Colloids and Sur/occr.

6 (1983) 35-42 35
Elsevier Scientific Publishing Company, Amsterdam - PrInted in The Netherlands

EMULSIFYING PROPERTIES OF PROTEINS AND POLYSACCHARtDES

1. METHODS OF DETERMINATION OF EMULSIFYING CAPACITY
AND EMULSION STABlLlTY

A.N.OUR0V.M.A. MUKHIN, N.A. LARICHEV*, N.V. LOZINSKAYA,

V.B. TOLSTOGUZOV
Laboratory of New Food Forms, A.N. Nesmcyaraot~ lnslitutc of Oruat~oclert~rnl Corn-
pounds, U.S.S.R. Academy of Scicwcs. 28 Vavifav Sir.. B-312 ~~loscow. I1 7813 (U.S.S.II.)
(Received 29 .Iprit 1982; accepted in final form 12 August 1982)

r\BSTR ACT

A new method of dctrrrnination of the emulsifying characterislics of protein and some

other substances, based on a rtudy of the dependence of the nonpolar emulsified phase
quantity at the phase inversion point upon the emulsifier quantity, is proposed. Emulsi-
fying characteristics of proteins under lhe conditions wheru? the amount of oil in emulsion
is tower than necessary for phase inversion may be compared by rn?z??cof the diagrams
p!ottcd in the coordinatcr of 51 separated phase fraction-ktitial nonpolar phase fraction”
(emulsion stability diagrams). L:ome changes in the terms denoting emulsifying charactcr-
istics are proposed.

INTROL)UCTiON

Functional propertics nf proteins and fame other nutritional substances

are comt,lcx characteristics evaluated in model systems and describe the
ability of the substances to fulfil certain structural function5 in red natri-
tional systems (produced or ready for use) (11. In this paper we shall primari-
ly dcaI with the functional properties of’ I>roteins, among which emulsifying
characteristics, namdy, emulsifying cap;ti:ity and amulsion stability, are of
much importance, describing jlrotcin be:?wiour 1.1a wide range of nutritional
syst-.:ms.
Ry emulsifying capacity (EC,) is impli&d the ratio of an oil quantity emulsi-
fM under specified conditions at the phase inversion point to a protein quan-
tity in the system. The concept of emulsifying capacity was originally intro-
duct d by Swift et al. 121. Later a number of workers (3-G) investigated
t!!s influence of other factors, such as the equipment design, oil addition
rate and blender speed, temperature, pH, ionic strength of the solution, on
the EC, value of different proteir,s. EC, was shown to depend, to some cx-
tent, upon these parameters. This led the authors 13-G) to think that EC,
of proteins may serve as a comparative characteristic of their emulsifying
properties, only tvhcn it is determined under the same conditions, for the

0166~6622/83/0000-0000/$03.00 0 1983 Ekvier Scientific Publishing Company

same nonpolar phase, with the same system parameters and emulsifying
regime. Other workers [ 7 ] conduded that EC, should be determined under
condit,ions optimized for all relevant parameters. Optimization of all para-
meters may, however, be very difficult.
Thus with existing methods EC, may not be used as a standard charackr-
istic for direct comparison of the emulsifying properties OF different pro-
teins or of results obtained by diffelr:nt authors [I].

hlRTEhlALS AND hlI3THDDS

The phase inversion point was determined by a sharp fall of the electric
conductivit.y during titration of 5 ml aqueous solution of protein or poly-
saccharide with a nonpolar phase. Emuslification was performed in a
cylindrical vessel. For each nonpoIar phase its addition rate (4.6 mI/min)
and blender spcud (3,400 2 150 r.p.m.) WLW adjusted so that determined
character&tics remained practically unaffected by lhcm.

The procedure was as follows. To II protein, polysaccharidc or their com-

plex solution, some amount of a nonpolar phase was added so that the total
volume was 26 ml, The system was emulsified in a square-sectioned vessel
for 3 min at the Mender sexed of 3,000 i 150 r+m. Tire choice of lime
nud splzd was dictated by the above discussed cansidcrations. Then emulsion
was transferred into u measuring test lube, 26 ml in volume, thormostattcd
for 1 It at 25°C. then the volumes of separated aqucuus and no~~polm phases
wcrc measurcrl. By the end of thcrmostntt,ing the phase sqaration was prrlc-
tically over. These dala were used ta illot. a stability diagram (Fig. 3) laying
off as abscissa lhc initial volume fraction of the nonpolar phase and as
orttinnte, m the left al~d on the tight, respectively, volume fractions of the
separated aqueous and nonpolar phases. In tiae CUSPof full decay of the
emulsion, the exRtximr?ntal points tie at tbc diagram diagonal.
Rovinc serum alhumin (MA) and ovalbumin (O-4) of the Oline plant of
chemical reagents jUSSR), sodium salt of dcxtrnn sulphak (DS), .md
beet pectin of the Krasnodar pectin plant (USSR) were used in tire work.
Dcuunc and cctton oil were used as ;1nonpolar phccm

RFSULTS AND DISCUSSION

Emulsifying capacity is an important clmracteristic. it is usually determined

from the equation
 37

EC, = P/P (1)

where EC, is apparent emulsifying capacity, F is maximum amount of emulsi-
fied oil, P is protein quantity in the system (2’). F is determined by phase in-
version when a nonpolar phase is continuously added to an aqueous phase: that
is, a protein solution or suspension of a definite concentration. the arbitrarily
chosen protein concent.ration remaining constant for ali proteins under com-
parison t&-12], whether the protein is fully or partially dissohwd. In our
opinion, this invalidates the method, inasmuch as F would depend on protein
concentration, i.e., the latter would be determinant for the EC, value, and
in additio)l F dependence on P may vary from protein to protein. Moreover,
EC, value of a partially dissolved protein is not indicat.ive of its emulsifying
properties; as the stabiiizing effect would, presumably, depend upon the
properties of both the dissolved protein and its suspension, dissolved protein
fraction and several other, generally uncontrolled factors (e.g., degree of
suspension dispcrsity). It may be noted that the correlation between solubil-
ity and EC, of proteins reported in [ 13,143 may be accounted for by the
fact that EC, depends primarily upon dissolved protein quantity in suspensions
under study.
Considering Eq. (1) again, it may be noted that if F is determined in a
dynamic regime (i.e., when the fact of emulsion esistencc is established dur-
ing the emulsification process), then
FP = 0 =FofO.
In other words,

where IrP is nonpolar phase quantity at the phase inversion point additionally
emulsified by the protein action. Fig. 1 shows a achemc of the expected F
depcndcncc upon P (or protein concentration, CP ), F,.,,* reached at some
P = P* (see Fig. 1) may bc accounted for by the frrci that generally F cannot
increase unlimitedly with an increase of P in the system. In the case of a
linear F growth with P (or CP ) at P < P* emulsifying capacity, EC, can be
easily dctcrmincd from the equation

(P < P*) (2)

where it is assumed that, similar to EC, F, is constant. The assumption of
EC independence of the emulsifier quantity is apparently based on the as-
sumption that emulsion dispersity and the structure of the adsorption layer
are invariable. It may be rewrirtcn in a more practicabk form:
F=EC*P+Fo (P c P*) (3)

According to eq. (3), EC is plot&d as the slope of a linearly increasing part

of the diagram, F = f(P). It may be rewritten in a more genera! form for the
 1 tgo=lxo
2- t*p’ = EC,
3 tgatEC

Fig. 1. The determination of emulsityin~ oapacity (KC), saturation concentration (P+)

values and emulsificetion Ihi: {Km, ).

non-linear F dependence on P:

(see rig. 1).

Expcrimuntnlly obtaillcd F dq~endence on Pin coordinates spccificcl
about?, for t~rrr proteins, puclin and I3SA-DS complex, is shown in Pig. 2.
WC can SL’Cthat. at sufficiently low conccrrlmtions of emulsifier, EC, dclor-
mined from eq. (4) is actually independent of the latter pnmmetcrs. WC
wn &o set that for ~albumin (cotton uil emulsion) the P-P dcpcndcnue
plot has the same shape % that in Fig. 1. E’or BSA, ho~cvcr, abnormal
P---P dcpcndcncc is displayed for reasons so far unknown. Ptxhaps the ab-
normality arises from emulsion dispxsity and/or emulsifier conccntration-
dqxnrlcnt. structural rearrangements in the adsorption layer. At any rate
it provce thnt. EC should not. be dctermincd for a single arbitrary conccn-
traticn of the emulsifier but should rather be detemlincd from eq. (6).
Thus emulsifying capacity (lhe intrinsic one inr!udcd) under given condi-
 39

F.9 ’ BSA

-l
Icconc

+/#---+--

20

k
E&A-OS
__+A--’
A’
r)

1
__
__
_---
15
P

GA

0 _ao’
20 ---x-- 63 100
P-lo’_9 - BSA.or.f3relse~t~
P- IO3.g - BSA-WT.

Fig. 2. Dependence of cmuisificd dccanc quantily at the plmsa invcrsion point (F) upon
~mulaifier quanlily (P).

tiom may bc evaluated only on &he basis of F (or EC,) dependence upon
the concentration, and only at I’ < P*. Foi example, P* vahe for BSA
(dccane emulsion) under the experimcntaI conditions was 0.01 g. At higher
quantities of the protein EC estimation has no significance. P* and F,,,
values (we shall refer to them as “saturation concent-ration” and “emulsifi-
cation Iimit,” respectively) may also be regarded as emulsifying character-
istics of the dissolved substance.

Characteristics of emulsion that contain a nonpolar yhase in quantities

smaller tha required for phase inversion, can he investigated as exemplified
by “cmuIsion stability” (ES) anal %mulsIfying activity” (EA ). ES is usually
exIwcssed as a fraction of oil separated from the emulsion during a given
period of time at constant tcmperaturc and gravitational field 1lS-181.
Essentially, EA was ovaluatcd in a similar way. (For example, Iasumatsu
et al. [ 141.) Franzen and Kinzella calculated emulsifying activity as “height
of emulsified layer divided by height of total contents in the tube after
centrifugation at 1300 g for 5 min” [IS).
Further, in this work, a method of evaluation of emulsion stability (ES)
as a function of the initial nonpolar phase fraction (8) at constant total
volume, is used. In the diagram in Fig. 3 a nor polar phase volume fraction,
ILC., is plotted as abscissa, while aqueous and nonpolar phase vohame fmc-
NE. 3, Emulsion stability diagram.

tiuns, separated under givenexperimental conditions, arc? plotted as orclinatq

at. the left and at the right, rcqxrtively. Emulsions with various Q wwc
transferred into mcasuriug test tubes and thcmrostatttid at 25°C till phase
scparalion was practically complete. Then volumes of the separaterl aqueous
and nonpolnr phases were measured, thus two poinbs for one vr?rticaI section
of the diagram wcrc obtailxd. Quite clearly, upon full emulsion decay there
will be one point lying at 11~ diagonal (Fig. 3). This fib’tlrc gives an cxan~ple
of snctiou AQ (for Q = 46), BCiYAC gives ES, while CD/CQ describes flotation
stability (IG’S)of emulsion of composition Q. The lines drawn stcross cxperi-
mental points dcfinc free phaws and emulsion regions. For example, for a
dcconc wntcr emulsion stnbiiizcd with BSA, ES = 100%, until Q = 30%. At
Q > 30% Ilartial decant separation occurs, at- Q = 66% a sharp dccre~~se of
the cmarlsion rluantit$ takes place. Diagrams likr? that depicted in Fig. 3 cn-
able the following quantitative charactcristIcs of emulsions to br? obtatncd:
(a) Q value at which separation of a nonpolnr phase occurs (point K in
tho diegram).
(b) c) value, above which emulsion stability decrcascs rapidly (point. F in
the diagram).
(c) Volume fraction of the nonpohr phu in a concentrated emulsion.
This is datermined by BC/BD.
(d) For each section of &he diagram, ES and FS arc determined as unscp-
arat. aqueous and nonpolar phase fractio1.s of their total amount in the
system (BWAC and CD/CQ, respectively). F~lr the diagram as a whole, a
 41

relative area corresponding to the unbroken emulsion (S) may serve as a

characteristic similar to ES. Likewise, an area corresponding to tho unsepa-
rated aqueous phase (part of S area under the diagonal) may ~crve as a
measure of flotation stability relevant to all emulsions described by a given
diagram and being similar to 8’23.

3. SOHM?propmals regarding the terminology

As mentioned above, the dynamic method of determination of emulsify-

ing characteristics of nutritional substances, which we propose, led us to
introduce special terms for P* and F,,.,=, namely “saturation concentration”
and “emulsificat.ion limit.” Furthermore, the term “emulsifying capacity”
upplkd to c&ii,‘W and Iim (dF/rlp) vdues does not. reveal their significance.
Two other terms: %r%%ficr activity” for dF/df and “intrinsic emulsifier
activity” for lim (dF/dP) seem to us more pertinent.
To SIIMII&~~, we propose the following terms and designations:
dF
- = A, activity of emulsifier,
cw
lim T&T= lim A = Ai, intrinsic activity of emulsifier,
I’-- o tW P-r o
P*, saturation concmitrMion
Ftnax, emulsification
F tlllct level and limit, respectively.

ACKNOWLEDGEMENT

The authors arc grateful to Dr. E.E. Sraudo for his participation in the
discussion of the paper.

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