
proteines telie que mesuree par fluorometrie. Cette hydro-
phobicite determinee en rapport avec la solubilisation pour les
proteines de soja, de tournesol et de colza traitees aux agents
tensio-actifs et pour la proteinase de B. subt;lis donna de bonnes
correlations avec la tension superficielie, la tension interfaciale et
l'activite emulsifiante des proteines. Cette correlation met en
relief l'importance de l'hydrophobicite des proteines dans leurs
proprietes emulsifiantes.
Introduction
When proteins are used as emulsifiers, the protein
molecules migrate from the bulk phase to the
oil/water interface due to their amphiphilic nature.
This surface adsorption lowers the interfacial tension
and hence reduces the mechanical energy required
to form an emulsion. The more hydrophobic the
protein, the higher the protein concentration at the
mterface, and hence, the lower the interfacial
tension. This relationship was reported previously
(Keshavarz and Nakai, 1979), using hydrophobic
chromatography and hydrophobic partition to evalu-
ate "effective hydrophobicity" of model proteins.
Later, a simpler fluorometric method was developed
for determination of effective hydrophobicity as the
·Present address: Department of Agricultural Chemistry, Yamaguchi University.
Yamaguchi, Japan.
hydrophobic probe cis-parinaric acid (PnA) was
titrated with proteins (Kato and Nakai, 1979). Good
correlations of So were found with interfacial tension
and emulsifying activity of model proteins.
We have been investigating the solubilization of
rapeseed, soy and sunflower protein isolates using
surfactant and proteinase treatments (Nakai et al.,
1980). The treatments with sodium dodecylsulfate
(SDS) and potassium linoleate were effective. For
example, potassium linoleate at a level of 8-10% of
protein increased the dispersibility of rapeseed
protein isolate from 55% to about 90% when the
mixture was treated at 65°C, pH 10 for 15 min. The
treatments were equally effective for soy and sun-
flower proteins. These proteins which were variously
treated were used for investigation of relationships
between hydrophobicity and emulsifying properties
of proteins.
This paper describes the results of determinations
of So, surface tension, interfacial tension, dispersibil-
ity, emulsifying activity and protein bound SDS and
linoleate. Efforts were made to correlate these
property values in an attempt to elucidate the
solubilization mechanism of surfactants and emulsi-
fying activity of proteins.
Materials and Methods
cis-Parinaric acid was purchased from Molecular
Probe, Roseville, MN. Corn oil was from Fisher
Scientific Co., Fair Lawn, NJ. Rapeseed meals were
by-products of oil extraction from rapeseed (Bras-
sica campestris var. Span and var. Tower), courtesy
of Canbra Foods, Lethbridge, Alberta. Soy protein
isolate (Promine D) and sunflower protein concen-
trate were the products of Central Soya Co. ,
Chicago, IL and Dr. F. Sosulski, Department of
Crop Science, University of Saskatchewan, Saska-
toon, respectively. Bacillus subtilis protease (Enzeco
bacterial protease concentrate) was a gift from
Enzyme Development Corp., Keyport, NJ.

0315-5463/80/010023·05$02.00/0
Copyright <0 1980 Canadian Institute of Food Science and Technology

23
Effective Hydrophobicity
According to the method of Kato and Nakai
(1979) ethanolic solutions of PnA (0.0036 M) were
purged with nitrogen and equimolar butylated hy-
droxytoluene (BHT) was added as an antioxidant.
Ten /-LI of PnA solution was added to 2 ml of protein
solution in 0.001 M phosphate buffer, pH 7.4
containing 0.002% SDS. The PnA-protein conjugate
solutions were excited at 325 nm and the fluores-
cence intensity was measured at 420 nm in an
Aminco-Bowman Spectrophotofluorometer, No.
4-8202. The initial slope (So, relative fluorescence
intensity!% protein) calculated from the fluores-
cence intensity vs. protein concentration plot was
used as an index of the effective hydrophobicity of
proteins.
Surface and Interfacial Tensions
Using a Fisher Surface Tensiomat Model 21, the
surface tension was measured for 0.2% protein
solution in 0.01 M phosphate buffer, pH 7.4, 5 min
after preparation of the protein solutions. Interfacial
tension was measured at the 0.2% protein solution!
corn oil interface, 5 min after preparation of the
interface. Tension readings steadily decreased fol-
lowing the preparation of protein solutions but
stabilized after 5 min.
Emulsifying Activity Index
Emulsifying activity index (EAI) was determined
according to the turbidimetric technique of Pearce
and Kinsella (1978). To prepare emulsions, 2 ml of
corn oil and 6 ml of 0.5% protein solution in 0.01 M
phosphate buffer, pH 7.4, were mixed and homoge-
nized in a Omnimixer with the micro-attachment
(Ivan Sorvall, Norwalk, eN) at the lowest speed
setting for 1 min at 20°C.
Protein Solubilization
Ten percent suspensions of rapeseed, sunflower
and soy protein isolates were treated with 0, 1,2,3,
4 and 5% SDS vs. protein and 0, 2, 4, 6, 8 and 10%
potassium linoleate according to the method de-
scribed by Nakai et al. (1980). The mixtures of
protein isolate and surfactant were heated for 10 min
at 65°C after adjusting to pH 10, then freeze-dried
after blending at pH 8.2. For proteinase treatment,
B. subtilis protease, 2.5% vs. protein, was added to
each protein suspension at pH 8.2, the mixture was
incubated for 10 min each at 35°C, 45°C and 55°C
successively and pasteurized at 65°C for 30 min, then
freeze-dried.
Bound Surfactants
The treated and control plant protein samples
were dialyzed exhaustively against phosphate buffer,
pH 8.2, I = 0.005, at 5°C and freeze-dried. The
concentrations of dodecylsulfate and linoleate in the
dialysate as well as in the undialyzed and dialyzed
protein samples were determined by the method of
Reynolds and Tanford (1970) and by the procedure
of Pro ss er et al. (1977), respectively. Lipids for
24/Nakai, Ho, Helbig, Kato and Tung
lino le ate determination were extracted from protein
samples by the procedure of Folch et al. (1957), as
follows: protein sample, 0.1 g, was homogenized
with 15 ml of 2: 1 chloroform-methanol mixture, then
filtered. To 10 ml of the filtrate, 2 ml of water was
mixed and centrifuged for 30 min at 100 xg. The
separated water phase was discarded. The washing
was repeated three more times using 1-2 ml of the
upper phase of 8:4:3 chloroform-methanol-water
mixture. The lipid-containing solvent layer was
directly used for linoleate determination.
Curve Fitting
Data transformations suggested by Mullen and
Stanley (1979) were applied using programs written
for a Monroe 1880 calculator and PL2 plotter manu-
factured by Monroe, Orange, NJ. Significance of the
Pearson product-moment correlation coefficient r
was assessed in the usual manner (Spence et al.,
1976).
Results and Discussion
Relationships of Bound Surfactants with
Dispersibility of Protein
The results of determinations of bound linoleate
and SDS are shown in Table 1. The major portion of
linoleate added for the solubilization treatment is
bound to the proteins while only about half of SDS is
bound to the same proteins.
The relationships of dispersibility of the solubil-
ized sunflower protein concentrates with bound
surfactants and So are significant and indicated in
Figures 1 and 2. The dispersibility is improved with
increased bound linoleate or SDS and a larger
quantity of linoleate than SDS has to be bound for
obtaining the same improved dispersibility. High
values of protein dispersibility are associated with
high indices of the effective hydrophobicity. Similar
significant relationships are observed for soy protein
isolate (Figures 3 and 4) and for rape seed protein
isolate (Figure 5). The different shape of the
regression lines for rapeseed protein is probably due
to lower dispersibilities attained in this particular
example and not due to species difference. Since So
was calculated based on a relative value of fluores-
cence intensity, the comparison of the values of So
between figures in unwarranted.
Table 1. Bound surfactant content of plant proteins solubilized
by surfactant treatment (%).
Added Sunflower protein Soy protein Rapeseed protein
surfactants concentrate isolate isolate
Linoleate
0 0.4 0.4
2 2.2 1.4
4 4.8 2.1
6 5.4 4.5
8 6.8 6.0
10 9.4 8.3
SDS
1 0.7 0.8 0.7
3 1.4 2.1 1.3
5 2.3 3.1 2.1
Can. Inst. Food Sci. Technol. I. Vol. 13, No. 1. January 1980
 ,.____________--------------------~5 60 __-----------------------------------,
700

/-
E
-
(/)
o
u
"Cso
Q)

>
. III
>-
'C

z-
C
l-
2.5 III
t) 500
.. C
o
ID Z en
o ::l z
J:
a. o w
I-
o ID
40
a: W
C
> U
J: <I:
LL.
a::
:J
en
300t/~------~6~0~--------~7~0--------~8:
50
DISPERSIBILlTY, Ofo
30L----4~0-0--------~6~00~------~80~0~------7.10~00
Fig. 5. Relationships of hydrophobicity and amount of bound HYDROPHOBICITV (So)
dodecylsulfate t,) dispersi~ility of/:reseed pr~tein ~o?­
ccntrate. RegreSSiOn equahons, So - 3314 x DlsperslbIi-
Fig. 6. Relationship between hydrophobicity and surface tension
ity + 5423: (r = 0.98~,'6g < 0.01); B~und SDS = (0.12 x of plant proteins. Regression equation, Surface tension =
DlsperslbIltty - 5.87) . - 0.2, (r - 0.988, P < 0.01).
e-, So; 0---, bound SDS -0.0383 x So + 67.664, (r = 0.851, p < 0.01).
. , Rapeseed protein; ., soy protein; .... , sunflower
protein. 1, untreated; 2 and 3, treated with linoleate and
proteins through hydrophobic interactions must be proteinase, respectively.
superseding the increased protein hydrophobicity
gained from surfactant binding. That the So in-
creased with bound surfactants can be interpreted as
a more intensive reaction of PnA with hydrophobic
sites on the surface of protein molecules or the sites
exposed as a result of interactions of surfactants with
10r------------------------------------,
proteins. The hydrophobic sites themselves must
have been extended due to the surfactants bound to
the protein molecules. E
u

~:.'
"-
Cl)
Relationships of Effective Hydrophobicity with c
>-
Surfactant Properties of Plant Proteins 'C
Figures 6, 7 and 8 show the relations of surface Z 8
tension, interfacial tension and EAI to So of all o
soy, sunflower and rape seed protein isolates en .3 -,~.!....
treated with linoleate and proteinase. All correla- z
w -2
tion coefficients are significant. Data of SDS- I-
treated samples were excluded from these figures ..J
since the effects of unbound SDS in the samples <I:
- 6
on surface tension, interfacial tension and EAI U
may not be ignored. Each dot in the figures repre- <I:
LL.
sents the average value of three determinations for a::
w
sample of different proteins treated differently I-
with linoleate or proteinase. Broader scattering of Z
Figures 6 to 8 than Figures 1 to 5 may be due to
the combination of all three different proteins in 4L---~--------~------~~----~7.
400 600 800 1000
each figure compared to data for single protein in HYDROPHOBI Cl TV (So)
one figure in the case of Figures 1 to 5. The So
appears to be closely correlated with all of these Fig. 7. Relationship between hydrophobicity and interfacial ten-
surfactant properties. A general correlation be- sion of plant proteins. Regression equation, Interfacial
tween emulsification capacity and protein solubility tension -4.9 = (0.0000744 x So + 0.01893t027, (r =
0.735, p < 0.05).
was discussed by Crenwelge et al. (1974). How- . , Rapeseed protein; . , soy protein; .... , sunflower
ever, "effective hydrophobicity" seems to be more protein. 1, untreated; 2 and 3, treated with linoleate and
directly correlated with emulsification capacity. proteinase, respectively.

26/Nakai, Ho, Helbig, Kato and Tung Can. Inst. Food Sci. Technol. J. Vo!. 13, No. 1, January 1980
 220~-------------------------------------'
surfactant treatment was more advantageous than
proteinase treatment for soy, sunflower and rape-
{ seed proteins due to an additional benefit of
emulsification activity improvement.
>< It is evident that the good correlation of protein
W
o effective hydrophobicity with surfactant properties
:z observed for model proteins by Kato and Nakai
., (1979) also holds true for some plant proteins. These
>-
I- results indicate that hydrophobicity may be an
::: 120 .3
important property in elucidating the mechanism of
l- protein functions in food processing.
t)
<I:
.,
(!)
:z Acknowledgement
.1
>- Research for this paper was conducted according to
U-
ti)
the terms of contract no. OSU78-00061 for Agriculture
..J Canada .
::::::>
~ .3
w
3
References
20 ~0~!0----------6-0~0-----------8~0-0---------1-0'00 Bigelow, C. C. 1967. On the average hydrophobicity of proteins and the relation
HYDROPHOBICITY (SO) between it and protein structure. J. Theoret. BioI. 16: 187.
Crenwelge. D. D., Dill, C. W .. Tybor. P. T. and Landmann. W. A. 1974. A
Fig. 8. Relationship between hydrophobicity and emulsifying comparison of the emulsification capacities of some protein concentrates. 1. Food
Sci. 39: 175.
activity index of plant proteins. Regression equation,
Folch, J .• Lees. M. and Sloane·Stanley, G. H. 1957. A simple method for the isolation
(EAI-1O)07 = 0.049 x So - 6.546, (r = 0.812, P < 0.01). and purification of total lipides from animal tissues. J. BioI. Chem. 226: 497 .
• , Rapeseed protein; . , soy protein; ,A, sunflower Kata, A. and Nakai, S. 1979. Hydrophobicity determined by a fluorescence probe
protein. 1, untreated; 2 and 3, treated with linoleate and method and its correlation with surfactant properties of proteins. Abstract 98. 39th
proteinase, respectively. Annual IFf Meeting. St. Louis. The manuscript was submitted for publication.
Keshavarz, E. and Nakai. S. 1979. The relationship between hydrophobicity and
interfacial tension of proteins. Biochim. Biophys. Acta 576: 269.
As shown in Figure 8, when the EAI was Mullen, K. and Stanley. D. W. 1979. Data transformations in statistical analysis. Can.
measured for the proteinase treated soy, sunflower Inst. Food Sci. Technol. J. 12: 98.
Nakai, S., Ho. 1.., Tung, M. A. and Quinn, 1. R. 1980. Solubilization of rapeseed, soy
and rapeseed protein isolates used in the solubiliza- and sunflower protein isolates by surfactant and proteinase treatments. Can Inst.
tion work of Nakai et al. (1979), the values were Food Sci. Technol. 1. 13:14.
generally lower than those for the surfactant-treated Pearce, K. N. and Kinsella. J. E. 1978. Emulsifying properties of proteins: evaluation of
a turbidimetric technique. 1. Agr. Food Chem. 26: 716.
samples, although they tended to be inconsistent; So Prosser, A. R .• Sheppard. A. J. and Hubbard. W. D. 1977. Modification of Canadian
did not markedly change by partial hydrolysis of Food and Drug Directorate lipoxidase method for polyunsaturated fatty acid
these proteins while the protein dispersibility was determination. J. Assoc. Offic. Anal. Chem. 60: 895.
Reynolds. 1. A. and Tanford, C. 1970. Binding of dodecylsulfate to proteins at high
improved from 55% to 95% by the proteinase binding ratios. Proc. Nat. Acad. Sci. 66: 1002.
treatment. This also indirectly supports a closer Spence, J. T .. Cotton. J. W .. Underwood. B. J. and Duncan. C. P. 1976. Elementary
Statistics. Prentice-Hall. Englewood Cliffs. NJ.
correlation of emulsification capacity with hydro-
phobicity than with solubility. Thus solubilization by Received May 22. 1979

1. Inst. Can. Sci. Technol. Aliment. Vol. 13, No. 1, Janvier 1980 Nakai, Ho, Helbig, Kato and Tung/27