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Thermodynamic Aspects of Biopolymer Functionality in Biological Systems,

Foods, and Beverages

Article  in  Critical Reviews in Biotechnology · February 2002

DOI: 10.1080/07388550290789478 · Source: PubMed

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 Criticol Reviews in Biorechnology. 22(2):89-174 (2002)

4141
Thermodynamic Aspects of Biopolymer
Functionality in Biological Systems, Foods, and
Beverages
Vladimir Tolstoguzov
Nestlé Research Centre, P.O. Box 44, Vers-Chez-les-Blanc, CH-1O00 Lausanne 26 Switzerland

ABSTRACT: Molecular mimicry and molecular symbiosis are proposed to be the main factors
controlling thermodynamic activity and phase behavior of macromolecular compounds in foods,
beverages, and chyme. Molecular mimicry implies a chemical resemblance of hydrophilic surfaces of
globular proteins with their chemical information hidden in the hydrophobic interior and low excluded
volume of the globules. The molecular mimicry contributes to the efficiency of enzymes. Molecular
symbiosis means that interactions attraction or repulsion) between biopolymer molecules greatly
differing in conformation (globular and rod-like) favor the biological efficiency of one of them at least.
The symbiosis is based on excluded volume effects of macromolecules in mixed solutions. Association-
dissociation of rod-like macromolecules can dictate thermodynamic activity of an enzyme in the mixed
solution. Thermodynamic incompatibility is typical of food macromolecules, whose denaturation,
association, complexing, and chemical modification reduce their mimicry and co-solubility. Foods are
normally phase-separated systems with highly volume-occupied phases. The phase-separated nature of
the gel-like chyme is important to the efficiency of digestion of mixed diets. Phase separation of
biopolymer mixtures, presumably, underlies mechanisms of nonspecific immune defense. The phase
behavior-functionality relationships is presented through concrete examples of some foods (such as
milk products, low-fat spreads, ice cream, wheat and rye doughs, thermoplastic extrudates, etc.),
beverages (tea and coffee), and chyme.

KEY WORDS: molecular mimicry, molecular symbiosis, thermodynamic incompatibility,

interbiopolymer complexing, exopolysaccharides, chyme.

I. INTRODUCTION: FUNCTIONAL was not a coincidence that forbidden fruit was

PROPERTIES OF FOOD
MACROMOLECULES
Historically, creativeness in food has been
a deep-rooted habit. Empirical searching of food
for survival, pleasure, and health went through
the turns of the epochs, natural disasters, wars,
epidemics, and other cataclysms. Both food
technology and biotechnology were empirically
developed, and nevertheless they became the
first high technologies. Permanent experimen-
tal findings fed into the selection of better pro-
cessing techniques and those food habits posi-
tive for longer healthier life. Survival was not
the only driving force in food development. It
0738-8551/02/$.50
O 2002 by CRC Press LLC
the first sin and regarded as a great pleasure.
However, not only satiety and pleasure but
health and time become of increasing impor-
tance nowadays. The result is a new area of
scientific research and industrial competition,
new trends in food science and technology.
The rapid development of formulated foods
has used an increasing number of individual
nutrients for food formulation, starting in the
1950s. Later, from the 1980s this process has
begun to focus on functional, quality improved
light, and instant foods. Continued success re-
quires a better understanding of functional prop-
erties of individual food components and man-
aging of composition-propertyrelationships in
89
foods, beverages, and chyme. Inasmuch as
macromolecules are the main construction
materials of formulated foods, the importance
of controlling structural functions of proteins
and polysaccharides resulted in a conception of
functional properties of food macromolecules
formulated in the 1950~l-~. A wide application
of this concept during 30 years showed, how-
ever, that functional properties of a food
biopolymer are only interpretable in systems
with artificially simplified compositions. The
need to revise this highly popular concept be-
came obvious quite ra~idly.4-I~
Functionality prediction of food macromol-
ecules is limited for the following rea son^.^-'^
Food processing implies a conversion of bio-
logical systems, whose functioning is based on
specific interactions of the components into
food systems with properties based on the non-
specific interactions of denatured components.
In other words, food functionality implies an
understanding of the physical properties of a
multicomponent physical system with the com-
ponents, interacting in a non-chemical way.
Exceptions include the Maillard reaction and
some nondesirable chemical processes, for ex-
ample, oxidation of nutrients. Therefore, de-
spite a lot of data available on the chemical and
physical properties of the best-studied food mac-
romolecules, such as soybean proteins, gluten,
casein, m i k proteins, and starch, controlling
the functionality of the most traditional sys-
tems, such as dough and milk, remains diffi-
cult. The reason for this difficulty is that prop-
erties of a food system reflect more the
interactions between its components than the
properties of the individual components them-
selves. Because there are several structural lev-
els of nonspecific intermolecular interactions
in any food, there are several reasons for the
low predictability of the structural contribu-
tions of individual food corn ponent^.^^-'^ The
f i s t important reason is the functional integra-
tion of proteins with polysaccharides. Gener-
ally, foods are mixed and multiple dispersed
systems. Foods contain a complex mixture of
proteins and polysaccharides, both of which
90
are multifunctional and fulfill several, similar
structural functions simultaneouslyI8 For in-
stance, they can simultaneously act as thicken-
ing, emulsifying, foaming, gelling, and water-
binding agents. Functional properties of food
proteins and polysaccharides could therefore
be only arbitrarily discussed separately. The
second reason is that both proteins and polysac-
charides change their functionality during food
formulation and processing due to conforma-
tional changes and complexing with one an-
other and with other food components, such as
lipids, surfactants, and metal ions. One more
reason is that foods are usually nonequilibrium
systems in which the functionality of macro-
molecules depends on kinetic factors and pro-
cessing conditions. Foods usually contain con-
centrated, highly viscous structural elements
formed by macromolecules and colloidal par-
ticles, whose functionality depends greatly on
slow processes of structure-formation and re-
laxation. Finally, the structural contributions of
a protein or a polysaccharide depend on its
place in the structural hierarchy of a food.I6-l8
A specific Structural hierarchy is typical of every
food. By analogy to the structural hierarchy of
a globular protein, the four levels of food struc-
tures are submolecular, molecular, supermo-
lecular, and macroscopic.16The macroscopic
structural elements of a composite food are the
most compelling concerning its structure-prop-
erty relationship.16-22
The objective of the article is to illustrate
that understanding how to control food system
functionality is, however, possible on the basis
of the interactions of macromolecular compo-
nents and understanding their contributions to
phase behavior and structural functions in foods.
The two types of nonspecific interactions be-
tween proteins and polysaccharides: the repul-
sion and the attraction, underlie two opposite
phenomena occurring in their mixtures. They
are (1) thermodynamic incompatibility, that is,
the limited miscibility of biopolymers at the
molecular level and (2) interbiopolymer
complexing. These two phenomena correspond
to the two types of phase separation, both of
 which usually result in two-phase liquid aque-
ous systems, that is, water-in-water (W/W)
emulsions.”~15~21-z3 Interbiopolymer complexing
concentrates the biopolymers in one of the
phases, while incompatibilityleads to their sepa-
ration and concentration within the different
co-existing phases. These two phenomena are
responsible for the functional integration of
food biopolymers, their competition, antago-
nism, and synergism.23Information related to
biopolymer incompatibility in both food and
biological systems is still limited.23-26 The hy-
pothesis on phase separation in cytoplasm was
proposedz7in 1995, the fnst monograph on this
subject has been published in 2000.28.29 Re-
cently it has become clear that there is a great
difference in phase behavior between foods
and biological systems.’9,30,31 Denatured pro-
teins, gelatinized starches, aggregated food pro-
teins, and polysaccharides are miscible in all
proportions at the molecular level only in very
dilute aqueous solutions.Most formulated foods
and chyme do not consist of dilute aqueous
solutions and, normally, are therefore phase-
separated sys tern^.'^-'^-^^ Biological systems are
of interest in this article because they are food
raw materials and active elements of food bio-
technology.
The general character of nonspecific inter-
molecular interactions of food macromolecules
underlies one more feature of food systems, the
thermodynamic similarity of most foods, which
is of applied importance for food formulation
and processing.” Actually, every cookery book
generally uses a very limited number of raw
materials and basic operations to produce a wide
variety of foods and dishes. An obvious reason
is the thermodynamic similarity of foods, which
recently has become The thermody-
namic similarity underlies the common scien-
tific basis of food processing and results in a
high efficiency of food technologies developed
empirically?’ Normally, most food technolo-
gies consist of three main steps: food formula-
tion, mechanical, and thermal tre at ment^.^^^^^.^^
These steps are designed (1) to produce a liquid
system of desirable composition; (2) to form the
structure and shape of this liquid system by its
heating, and mechanical treatment, and (3) to fix
the food product’s structure and shape, for ex-
ample,by heating, and/or cooling, and/or chang-
ing pH. The first stage, food formulation, pro-
vides both functional properties and nutritional
qualities of a processed food system. In the sec-
ond and third stages, a typical food system is
subjected to a shear flow and a heat treatment to
provide the texture, flavor, and other important
qualities of a food. By understanding the causes
of palatability and digestion efficiency of tradi-
tional food, it becomes possible to develop truly
novel functional foods.
The thermodynamic similarity of food sys-
tems could, presumably, be extended to the
chyme, that is, the mass of digesting food trans-
ported along the intestines. Actually, the chyme
is produced by physical and physico-chemical
treatments of food, which are also based on
nonspecific interactions of macromolecules. In
other words, the processes that form structure
in foods and chyme can have most features in
common. Both foods and chyme are phase-
separated systems, whose mechanical treat-
ments form their structure, shape, and func-
tionality. As in the formation of food structures,
the formation of chyme includes the similar
three main treatments: (1) grinding and mixing
of mixed diet ingredients, ( 2 )mechanical treat-
ments and changes in pH, and (3) mixing of the
comminuted food and digestive juices to form
a composition and structure of the chyme.
Analogous to food processing, foods are con-
verted into chyme by three steps, except the
denaturation of food components by thermal
treatment is replaced by treatments with a strong
acid, surfactants, and enzymes.
Because the basic concept of paper is to
illustrate the basic thermodynamic similarity
of foods, and food and the chyme, the first
section is devoted to nonspecific
intermacromolecular interaction and phase
separation as the factors involved in the struc-
ture formation processes. The objective of the
article is to consider thermodynamic aspects
of biopolymer functionality in biological sys-
91
tems, foods, beverages, and chyme. This in-
cludes probable structural functions of phase
separation in food and chyme. Thus, the ar-
ticle is focused on (1) the complexing and
thermodynamic compatibility of biopolymers
in different systems; (2) their possible role in
phase behavior of food and chyme; ( 3 ) and in
formation of structure and quality of foods
and drinks.
II. ELECTROSTATIC
INTERBIOPOLYMER COMPLEXES
Formation of interbiopolymer complexes usu-
aìly results from electrostaticinteractionsbetween
oppositely charged macromolecules.11J5~23 At pHs
below the isoelectric points (IEP), protein mol-
ecules have a net positive charge, that is, behave
as polycation. Typical food proteins, such as seed
storage proteins, milk, and meat proteins, usually
have E P s from 4to 7. Proteins as polycations and
anionic carboxyl-containing polysaccharides as
polyanions form electrostatic interbiopolymer
complexes at pHs varying from 2 to 5.
Interbiopolymer complexes usually dissociate
when the ionic strength exceeds 0.2 to 0.5, and
when the pH value is above the protein's E P .
Oligomericproteins can still form complexes with
anionic polysaccharides above the IEP, due to
different charges of their subunits.11J5,23 Sulfated
polysaccharidesmay form electrostatic complexes
with proteins above the IEP, from a mild acid to
a mild basic range of p H ' ~ , 3which
~ ~ ' is typical of
both formulated foods and chyme. An additional
cross-linking of macromolecular reactants, for
example, by multivalent cations, such as Ca, Fe,
Cu, and by other bonds may increase the stability
of protein-anionic polysaccharide com plexe^.^*-^
The triple protein-multivalent cation-anionic
polysaccharide complexes are of anonequdibrium
nature!' The thermal denaturation of protein mol-
ecules that are bound by an anionic polysaccha-
ride promotes subsequent protein-protein interac-
tions stabilizing the overall ~ o m p l e x e s . ~ ~ . ~ ~ . ~ ture
The stoichiometry of an electrostatic com-
plex (i.e., proteinhnionic polysaccharide weight
92
fraction ratio) depends on the conformation
and net charges of the reactants. Owing to to-
pological limitations, protein globules, and rigid
anionic polysaccharide chains cannot achieve
contacts among all their charged groups. Un-
like rigid protein globules, unfolded structure
proteins, such as gelatin, casein, and seed stor-
age globulins denatured in acid media, tend to
form a maximum number of contacts with an
oppositely charged polysaccharide. Therefore,
proteins with an unfolded structure usually form
electrically neutral insoluble complexes where
the charges of the components are completely
mutually neutralized. In other words, as shown
in Figure 1, the weight fraction ratio (N) of an
unfolded structure protein to an anionic polysac-
charide in the insoluble complexes equals the
ratio of their charges (Z). This means that the
stoichiometry (N = Z polysaccharide@ pro-
tein) of a complex depends on the pH value of
the system, but not on the initial ratio of mac-
romolecular reactant concentrations. At a given
pH the stoichiometry of the electrically neutral
insoluble complexes is constant and is equal to
the ratio of net charges of the macromolecular
reactants, whose excess remains in solution
over a wide range of their weight fraction ratio.
Figure 1 also shows that an insoluble complex
is enriched in polysaccharide when pH is de-
creased below IEP of the protein. This is due to
an increase in the net charge of the protein and
a decrease in the net charge of the polysaccha-
ride.39-41,48-50
Soluble charged complexes are
usually formed when the ratio of the reagents is
far from equivalent. An equivalent ratio of
macromolecular reactants corresponds to a
maximum yield of insoluble electrically neu-
tral complex. Insoluble interbiopolymer com-
plexes are formed even in dilute solutions (less
than 0.01% wt) of macromolecular reactants.
The stoichiometry of insoluble electrostatic
complexes formed by globular proteins depend
on the ratio of macromolecular reactants. Thus,
insoluble complexes formed by unfolded struc-
" ~ 6proteins are electrically neutral, while those
of globular proteins are usually charged and of
variable c o m p o ~ i t i o n . ' ~ Well
, ~ ~ , below
~ ~ - ~ the
~
 Protein tieight fraction in
the initial mixed solution,
w=c,,/c,, +cps

FIGURE 1. Weight ratio (N) of gelatin and pectin in an insoluble complex vs: (A)
gelatin weight fraction W ( W = C gelatinic gelatin + C pectin) in the initial mixed
solution, and (B)pH of the system. (From Ref. 35.)

Protein Solution
+
Polysaccharide Solution
R

f M-Complex Protein solution

! Solution

I c
Isoelectric pH
Point of
the Protein

FIGURE 2. M (mixing)-complex prepared by mixing biopolymer solutions at

a pH below the protein's IEP is insoluble. A mixture of the same biopolymer
solutions in the same proportion is stable, when is prepared at the pH above
IEP of the protein. Lowering its pH to the same value below the protein's IEP
leads to a soluble T(titration)-complex. Turbidity (arbitrary units) hysteresis
during nephelometric alkali and acid titration of an aqueous dispersion of the
M-complex. (From Ref. 15.)

93
IEP, that is, under conditions of strong protein-
polysaccharide interaction, interbiopolymer
complexes are of nonequilibrium nature. Un-
der these conditions the redistribution of pro-
tein molecules between oppositely charged
Figure 2 shows that prop-
chains is frozen.34.42,43
erties of a nonequilibrium complex depend on
the way of its preparation. For example, com-
plexes with the same overall composition but
differing greatly in their properties can be pre-
pared by mixing the macromolecular reagents
under two different conditions: ( I ) at a pH well
below the IEP of the protein (so-called com-
plexes of “Mixing”) or ( 2 ) at a neutral (or a
mild basic) pH following with titration to the
same pH well below the IEP (so-called com-
plex of “Titration”). These two types of (i.e.,
mixing and titration or M and T) complexes
can greatly differ in the size and structure of
their particles and their overall heterogene-
ity.23-34%42,43
Normally, the complexes of mixing
are heterogeneous polynuclear nonequilibrium
complexes. M-complexes can be regarded as
polysaccharide chains crosslinked by proteins.
In contrast, the complexes of titration are usu-
ally mononuclear. Under pH values near the
IEP and a sufficiently high ionic strength, equi-
librium T-complexing results in a uniform par-
titioning of protein molecules among polysac-
charide On the contrary, an
enhanced attraction between protein molecules
in the vicinity of the protein’s IEP may result in
their nonuniform distribution on the polysac-
charide matrix. Some polysaccharide chains
tend to be completely covered by protein mol-
ecules, while others tend to be almost free of
them.34.4*s43The cooperative binding of protein
molecules is due to the attraction between pro-
tein molecules bound on the polysaccharide
matrix, which makes each free site near the site
already occupied by a protein molecule prefer-
able for binding the next protein molecule. This
disproportionation of interbiopolymer com-
p l e x e results
~ ~ ~ in two fractions of the complex
greatly differing in the proteirdpolysaccharide
ratio. The cooperativity of biopolymer interac-
tions, which reflects an increase in stability of
94
interbiopolymer complexes compared with the
similar complexes of low-molecular-weight re-
agents, is more typical of proteins of unfolded
structures.This results from a low entropy change
accompanied the complexing between macro-
molecules. Weak intermacromolecular(e.g., van
der Waals) interactions therefore can stabilize
complexes of unfolded macromolecules.23~36
Interbiopolymer complexing controls the
functional properties of proteins and polysac-
charides, the conformational stability of pro-
teins and the pH dependence of enzymatic ac-
t i ~ i t y . ~ Functional
- ~ ~ , ~ ~ properties
-~~ (such as
solubility, emulsifying, foaming, and gel-form-
ing) of interbiopolymer complexes depend on
the stoichiometry and structure of com-
plexes’ 1,23,34-38 and differ greatly from those of
the initial macromolecular
A gradual attachment of each successive pro-
tein macro-ion progressively neutralizes the
polysaccharide-anion,reducing the net charge,
hydrophilicity, and solubility of the resultant
~ o m p l e ~ . 1 The ~ . ~neutralization
~ , ~ ~ - ~ ~ of charges
of anionic polysaccharide can also reduce the
rigidity of backbone chains due to a decrease in
the charge-chargerepulsive interactions of like-
charged-groups and a “kinked” function (be-
havior) of junction zones. Figure 3 illustrates
the consequence of the mutual neutralization of
macromolecular segments and a decrease in
hydrophilicity of junction zones formed. Com-
paction of the complex particles in solution
decreases its viscosity, which does not depend
on the molecular weight of the reagents and
mainly obeys Einstein’s viscosity law?
The compact conformation with a hydro-
phobic interior and the reversibility of
interbiopolymer complexes (against pH and
ionic strength) underlie their application for
flavor binding and release, encapsulation of
nonstable nutrients, and drug d e l i ~ e r y . 5Pro-
~-~~
tein-polysaccharide complexes and the com-
plexes of oppositely charged proteins are also
used to recover proteins from dilute solu-
t i o n ~ , ~ to ~ control
, ~ ~ . rheological
~-~ properties
of solutions, food fibers, and gels?-15,37s4849,59
These complexes are effective stabilizers of

Under conditions where interbiopolymer
complexing is inhibited. incompatibility of
biopolymers is rather a rule than an exception.
'Fhe entropy of mixing of macromolecules is
smaller by several orders of magnitude than
that of monomers. This results in a highly pro-
nounced tendency for demixing polymer solu-
A higher co-solubility is typical of
polyelectrolytes.81 Normally, however, poly-
mers differing in structure and composition have
very low co-solubility and tend to be com-
pletely separated into individual, nearly pure
phases. Recent advances showed that the co-
solubility of many biopolymers with each other
is significantly higher,11J9*.30,31 than that of
synthetic polymers. Limited co-solubility is
typical of the following biopolymer mixtures:
(i)proteins and polysaccharides; (2) structur-
ally- dissimiisrr polysacchaiides: ( 3 ) proteins
belonging to different classes accordkg to
Osborne' classification, that is? albumins (wa-
ter-soiubie), globulins (soluble vi sal: solutionj,
gintehes (solnble in alkali and acid solluiions),
and prolamines (sokbie in wats-alcohol mix-
tues); and (4) native and denamred forms and
aggregated and nonaggregated forms of the
same protein (Sable 1). Unlike synthetic poly-
mers, the incompatibility of biopolymers 5c-
curs ody under cernin conditions:".2j.j"-36.e2-90
which are typical of formulated foods. Xor-
mally, dissiniilar biopolymers are miscible only
in sufficiently diluted solutions either below
their co-solubility threshold or when soluble
complexes are The phase separa-
tion threshold is itself sensitive to the excluded
volume of the macromolecules.
A. Excluded Volume Effects
Excluded-volume effects arise from the
nonpenettable nature of molecules that cannot
occupy the same solution volume. Figure 4 il-
lustrates the principle of the excluded volume
for the simplest case of a globular protehm The
h t molecule (A), present in a dilute solution,
95
TABLE 1
Phase Diagram Parameters for Some Biopolymer Solution Mixtures

Siopolymer pair Conditions Critical point Sepa- References

Temperature," coordinates, % ration

C, pH, salt wt. thre-

concentration P o l p P o l p shold,

1 2 % wt.

Jelatin (MW. 170 35°C and 45°C (153) Grinberg,V.Ya.,

rD) + starch (freshly et al. (1968)

:elathized at 95°C for

!O min)

3elatin + starch 25-80°C. pH (1 54) Khornutov,L.I.,

5.82-6.5 et al. (1995)

3elatin-potato + 3OoC, 37T, (1 55) Doi, K. (1965)

mylopectin p H 5.2,7.0

3elatin-potato + 51°C (156) Durrani, C.M.,

mylopectin et al. (1993)

Qyrolysed waxy

naize starch)

Jelatin + maltodextrin 45°C (157, 158) Kasapis,S.,

P E 6 and 2) et ai. (1992)

96
TABLE 1 (contiued)

Gelatin (MW. 170 $ O T , 0.0 - (159) Grinberg,V.Ya.,

+ dextran (MW.
0) 3.60 M NaCl. et al. (1970)

65 kD) 3.0 - 4 M urea,

3.0 - 0.80 N

HC1

Gelatin (MW. 170 Q2S°C, pH (82, 11) Grinberg,

kD) + dextran (MW. Q.9; pH 4.6- V.Ya. et al. (1972

2000 kD) 5.4.

4O"C, pH 6.0,

3.2 M NaCI

Gelatin (MW.23 kD, 40°C - 60°C (160,ll) Grinberg,

IEP 4.9) + dextran V.Ya., et ai. (1970)

(MW. 83 kD)

Gelatin (80 kD, E P IOOC, 40-60°C (161,ll) Antonov,

4.75) + pectin (MW. p H 3.5; 6.0; Yu.A., et al. (1996)

21.5; 23.0; 23.6; 4.9; B.0; 0.5 M

33.1; 330 and 390 kD, NaCl

esterification degree

62.7%, 35%; 14.6 -

56%)
- -
Gelatin (80 kD, IEP QO"C,pH 6.0 l.6 1.o I .4

4.75) + pectin (MW. 40°C, pH 8.0 !.4 0.95 3.3

330 kD; DE=67.2%)

97
TABLE 1 (contiued)
--
3.25 r67
Gelatin (80 kD, IEP 4OoC, pH 6.0; 1.65

4.75) + alginate ( M W . 0.2 M NaCl

400kD; MG=50%)

D.3
-
Gelatin (80 kD, IEP 4O"C, pH 6.0; 1.25 1.36

4.75) + alginate (MW. 0.0-0.5 M

400kD; MG=20%) NaCI

Gelatin (MW. 170 40"C, pH 6.0;

kD) + alginate (MW. 0.2 M NaCl

400 kD;MG=20%)
- -
Gelatin (MW. 170 40°C, pH 6.0;

kü)+ methylcellulose 0.2 M NaCl

(70 w
- -
Gelatin (MW. 240 35"C, pH 3.0 3.43 0.57 1.98 (i62) Grishchenkova,

kD) + methyl 35"C, pH 4,75, -

1.86 0.26
-
1.36
E.V., et aL(1984)

cellulose (70 kD) - --

35°C pH 4.75, 2.06 0.42 1.49

0.5 M NaCl

70°C and (163) Michon, C., el

+ iota- 20"C, pH 6.5, al. (1995)

carrageenan (MW. 0.0 and 0.2 M

700 kD) NaCl

Gelatin (243 kD)- k- 40°C; pH 5.0: 0.75

- 0.84 -
1.24 (164) Antonov, Yu.A

carrageenan (MW. ionic strength et al. (1999)

356 kD) 0.35 NaCl

- --
98
TABLE 1 (contiued)
Gelatin alkaline (MW. 4OoC, pH 5.0;
-
1.47 165) Alves, M.M., et

243 kD) and locust ionic strength il. (1999)

bean gum 0.002

Locust bean

gum MW.

1510 kD
-
Locust bean 1.87

gum MW.

1470 kD)
-
Locust bean 2.25

gum Mw*
1770 kD)
-
Gelatin acid (EP 7.9) 18"C, pH 9.0. D.17 J66) Alves, M.M., et

[MW. 100 kD) + ionic strengîh il. (1 999)

locust bean gum 0.002

-
(Mw. 1500 kD) 18"C, pH 4.3; 0.57

ionic strength

0.002
-
18"C, pH 4.0; 0.59

ionic strength

0.002
-
1S0C, pH 5.0; 0.433

ionic strength

o. 1

99
TABLE 1 (contiued)

Gelatin alkaline (IEP ]BOC, pH 5.0; 0.64

--
0.11 0.29

4.9) (Mw. 100 kD) + ionic strength

locust bean gum 0.002M

(MW. 1500 kD)

---
Gelatin alkaline ( E P 40°C, pH 5.0; 2.67 0.51 3.13

5.4) (MW. 243 kD) + ionic strength

locust bean gum 0.1

---
(MW. 1500kD) 4OoC, pH 5.0; 1.66 0.35 1.47

L
ionic strength

0.002M
---
18"C, pH 5.0; 0.92 0.015 0.18

ionic strength

0.002M
---
Soybean globulins + 20°C, pH 9.0 7.7 0.2 4.3 (86) Antonov, et al..

pectin (MW. 40 kD) (1979)

---
Soybean globulins + 2OoC,pH 9.0 6.7 0.36 4.9 (85) Antonov, Yu. A.

sodium alginate (MW. et al. (1979).

150 !a)
---
Soybean globulins + 20°C, pH 9.0 3.5 0.32 4.5 :86) Antonov, et al.,

carboxymethyl J979)

cellulose (MW. 136

w
---
Soybean globulins + 20°C, pH 9.0 18.0. 0.35 7.2

arabic gum (MW. 497

kD)

1O0
TABLE 1 (contiued)
~ - --
12.63 D.37 5.6

dextran sulfate (MW.

500 kD)

2OoC, pH 9
-
12.2
-
3.35
-
7.3
dextran (MW. 500) 0.25 M NaCl
- -
I11s soybean globulin 25OC, pH 8,0

(glycinin) + sodium O.OlM, 0.1M

?.O 3.28 (98) Semenova, M.G.

et al. (1990)

pectinate (MW. 240 and 0.3M

kD, esterification NaC1

degree 58%) mercaptoethan

ol

+
-
11s globulin k- looc,pH 7.8, 1.001 (99) Semenova, M.G.

carrageenan (470 kD) o. 1 M et aí. (1991)

phosphate

buffer.

40°C, pH 7.8,
-
).o05

o. 1 M

phosphate

buffer
-
Broad bean (Vicia 55"C, pH 6.0, (167) Andersson, O. e'

faba) protein isolate 6.5,7.0. al. 1983

(acetylated and

nonacetylated + agar

agar

1o1
TABLE 1 (contiued)
11s Vicia fabr 25"C, pH 7.8, 3.1
--
1.3 (100) Tsapkina, E.N.,

globulin (legumin) i 0.1 et al. (1 992)

dextran (MW. 48 kD M NaCI. (103) Semenova,

270 kD and 2500 kD) dextran: MW. M.G. et al. (1998)

270 kD
-
Milk whey proteins t 25°C (168) Syrbe, A., et al.

maltodextrin (DE 06) (1997)

Milk whey proteins 25°C

+dextran

Milk whey proteins + 2S°C, 4"C, pH

methylcellulose 5-7, 0.5%

NaCI
- -
Caseinate (sodium) t 25"C, pH 7.2 5.0 2.8 (169) Suchkov, V.V.,

alginate (sodium, rt al. (1981)

MW. 124 kD; content

3f MM,GG and MG

docks: 30%, 20%,

5 0%)
- -
laseinate (sodium) + 20°C; 0.1 M 5.24 3.0 3 8 ) Antonov, Yu.A.,

;odium alginate (MW. NaOH :tall (1975).

150 kD)

Zaseinate (sodium) + 20°C; 0.1 M

-
i.8
-
9.0
irabic gum (MW. YaOH

!OO-300kD)

102
TABLE 1 (contiued)
-- -
laseinate (sodium) + 2OOC; 0.1 M 4.25 ! ,65

:arboxymethyl NaOH. 25"C,

:ellulose (MW. 135 3.15 M NaCl;

pH 6.5
~
--
Saseinate (sodium) + 20°C; 0.15 M 4.30 1.90 3.0 :87,170) Antonov,

mylopectin (MW. NaOH; 25"C, Yu.A., et all (1976,

38000 kD) 0.15 M NaCl; 1977)

pH 6.5
- -
Caseinate (sodium) + 20" - 40°C; pH 4.50 1.65 7.8

lextran (MW. 2000; 6.5; 0.15 M

184; 154 and 34.5 kD) NaCl;

20°C; 25°C;

O. 15 M NaOH;

0.0; 0.15; 0.30

and 0.5 M

NaCl
--
Mik proteins 2OoC, pH 6.4 D.9 (171) Antonov, Yu.A.,

[micellar-casein and et al. (1982)

whey proteins) +
apple pectin (MW. 69

kD, Esterification

iegree 62.7)

103
TABLE 1 (contiued)
Milk proteins Z O T , pH 6.4 6.7

(micellar-casein and

whey proteins) + gum

arabic (MW. 219 kD).

-
Milk proteins 20°C, pH 6.4C 24

(micellar-casein and

whey proteins) +
arabinogalactan (MW.

29 kD)

Milk proteins + locust SOC, pH 6.8, [172) Schorsch, C., et

b a n gum ionic strength al. (1999)

0.08M +

20%, 30% or

40%sucrose

Micellar-casein + 20°C, pH 7, 1.4 0.6 4.8 :173) Bourriot, S., et

dextran (MW. 519 0.25M NaCI il. (1997)

Micellar-casein +
- -
20°C, pH 7, '5.8

dextran sulphate 0.25M NaCl

(Mw.500 kD)
Micellar-casein + K- 5 0 T , pH 7, 1.o 0.12 3.4 :174) Bouniot, S., et
carrageenan (sodium) 0.25 M NaCl, il. (1 999)

0.05 M

NaCl+O.Ol M

KCI

104
TABLE 1 (contiued)
- -
I Micellar-casein + K- SOOC, pH 7,

0.25 M NaCl.
3.5

I (potassium)
- -
Micellar-casein + guar 2OoC, pH 7, 3.8 3.4 (175) Boumot, S., et

0.25 M NaCl ai. (1999)

Micellar-casein 20"C, pH 7, (176) Boumot, S., et

galactornannan (guar 0.25 M NaCl ai. (1999)

I gum and locust bean

-
Clover leaf protein + 2OoC,pH 6.2 (64) Antonov, Yu.A.

apple pectin et al. (1990)

-
Amylose (MW. 700 75°C 5 .O (177) Kalichevsky,

kû) + dexîran (MW. M.T., et al. (1986)

472 kD)

Amylose (from pea, 70°C and 90°C (178) Kalichevsky,

leached at 7OoC, MW. M.T. et al.

560 kD, 1100 kD, (1987)

fiom potato leached at

70-90°C) + waxy-

maize amylopectin

(MW. 65-400000 kD)

-
Maltodextrin (potato) 45°C. 0.1 M 5.3 (179) Annable, P., el

+ locust bean gum NaCl al.. (1994)

-
Maltodextrin (potato) 45"C, aqueous *7

+ gum arabic solution

1o5
TABLE 1 (contiued)
16

-
Maltodextrin (potato) 0.8

t carboxymethyl

;eiiulose

Dextran (MW. 25,40, 80°C 180) Medin, A.S. ei

70 kD) + agarose 1. (1993)

-
Dextran (500 kD) + 20°C 181) Gamier, C., ei

locust bean gum (1700 1. (1 995)

-
Pectin (MW. 69 kD; 2O"C, pH 5.0, ).58 1.62 I .20 i 1 i) Antonov, Yu.A.

esterification degree 0.5 M NaCl :tal. (1987)

62.7%) + methyl

cellulose (MW. 70

0)
-- -
Pectin (MW. 69 kD; 2OoC, pH 5.0, 1.42 0.45 3.86

esterification degree 0.5 M NaCl

62.7%) + locust bean

gum
-- -
Pectin (MW.69 kD; 2OoC, pH 5.0, 3.26 4.10 3.10

esterification degree 0.0 - 0.5 M

62.7%) + arabic gum NaCl; pH 2.5 -

(MW. 219 kD) 7.0

106
TABLE 1 (contiued)
Pectin ( M W . 69 kD; 20°C, pH 5.0,

esterification degree 0.5 M NaCl

62.7%) + dextran

sulfate

Pectin (MW. 69 kD; 20°C, pH 5.0,

esterification degree 0.5 M NaCl

62.7%) +
arabinogalactan (MW.

29 kD)

Methyl cellulose 20°C, pH 5.0,

(MW. 70 kD, 29.5% 0.5 M NaCI

methyl groups) +
arabinogaiactan (MW.

29 kD)

Alginate (sodium, 20°C, pH 5.0, '110) Antonov, Yu.A.,

MW. 150 kD) + 0.5 M NaCl :tal. (1987)

arabic gum (MW. 219

W)

Alginate (sodium, 20°C, pH 5.0,

MW. 150 kD) + 0.5 M NaCl

dextran sulfate

107
TABLE 1 (contiued)

Alginate (sodium, 20"C, pH 5.0

- -
MW. 150 kD) + 0.5 M NaCI

methyl cellulose

(Mw. 70 kD, 29.5%

methyl groups )
- --
Gelatin (160 kD) + 40°C, pH 7.0 2.4 11.6 9.0 (182) Andersson, O.,

Vicia faba globulins et al. (1 985)

(280 kD)
---
Gelatin (160 kD) + 40°C, pH 7.0 2.0 12.0 7.6

11s vicia fabu

globulin (350 kD)

Gelatin (160 kD) + 40T, pH 6.0; 3.4 15.4 12.7

11s Vicia faba 0.5 M NaCI

globulin (350 kD)

---
Casein + ovalbumin 20°C, pH 6.6. 6.5 13.9 19.7 (1 07) Polyakov, V.I.,
--
Caseinate (sodium) + Z O T , pH 11 .O >10 3.8 et ai. (1985)

gliadin

Casein + soybean seed 25T, 10°C

--
- 8.0 12.0 (106,108) Polyakov,
storage globulins (290 35°C. pH 6.9, V.I., et al.

3.0-0.1 M (1980,1985)

VaCl.

IH 6.0 - 8.0.
).O - 0.1%

:ystein

1O8
TABLE 1 (contiued)

Ovalbumin + soybean pH 6.6, 2OoC >15 15,4 (107) Polyakov, V.I.,

seed storage globulins et al. (1985)

11s Vicia faba pH 7.0; 7.8, 19 (102) Wasserman,

globulin (legumin) + ionic strength L.A., et al. (1997)

ovalbumin O.l,25OC (103) Semenova,

M.G. et al. (1998)

Ovalbumin + 20°C, pH 6.6- 3.6 13.3 (108) Polyakov, V.I.,

themodenatured 6.8 et al. (1986)

ovalbumin

Bovin serum 20°C, pH 6.6- 215 17.2

albumin+ 6.8

thermodenatured

*@U
length -L
radius R
diameter D !!i diameter D -
FIGURE 4. Schematic representation of excluded volume for spherical globular
protein molecules and the rigid rod-like macromolecules. (From Ref. 20.)
diminishes the volume available for the second
molecule for a certain volume, U, called the
excluded volume. In other words, the volume of
a solution available for the second protein mol-
ecule (B), u2 = A(V - U), for the third molecule,
u3= A (V - 2U), and other molecules are less
than the entire volume V of the solvent. A is the
proportionality factor. The radius of the excluded
volume, from which the centers of other protein
molecules are expelled, equals a minimal dis-
tance (D) between two adjacent protein mol-
ecules (A and B), that is, equals the sum of their
radii. This means that the excluded volume (U)
around each spherical molecule in the solution is
eight times greater than that of the protein mol-
ecule itself. For a rigid Spherical molecule of
radius R, the excluded volume is U = (32/3) a R3
=8M2v2/N, where M2 is the molecular weight
and v2 is the thermodynamic specific volume,
which is close to the effective hydrodynamic
specific volume and N is Avogadro's number.
For rigid rods the excluded volume is U = 1/2 a
DL2= 2L M2v2/DN according to T a n f ~ r dIn .~~
dilute solution stiff rod-like macromolecules are
relatively independent when the distance be-
tween them equals to or is larger than their
length. High excluded volumes are especially
typical of linear rigid polysa~charides?l-~~ A like polysaccharide molecules; (2) about 2 to
decrease in the excluded volume with the con-
centration of macromolecular solutes results in
v)
Polysaccharide
5
I
i"
m
O
V
m
5 50
O 10
FIGURE 5. Schematic representation of the effect of concen-
tration on viscosity oi biopolymer solutions. (From Rel. 20.)
110
small repulsive interactions between macromol-
ecules. Excluded volume effects reflect mutual
spatial limitations and competition between mac-
romolecules for solution space.
There are many manifestations of excluded
volume effects that influence the buik, the surface
properties, and the phase behavior of mixed
biopolymer solutions.11,12,17-24.30,31,92-L04 For in-
stance, Figure 5 shows a steep increase in viscos-
ity with concentxation of a biopolymer solution.
This increase occurs at the critical range of the
biopolymer concentration corresponding to the
transition from a dilute solution, where macro-
molecules are kinetically independent, to a
semiconcentrated solution, where the biopolymer
molecules enter into physical contact. At higher
concentrations molecules forn a fluctuating net-
work that makes the change of the partners and
the flow process slower and more difficult. This
critical range of concentration is usually below
1%for polysaccharides and varies from 10%to
40% for globular p r ~ t e i n s .At
~~a certain
. ~ ~ degree
of higher occupancy of a mixed solution phase
separation normally occurs.
The phase separation threshold reflects the
competition for space between macromolecules
and is about: (1) 1 to 3% for mixtures of rod-
4%for mixtures of gelatin or casein with linear
polysaccharides, (3) about 4% or higher for
20 30 40
Concentration, ( w t %)
 globular protein-polysaccharide mixtures; and
(4)usually exceeds 12% for mixtures of globu-
(Table 1). It should
larproteins11-16~24~34~75-8*~10s-111
be stressed that denaturation, aggregation, and
chemical modification of food macromolecules
during food processing usually decreases the
value of the phase separation threshold. Impor-
tantly, phase separation threshold values are
below the biopolymer concentrations typical of
most foods, which are therefore normally phase-
separated systems11-21~89~105~106(Table I).
Both thermodynamic and kinetic aspects of
phase separation are of great importance for
the structure and stability of food~.~2JI4J*6 Coa-
lescence of the dispersed phase particles and
separation of the phases, that is, minimization
of the interfacial surface area, can be stopped
by an increase in viscosity and/or gelation of
one or both phases of the system. The gel point
(i.e., the critical biopolymer concentration re-
quired to form a continuous gel network) var-
ies greatly from less than 0.1 to 0.5% for many
anionic polysaccharides and 1% for gelatin and
to 2 to 15% (and higher) for heat-set globular
proteins. Because of excluded volume effects,
the gel point decreases for mixed solutions with
concentration of an added biopolymer includ-
ing non-gel-forming b i ~ p o l y m e r s . ~ ~ J ~ . ~ * *molecular
B. Phase State of Biological Systems
The phase behavior of processed biomate-
rial systems is of importance for both food
technology and biotechnology. Because the
phase behavior of polymers and biopolymers
are markedly different, a wide experience of
polymer physics is of limited application. The
chemical structure and molecular weight are
the most important characteristics determining
the physical properties of polymers. However,
this is not the case with biopolymers. The most
surprising feature of proteins compared with
all other types of macromolecules is their high
co-solubility with each other and with other
bi0polymers.~~2~ Globular proteins have greater
than tenfold-higher phase separation threshold
values compared with those of polymers and
unfolded chain proteins such as gelatin and
casein30 (Table 1). Moreover, many globular
proteins belonging to the same class according
to Osborne are presumably co-soluble in all
proportions, despite great differences in amino
acid composition. This feature is obviously of
key importance for the biological functions of
proteins. To fulfill their catalytic role, enzymes
must be miscible at the molecular level with
their various macromolecular substrates.19.30,31
However, how could protein avoid immiscibil-
ity typical of polymers?
1. Macromolecular Mimicry
According to an hypothesis recently pro-
posed, the unusually high co-solubility of pro-
teins with one another and other biopolymers is
due to a “molecularm i r n i ~ r y ” . ’The
~ . ~molecu-
~
lar mimicry of proteins is their mutual camou-
flage close resemblance or likeness (for the
viewpoint of interactions with each other and
the medium). This molecular mimicry is pre-
sumably a direct result of their globular confor-
mation. The latter is able to achieve effective
~ ~ * ~ ~ * mimicry
- ’ ~ ~ via three principle reasons.
The fust reason is that the formation of densely
folded compact protein globules of quite sphen-
cal shape effectively hides the chemical infor-
mation within the molecular interiors imper-
meable to the solvent water. The second
consequence of forming hydrophobic interiors
inaccessible to the medium is that the hydro-
philic surfaces of the globules sufficiently
chemically similar to each other. The third
consequence of the globular conformation is a
reduction of the excluded volume and an in-
crease in the phase separation threshold.
Globules with chemically similar surfaces
and shapes, and with their chemical differences
hidden in the interiors, mimic each other, and
therefore are better co-soluble. Globular pro-
teins typically have quite similar physico-chemi-
cal properties, such as viscosity, surface activ-
ity, conformational stability, and gelation. The
111
polyelectrolyte nature of globular proteins and
most biopolymers enhances their co-solubility
due to the contribution of low-molecular-weight
counterions to an increase in mixing entropy.
One more benefit of a globular conformation is
an increase in the chemical stability of globular
proteins, whose interior is inaccessible or of
limited access to the solvent and solutes. In
other words, the encapsulation of chemical in-
formation inside a densely packed rigid glassy
globule provides the best chemical security to
proteins.I9Examples of a near complete mim-
icry may be isomeric seed storage globulins.
These oligomeric proteins can form highly
concentrated mesophases without crystalliza-
tion or semicrystalline protein bodies in
seeds.’1,12.89,119-124
Moreover, the occurrence of
molecular mimicry seems to be typical of
biopolymers including those of helical confor-
mations, that is, cylindrically shaped molecules.
The latter property provides control over the
hydrophilicity and wettability of biological
structures. These effects could cause adsorp-
tion of hydrophobic compounds, for example,
fatty acids, within the helices of polysaccha-
rides (i.e., the hydrophobic interiors) on the
one hand and a great difference in wettability
between the gel surfaces formed in the contact
with air, oil, or with ate er.'^.^'^
It was assumed that the ability to control
the phase behavior of biopolymer mixtures is
one of the fascinating issues of the molecular
evolution of protein^.^^^^' A large number of
enzymes can be regarded as mimetic. This
mimicry achieves the co-solubility of enzymes
with their substrates and inhibitors, for example,
proteases with other proteins. The occurrence
of mimicry could provide a selective advantage
to protein evolution in space-overloaded bio-
logical systems. Both host mimicry (including
aggressive mimicry of different parasites and
viruses) and host defense against the mimicry
of foreign agents (as distinct factors) could
presumably have evolved competitively. In
terms of mimicry, a specific interaction pattern
of chemically heterogeneous molecular surfaces
(a “spectrum” of molecular interactions) with
112
each other and with the aqueous medium is
presumably functionally similar to the mimicry
of color patterns (resemblance in coloration
and shape) of some species of plants and ani-
mals with their medium. Exploiting such a tool
as mimicry would promote the development of
a complimentary tool for molecular recogni-
tion and protection of organisms against the
mimicry of hosts. Mimicry makes it necessary
to label similar globules by some side groups
and/or facets to provide the living organism
discrete functions. The signaling elements on
the globular surface specify intermolecular in-
teractions. In other words, molecular mimicry
makes the development of specific biopolymer
interactions both possible and necessary. The
mimicry and signaling domains are important
for specific interactions of the cells and the
basis of evolution of multicellular organisms.
2. Molecular Symbiosis and Mutualism
The efficiency of mimicry at the level of
individual biopolymers and macromolecular
interactions is obviously accompanied by some
disadvantage at the level of the biopolymer
systems. The likeness of macromolecules im-
poverishes properties of their mixtures, which
thermodynamic properties become difficult to
control by changing the system composition
and physical conditions. Therefore, the like-
ness of macromolecular components was evi-
dently not sufficient for prebiotic molecular
evolution. An additional thermodynamic tool
acting in the direction opposite to molecular
mimicry must have been involved in order to
control the properties of biopolymers in prebi-
otic systems. This supplementary principle
might be called “molecular This
term (symbiosis, Greek the state of living to-
gether) implies that interactions (an attraction
or a repulsion) between biopolymer molecules
greatly differing in conformation (globular and
rod-like) favor the biological efficiency of one
of them, at least. In other words, molecular
symbiosis implies a mutual influence of dis-
 similar mutually dependent species of macro-
molecules (symbionts), which is (including
incompatibility and complexing) beneficial to
a thermodynamic and/or a biological activity
of either one or all of them (symbiosis, mutu-
alism) and the system as a whole.31
A mixed solution of limitedly co-soluble
biopolymers is richer in properties compared
with that of mutually mimic macromolecules.
Moreover, the greater the difference between
macromolecules in shape, size, and composi-
tion, the stronger their mutual influence. Globu-
lar and cylindrical conformations provide an
extremely large difference in excluded volume
of biopolymers (Figure 4).Molecular symbio-
sis is suggested to underlie the predominance
of these two extreme macromolecular confor-
mations (globular and helical, rod-like) typical
of biological macromolecules, proteins, nucleic
acids, and polysaccharides.19,30.3'
Molecular symbiosis is based OR excluded
volume effects. Owing to these effects macro-
molecules behave as if they were in a more
concentrated mixed solution. The contribution
of rigid chains to macromolecular symbiosis is
predominant, because the association-dissocia-
tion of rod-like macromolecules can greatly
and rapidly change both the free volume in a
mixed solution and the effective concentration
of other macromolecules. For instance, the dis-
sociation of supermolecular structures formed
by rod-like macromolecules can increase the
effective concentration of an enzyme without
additional synthesis. Moreover, at concentra-
tions above the phase separation threshold,
spontaneousreversible transition to a two-phase
system is accompanied by an abrupt change in
both the concentration and the weight ratio of
dissimilarmacromolecules in co-existing phases
(see Section V.A, V.A.1). The phase separa-
tion of biopolymer mixtures would contribute
to: ( I ) collective transport of protein molecules
newly produced in the cell,19 (2) storage of
proteins in the cell, (3) natural selection of
macromolecules for nonspecific immune de-
fense, and (4)d i g e s t i ~ n . ' Phase
~ , ~ ~ separation
caused by an insufficient mimicry would pro-
vide for evolutionary improvement of protein
mimicry.
It can be assumed that molecular evolution
was not entirely based on purely random events.
The principles of molecular mimicry, symbio-
sis, and self-organization of macromolecules
in mixed solution would all provide potential
selective advantage of more functional molecu-
lar constructions and system compositions.
Macromolecules of high and controllable ex-
cluded volume, such as DNA and RNA, could
determine functional efficiency of their mix-
tures with globular proteins. The evolution of
viruses was reviewed recently within the con-
cepts of the evolution of the lipid membrane in
the prebiotic soup. In this context the formation
of lipid layers, lipid-covered dispersed particles
(e.g., encapsulated organic and inorganic mol-
ecules by lipid layers), and other lipid-contain-
ing structures in the prebiotic soup on the early
Earth125J26 are very important. By analogous
arguments, one can propose that the phase sepa-
ration of RNA-protein mixtures with the for-
mation of phases enriched in RNA and in pro-
teins was followed by the adsorption of lipids
between the immiscible aqueous phases (Sec-
tion V.F). The phase separation and adsorption
processes could result in the formation of thin
lipid layers and partitioning of other compo-
nents of the environments between the immis-
cible aqueous phases. These processes could
lead to prebiotic cell-like structures that would
compete for material exchange with the sur-
roundings.
IV. MOLECULAR MIMICRY AND
SYMBIOSIS IN FOOD DIGESTION
The digestive system faces a great variety,
the greatest amount and concentration of for-
eign biopolymers. As a large surface exposed
to the environment, the gut is adapted to both
defend against, decompose (per hydrolysis),
and reutilize host macromolecular species for
the needs of the organism. In effect, at the
earliest evolution stages both nonspecific im-
113
mune defense and nutrition were the same pro-
c e s ~aimed~ ~ at:
%(1)
~ precipitation
~ and hydroly-
sis of foreign biopolymers to make them harm-
less, and (2) use of the hydrolysates as raw
materials for the needs of the organism (nutri-
tion). Figure 6 illustrates this idea. The
exopolysaccharides secreted by bacteria to-
gether with the polysaccharides forming the
vegetable cell walls constitute an exocellular
layer acting like a passive alimetary system
(canal), that is, as both a universal nonspecific
immune defense against foreigner proteins
(from the environment) and the volume within
which hydrolysis and adsorption of hydroly-
sates occurs. The nonspecific immunological
hurdles of cell protection could be formed by
the two mechanisms of (I) the rejection of
foreigner proteins due to incompatibility and
( 2 ) the seizure of foreigner proteins due to
interbiopolymer complexing. Both types of
interactions of a protein could be fulfilled by
the different segments of the same cell wall and
exopolysaccharides. Accordingly, exopolysac-
charides are various copolymers, including pro-
tein-polysaccharideconjugates. Most published
data on phase separation thresholds for protein-
polysaccharide mixtures (Table 1) relate to
vegetable food polysaccharides (e.g., pectins,
alginates, etc.), that is, products of mechano-
chemical and thermal decomposition of cell-
wall materials. Their molecular weight and
excluded volume therefore are, lower than that
of native exopolysaccharides.The solution layer
around the cell containing an exopolysaccharide
of high local concentration can be regarded as
a buik phase enriched in polysaccharide encap-
sulating the cell. This phase or the solution
layer surrounding the, cell, presumably has a

Non- Specific Immunity

- Exopolysaccharide
-decules

Barrier solütion layer - a mimic

of polysaccharide-rich phase
FIGURE 6. Schematic representation of the barrier layer formed by the exopolysaccharide solution encircled the cell.
A schematic model for the alimentary and nonspecific immunity layer protecting the cell. (From Ref. 31.)

114
 low co-solubility with some proteins and is
immiscible with other proteins of both colloi-
dal and molecular degree of dispersity. The
barrier solution layer formed by an exopolysac-
charide around the cell (acting as “alimentary”
capsule) is penetrable by low-molecular-weight
substances and impenetrable for most protein
molecules, and thus provides a universal de-
fense of the cell during its n ~ t r i t i o n . ’ ~The
complexing of other foreign proteins with
exopolysaccharide segments can be accompa-
nied by a cooperative cross-linking of
exopolysaccharidechains by protein molecules
and by an increase in their local concentration.
The foreign-protein-exopolysaccharide com-
plexing leads to a loss of mimicry, a local
decrease in solubility (of junction zones mac-
romolecular complexes, like co-precipitation
of antigen-antibody complexes), and hydroly-
sis into low-molecular-weight substances. The
latter can diffuse through the barrier solution
layer to be used for the needs of the cell. The
structure, composition, molecular weight, and
concentration of the exobiopolymers determine
their excluded volume effects and their adhe-
sion to proteins. Exomucopolysaccharides, pro-
tein-polysaccharidecomplexes bound to the cell
surface, could fulfill larger functions of con-
trolling the pH of the microsurroundings,enzy-
matic hydrolysis, and antimicrobialdefen~e.’~~-I~9the following principles: (1) the ability of an-
Exocellular polysaccharides are elaborated by
most or all bacterial species.’31Extracellular
polysaccharides secreted by cells usually pro-
vide the cell-substratum and cell-to-cell adhe-
sive and signalingfunctions.132Such molecules
can presumably act as thickening and gel-form-
ing agents, metal-chelators, modify the short
distance environment, provide defense, nutri-
tion, and affect the fate of the ce11.131-137
instance, lactic acid bacteria are capable of produc-
ing exopolysaccharides,exomucopolysaccharides,
proteases, and antimicrobial compounds, such
as the nisin-like bacteriocins. Their biosynthe-
sis can be activated by changes in the environ-
ment.’39It was shown that a wide rage of anti-
microbial, antitumor, and immunomodulatory
activities are typical of extracellular protein-
polysaccharide complexes of bifidobacterium
and some mushroom^.^^^-'^^ The biological pro-
tective activity of polysaccharides has been
recognized and exploited, for example, as
wound protection materials has been demon-
strated using a wide range of polysaccharide
sources.130In the intestine exopolysaccharides
can also contribute to the attachment of bacte-
, ~ ~ , ~ rial
~ species to solid substrata and to the protec-
tion of intestinal walls.
Exomucopolysaccharide layers, as natural
protective coverings and alimentary capsules
of single-cell organisms, could have evolved
into the mucopolysaccharide membrane lining
the gut. Thermodynamic compatibility of
exopolysaccharideswith mucopolysaccharides
of the mucosa could be of importance for the
competitive selection of the microflora intesti-
nal. As in the case of microorganisms,
interbiopolymer complexing and incompatibil-
ity could underlie the efficiency of both essen-
tial processes within the alimentary canal, that
is, the hydrolysis and the protection of the ali-
mentary canal against its own proteolytic en-
zymes and food macromolecules. The nonspe-
cific immune defense of the digestive system
must: destroy foreign proteins and other food
macromolecules, viruses and cells, including
pathogens. These properties could derive from
ionic polysaccharides and other negatively
charged fractions of food fiber to precipitate
proteins, viruses and cells; (2) the complexing
with oppositely charged biopolymers, with lip-
ids, surfactants; (3) a strong acid medium in the
stomach and finally (4) gastric juices in the
stomach, and the digestive enzymes in the in-
testine. These techniques could form nonspe-
For cific immunological protective hurdles and si-
multaneously increase the extent of proteolysis.
Both biopolymer complexing and incompat-
ibility thus could contribute to efficient diges-
tion of mixed diets.
The phase-separated nature of chyme is
likely of importance for the efficient decompo-
sition of food macromolecules into substances,
which the body can adsorb. Figure 7 shows that
115
the chyme phhases cm be regarbed as 'micro-

of the chyme.17J9.31h the inteshe, the greater

excluded volume m d higher affuuty for water
of polysacchides (eespecially food fi-
ented only in the colon) compared
proteins (de
acid medium m
may result in a chyme continuous phase en-
riched in polysacchazide (SectionV.A.1). This
could underfie protection of the
w d s against self-digestion because the pro-
teolytk enzymes would be concentrated in the

,
it was found that complexing with oppositely
charged macromolecules makes denaturation
of the Kunitz trypsin inhibitor irreversib1e,"J3Ja
that anionic polysaccharides accelerate the di-
gestion rate of slow proteins,147and iron and
copper ions bound by anionic polysaccharides
decrease the solubility of protein-polysaccha-
ride c0mplexes,4~contributing to an increased
rate of protein digestion.I4*Thus, interbiopoly-
mer complexing, in those examples tested, in-
crease the hydrolysis rate of the protein.19J1
The mutual concentration of proteins and
polysaccharides by excluded volume effects,
their complexing and gelation of the chyme
phases could also favor the efficient digestion
of mixed The co-precipitation of
food proteins and enzymes together with
charged polysaccharides leaving the stomach
would contribute to their hydrolysis in the in-
testine. It has been shown that the main part of
chyme is a dense gel-like f r a ~ t i o n . ' ~The
~,'~~
remainder is dispersed particles of the initial
foods. It has been proposed that the gel-like
chyme is formed by mixing the duodenum juice
(pH about 7.2) with the acid (pH about 2.0)
products of stomach dige~ti0n.l~~ Morphologi-
cal studies and chemical analysis of the chyme
dense phase formed during the change of the
pH value of enteral milieu have shown that this
chyme fraction contains mainly proteins,
polysaccharides, and lipids as well as enzymes
concentrated and activated within this phase.
This concentration (complexing and co-precipi-
tation) results in an increased rate of protein
hydrolysis.
Incompatibility would decrease the co-solu-
bility of interbiopolymer complexes in the
mixed dispersion with other food biopolymers.
For example, the general nature of biopolymer
incompatibility and the low-phase separation
threshold of polysaccharide-food protein mix-
tures could underlie a change in the mechanism
of phase separation in chyme, which occurs
(during maturation) when mammals become
mixed-diet consumers. Milk presumably could
not contain dissolved polysaccharide as a ma-
jor source of energy, because of the low pro-
tein-polysaccharide phase separation threshold
typical of casein-polysaccharidemixtures. Lip-
ids therefore are the main source of energy in
milk. Accordingly, newborn mammals achives
effective phase separation via the enzyme ren-
nin, which causes casein precipitation. At later
stages of life renneting is no longer necessary,
when mixed diets include polysaccharides pre-
cipitating casein (due to both depletion and
bridging flocculation). Again, this principle of
using polysaccharides to precipitate casein and
for the clotting of milk could replace its
renneting, and thereby improve the cheese-mak-
ing process.238
Many questions related to the digestion
mechanism arise from the fact of the incompat-
ibility of proteins and polysaccharides in solu-
tion. First concerns are the partitioning of pro-
teolytic and amylolytic enzymes and the
hydrolysis products between the coexisting
protein-rich and polysaccharide-rich phases of
the chyme. Partial protein hydrolysis in two-
phase protein-polysaccharide (polymer)-water
model systems offer the means to: (1) control
protein co-solubility with other biopolymers
and (2) selectively limit enzymatic hydroly-
sis.LIJ51
The gel state of chyme (due to formation
complex and liotropic gels32) can also favor
food digestion by additional mean^.'^.^^ The
protein-rich phase and the co-existing polysac-
charide-rich phase of the chyme are better sol-
vents for the enzymes that catalyze the hy-
drolysis of proteins and polysaccharides,
respectively. The soluble hydrolysis products
continuously separate from the chyme phases.
The concentration and the substrate-enzyme
ratio within the chyme micro-reactors would
be preserved due to their gel state. The latter is
the expected reason for the absence of any
negative consequences of consuming liquids
during eating and the expected dilution of the
continuous phase of the chyme. Both complex-
ing and gelation decrease the amount and mo-
tion of the macromolecules, that is, reduce the
excluded volume effects and increase the solu-
bility of enzymes in the chyme. Therefore,
117
normally the dispersion medium of a biopoly-
mer gel is a better solvent than the liquid sys-
tem before It should, however, be
noted that the increased free volume, diffusivity,
and miscibility of enzymes is mainly related to
the presence of physical gels with extended
junction zones, which are more typical of food.
On the contrary, the formation of chemical gels
with local (covalent or coordinate) cross-links
is presumably not accompanied by an increase
in free volume because the chain segment
motion is usually preserved between knots of
chemical networks. This would explain the rela-
tively low digestion rate of casein and gluten
proteins.
Thus, the general nature of biopolymer in-
compatibility and interbiopolymer complexing,
based on nonspecific interbiopolymer interac-
tions, can achieve both nonspecific immune
defense and the digestion efficiency of mixed
diets. These phenomena presumably underlie
many digestion features, for example, the facts:
(1) that hydrolysis of starch starts in the mouth
during mixing food with saliva to control pro-
tein digestion and provide lubrication for the
swallowing of food, ( 2 ) that peristalsis effi-
ciency of the gel-like chyme is preserved for a
wide range of diet compositions.
V. FOOD FORMULATION AND
PROCESSING. BEHAVIOR FEATURES
OF WATER-IN-WATER (WMI)
EMULSIONS
Because aqueous phase-separated systems
are typical of formulated foods,I1-l6their struc-
tural and physico-chemical features are of great
importance for food p r o c e ~ s i n g . ' ~Struc-
, ~ ~ J ~ ~ Biopolymers are limitedly co-soluble, and, for
ture-property relationships in liquid phase-sepa-
rated aqueous systems (i.e., W/W emulsions)
has been studied mainly using model systems
containing proteins, polysaccharides, and their
mixtures.1l,23-25.82-89~104-106,110
The role of phase-
separated systems in food processing has been
mostly considered using as examples thermo-
plastic extrudates, wheat flour dough, ice cream
118
mixes, low-fat spreads, and be vera ge^.'^-^^-^'-
33.71-73,112-114
A. Phase Equilibrium
The first feature of W/W emulsions to un-
derstand (compared with O/W and W/O emul-
sions) is the nature of the equilibrium phases
and the thermodynamic stability.'4J9,22-24 Phase
behavior and phase equilibrium in mixed
biopolymer solutions is quantitatively described
by the phase diagram shown in Figure 8. The
binodal curve, DGFE, separates the composi-
tion regions of the single- and two-phase aque-
ous biopolymerl-biopolymer2-watersystems.
Compositions lying under the binodal curve
correspond to single-phase solutions, whereas
compositions of biphasic systems are above the
binodal. For instance, on mixing of a protein
solution A with a polysaccharide solution B a
mixture of composition C spontaneously breaks
down into two liquid phases, phase D and phase E.
The phase D is rich in protein and another, E,
is rich in the polysaccharide. The thin line DE
is a tie-line connecting the compositions of the
co-existing phases. Maximal co-solubility of
biopolymers can be characterized by the phase
separation threshold F, which corresponds to
the minimal total concentration of biopolymers
required for phase separation. The critical point
G gives the composition of the system demixing
into phases of the same volume and composi-
tion.
Figure 8 illustrates a typical mistake of
food formulation, for example, at the design of
a novel food. It is widely believed that mixing
of solutions results in their mutual dilution.
instance, a mixed solution of composition C is
the biphasic system with the phases correspond-
ing to point D and point E. The phase D to
phase E volume ratio is estimated by the in-
verse lever rule, that is, by the ratio of the tie-
line segments: EC/CD. When the composition
of the system C is shifted along tie-line DE, the
volume ratio of the co-existing phases D and E
 A
FIGURE 8. Phase diagram for protein-polysaccharide-water system.
is changed, but their compositions remain con-
stant. The line passing through the mid-tie-
lines and the critical point G is the rectilinear
diameter. It gives the composition of systems
splitting into phases of the same volume. Phase
inversion usually occurs in the vicinity of the
mid-tie-lines and greatly changes the system
For instance, phase
prop"ies.33-35,83,'L2-''6,152
inversion can have either synergistic or antago-
nistic effects on food texture.22The reasons are
the pronounced difference in composition be-
tween the coexisting phases and the fact that
the continuous phase is typically responsible
for the properties of a dispersed system, that is,
the properties of composite foods.
Figure 9 presents examples of phase dia-
grams for several model food systems. Phase
diagrams are also used to describe the
effects of other food components and pro-
cessing variables on the phase equilib-
rium.'1~24~105~'06 Generally, incompatibility
increases with polysaccharide molecular
weight, salt concentration, and tempera-
t ~ r e . ~ ~ Adding
, ~ ~ J salt,
~ ~ fatty
- ' ~ and
~ amino
acids, surfactants, sucrose, and other sugars
controls the co-solubility of many biopoly-
mers. Phase behavior and equilibrium in
mixed biopolymer solutions have been con-
sidered in several reviews.24~34~105~106~110
Table
1 shows the phase separation conditions,
Protein, YOwt.
the coordinates of critical point, and phase
separation threshold for studied biopolymer
mixed solutions.
1. Phase Diagram Asymmetry:
Membraneless Osmosis Process
The relative competition of dissimilar mac-
romolecules for space in the mixed solution
can be characterized by the critical point G
coordinates or the angle made by the tie-lines
with the concentration axis of one of the
biopolymers. 124,71,89The greater the difference
in biopolymer molecular weight, the greater
the shift of the binodal toward the concentra-
tion axis of the biopolymer of lower excluded
volume. The tie-lines on a phase diagram can
be nonparallel to each other because of struc-
tural changes of the biopolymer solutions with
concentration. For instance, the self-associa-
tion of proteins changes the excluded volume
and their affinity for the solvent. The asymme-
try of a phase diagram describes the method of
biopolymer concentration called membraneless
F i p y 10 illus-
~s~~~~~~14-16.34.63,64,71,89,17L,183-185
trates the principle of membraneless osmosis
and specific features of liquid-liquid phase sepa-
ration that follows the gelation of one of them.
Diffusion is the process of the mixing of a
119
 s 4)

g 10
I

f
Ê 5
f
h 5 10 20
Sodium Caseinate, Y*w t
82.0
O1.0
5 15
Sodium Caseinate, % wt

.
5 10 20 O 5 10 15
Ovalbumin, % Gelatin, %

8
n
O 5 10 20 30 3
rn O 10 25
Legumin, 'XO Glycinin, %

%l.o
a
W
U
o
8 3 10 20
Skimmed m i k proteins, % Skimmed milk proteins, YO
FIGURE 9. Phase diagram for systems: A - amylopectin-sodium caseinate-water; B - sodium
caseinatesodium alginate-water; C -casein-ovalbumin-water; D - legumin (i1 S Broad bean globu-
lin)-gelatin-water: E - dextran-legumin-water; F -sodium pectinate-glycinin (1 1S Soybean globulin)-
water: G - skimmed milk-pectin high ester (65%)-water; H - skimmed milk-gum arabic-water.

120
 Q Diffusion

Osmosis

Membraneles
Osmosis
I Cenírifupation:
Two immiscible
aqueous phases I
V

Elasto-osmomet
~ Solution
Gei swelling
mùDesweüing

FIGURE 10. Schematic representation of diffusion, osmosis, membranelessosmosis, and elasto-osmometry. (From
Ref. 20.)

121
 solvent with a solution (or of two miscible
solutions) by the random Brownian motion of
the molecules until mixture becomes uniform
(throughout the whole volume). Osmosis is the
passage of a solvent through a semipermeable
membrane from a solvent or a dilute solution to
a more concentrated solution until equilibrium
is established. The osmotic pressure is the pres-
sure that has to be applied to the solution to
stop the movement of the solvent through the
membrane. Being a colligative property, os-
motic pressure depends only on the concentra-
tion of the solution and not on the nature of the
solute. A semipermeable membrane is one
through which the molecules of a solvent can
pass but the macromolecules cannot. In
membraneless osmosis two macromolecular
solutions are immiscible, so that a semiperme-
able membrane is no longer necessary. The
membrane in effect is replaced by an interface
between solutions. Water passes through the
interfacial surface from a solution to another
immiscible solution until the osmotic pressure
of both solutions (co-existing phases) becomes
e q ~ a 1 . ~ ~Owing- ~ ~to, a~great
~ , difference
~ ~ , ~ ~in~
the concentration dependence of osmotic pres-
sure between dispersions of different biopoly-
mers, phase separation of their mixtures can be
accompanied by a severalfold increase in the
concentration of one of them and the dilution
of another. For instance, Figures 9 A, 9B, 9G,
9H, and 11 show that systems containing about
2.5% milk proteins and 0.9% pectin (molecular
weight 69 kDa, degree of esterification about
62.7%) obtained by mixing skimmed milk with
pectin solution (2OoC, pH 6.4) break down into
two phases: phase D-rich in protein and E-rich
in polysaccharide. Phase D contains 24% casein
(which corresponds to a IO-fold concentration
degree) and about 0.01% pectin. Phase E con-
tains about 1% pectin and about 0.3% milk
whey proteins. Assets to the membraneless
osmosis technique for biopolymer concentra-
tion include a high efficiency and low energy
The passage of a solvent
requirement~.~*5.*~*~~~~
through the interface separating the two solu-
tions of immiscible biopolymers is, however,
122
stopped and the separation of the phases cannot
be accomplished (Figure 9H) when the con-
tinuous phase or both system phases are rap-
idly gelled.21-24,112,17I,l84.185
2. Fractionation of Components
Between Coexisting Phases
Phase separation of a mixed biopolymer
solution is caused by its less-compatible
biopolymer components. Separation results in
the partitioning of other components between
the two immiscible aqueous phases as between
two complex solvents. This usually leads to an
uneven distribution of the components between
the coexisting phases, that is, leads to the frac-
tionation of system components. For instance,
a preferential concentration of flavor and gel-
forming agents in the continuous phase is im-
portant for flavor release and texture of the
system. Because both the bulk and the surface
properties of a food system are dependent on
its content of functional macromolecules, their
partitioning between the phases is an important
aspect of food formulation.19-21 Fractionation
between the separate phases and the interfacial
layers can cover all food components and in-
gredients of molecular and colloidal dispersity,
such as lipids, insoluble proteins, starch gran-
ules, food fibers, pigments, metal cations, and
macromolecules differing in molecular weight
and structure, etc.22-24~71~83,183-185
Actually, most
foods are filled composites where fillers affect
the nutritional, structural, and physical proper-
ties, including turbidity, color, and mechanical
properties. Fractionation is of great importance
for the structure formation in foods. Partition-
ing of colloidal particles depends on the rela-
tive wettability by the aqueous phases and in-
terparticle interactions and is still very poorly
studied. The first systematic investigation in
the field was not related to food. Albertsson
and Walter studied the partitioning of cells and
cell organelles between two aqueous polymer
phases.25.28,201,209
They showed that cells and
organelles are concentrated within the volume
u

123
of either one of the phases or the interfacial
layer between the aqueous phases. It was found
that lipid droplets can be concentrated and coa-
lesce within the interfacial layer separating
aqueous biopolymer phases. This lipid adsorp-
tion increases the rheological interaction be-
tween the aqueous phases and replaces aque-
ous continuous phases by a newly formed
continuous lipid phases. This phenomenon was
used to form low-fat spreads, to control the
consistency of a caviar analog, and to manufac-
ture many other foods'8J9~21322~200
(Section V.F).
3. Excluded Volume Effects on
Structure Formation
The main question concerning food formu-
lation therefore is what is the structural reac-
tion of highly volume occupied biological and
food systems on the additional increase in ex-
cluded volume? In other words, what is the
structural result of an increase in the concentra-
tion of a biopolymer, which is added to (or
synthesized in) a system? The consequences,
presumably, correspond to Le Chatelier's prin-
ciple that states that any system in equilibrium
shifts the equilibrium when subjected to any
constraint, in the direction that tends to nullify
the effect of the constraint. According to Le
Chatelier's principle, the reaction of a system
to a decrease in free volume is to reduce the
excluded volume. This result can be achieved
by several physico-chemical processes. Such
processes include the self-association of mac-
romolecules,interbiopolymercomplexing, crys-
tallization, and g e l a t i ~ n . ' ~An
- ~ increase
~ in
biopolymer concentration favors these pro-
cesses. For instance, at the buik biopolymer
concentration below the phase separation thresh-
old, the addition of a polysaccharide, dextran,
to a solution of the 11S broad bean globulins
increases protein adsorption at the oil/water
interface.11.'2.100.'o'
Other examples are syner-
gistic gels. Because the shear modulus of a gel
is typically proportional to the square of the gel
concentration, a relatively small increase in
124
effective concentration of gel-forming macro-
molecules due to an added biopolymer, can
result in a few-fold increase in the elastic modu-
lus of the gel. For instance, at the bulk biopoly-
mer concentration below phase separation
threshold, a small additive of dextran to a gela-
tin solution increases the elastic modulus of
gelatin gel by 2- to 5-fold and strongly acceler-
ates gelation process.15.32,3~,115,117.191,192
An in-
crease in the crystallization rate of polysaccha-
rides (e.g., amylose, amylopectin, and dextran)
is one more example. Doi first studied crystal-
lization of amylopectin in highly concentrated
gelatin gels'ssJ93as a model of the starch pre-
cipitation in plastids of plant cells. Later
Kasapis, Moms, and Norton described the crys-
tallization of maltodextrin in mixed solutions
with gelatin.'57J58It can be assumed that the
crystallization of amylopectin in gelatin solu-
tions and gels is of interest as a model of amy-
lopectin retrogradation in the process of bread
staling.20 Such a model system is useful for
studying the effect of gluten, added polysac-
charides, surfactants, and lipids on the staling
the bread. Any additional increase in the con-
centration of such a highly volume-occupied
system as dough favors the crystallization of
the starchy polysaccharides.20.2'Presumably,
this is the reason for the increased staling rate
of Chinese steamed bread, a dough contain-
ing less water. One more example is the for-
mation of crystalline-resistant starches in
pasta p r o d u ~ t s . ' ~It~ was
J ~ ~found that heat-
ing a concentrated aqueous suspension of high
amylose starches favored crystallization of
the starch. Thermodynamic incompatibility
between the amylose and amylopectin in
aqueous media underlies this technique178and
provides higher crystalline-resistant starches
for manufacturing low-fat speads.196Decreas-
ing the rate of hydrolysis of amylopectin in
the small intestine, which is of nutritional
and pharmaceutical interest, for example, for
a slow release of the active compounds, would
also be achieved by a special thermal treat-
ment (temperature cycling) of starchy
f0ods.l9~
 The last question concerning structural con-
sequences of excluded volume effects is how
can immiscible food biopolymers (e.g., in bread)
form mixed gels with interpenetrated networks?
Gels containing several networks can be formed
within both composition areas lying below and
above the binodal of the gel-forming agent
mixture. The reason is that gelation always
reduces the amount and mobility of space-fill-
ing particles (i.e., their excluded volume) in-
creases the co-solubility of biopolymers at the
gel state of one of them, and forms interpen-
etrated networks.'5,22The increased co-solubil-
ity of enzymes with biopolymer gels (more
precisely, with physical gels as it was men-
tioned in Section IV) underlies the importance
of the gel state of chyme.
Gelation of phase-separated systems can
lead to gels filled with liquid or gel-like dis-
persed particles. Features of phase separated
gels are ( I ) a low adhesion between two coex-
isting phases because of their immiscibility and
( 2 ) a great difference in the weight ratio of the
gel-forming agents between the l7
4. Excluded Volume Effects on
Thermal Denaturation of Globular
Proteins
According to Le Chatelier's principle, an
increase in the concentration of biopolymers
does not affect those processes that are not
accompanied by changes in excluded volume.
For instance, thermal denaturation does not
markedly change the volume of globular pro-
tein molecules. A tight packaging of globular
protein molecules and the ''dry'' state of their
interior correspond to the glassy state of native
and denatured protein molecules in solutions
and gels.19,33,186-188
The compact stable native
molecules are transformed into the compact
denatured molecules and their aggregates. The
loss of entropy associated with an increased
packaging density and positional ordering of
macromolecular segments is compensated by
the gain in entropy due to an increase in the
free volume of a space-overloaded system.
Accordingly, it was found that both the addi-
tion of a polysaccharide and an increase in the
concentration of the protein have no effect on
the denaturation equilibrium of both oligomeric
and small globular proteins.11.13-53 Although the
conformational stability of proteins is quite
sensitive to the environment of low-molecular-
weight solutes, including salts, fatty acids, al-
cohol, and ~ r e a , l l .it* ~is not typical of macro-
molecular co-solutes. It was shown that a high
increase in the local biopolymer concentration,
even the adsorption of a protein on an oppo-
sitely charged polysaccharide (protein-polysac-
charide complexing) or the covalent binding of
a protein with a polysaccharide (a protein-
polysaccharide conjugate) does not decrease
the conformational stability of the bound pro-
tein.'1J3,53
On the contrary, both the addition of
a polysaccharide and the increase in concentra-
tion of a protein favor the aggregation of dena-
tured protein molecules. The aggregation re-
duces the excluded volume of solute particles.
This is typical of both macromolecules and
colloidal p a r t i ~ l e s . ' ~ ~ J ~ ~
5. Effect of Temperature
The phase behavior of mixed biopolymer
solutions is sensitive to temperature. Biopoly-
mer solution mixtures can exhibit an upper
(superior) or a lower (inferior) critical tempera-
ture of demixing. A lower critical temperature
implies that the co-solubility of dissimilar
biopolymer decreases with an increase in tem-
perature. On the contrary, the co-solubility of
macromolecular solutes increases with increas-
ing temperature in biopolymer mixtures with
an upper critical temperature. Normally, poly-
mer mixtures with positive values of both en-
thalpy (endothermic effect) and entropy of
mixing have an upper critical temperature, while
mixtures with negative values of both enthalpy
(exothermic effect) and entropy of mixing have
a lower critical point of mixing. In systems
with a lower critical temperature, its value nor-
125
mally decreases with an increase in the mo-
lecular weight of macromolecules. In contrast,
in a system with an upper critical point, the
critical temperature increases with an increase
in molecular weight. Mixtures with a lower
critical temperature seem to be more typical of
polymer^.^^.^*
Figure 12 illustrates phase diagrams for
systems with a low critical point1IJm(A and C)
and an upper critical point (B and D).
11~12,1233124
The reversible phase separation shown in Fig-
ure 12B and 12D takes place in the vicinity of
IEP of 11s seed storage globulins. The phase
diagram is symmetric. The concentration of
dispersed phase generally varies from 18% to
40 to 50%, that is, approaches concentrations
characteristic of protein crystals. Figure 13
shows the phase diagrams for the broad bean
globulin-NaCl-watersystem and illustrates the
difference between seed storage proteins in a
critical phase separation temperature and its
dependence on the salt concentration used for
their fractionation and isolation.LLJ22
This fig-
ure presents the stages of the broad bean (and
peas) globulin (1 IS and 7s globulins, i.e., le-
gumin and vicilin) isolation process yielding a
degree of purity greater than 95%. According
to this method, seed storage proteins are ex-
tracted with an aqueous, 0.6 M NaCl solution at
Geiatin + Dextran
-ID%zc,
pH 6.5, 0.15 M NaCI
A
40 A
0.2 8.4 cIG+D
fludon
-
Legumio NaCl H,O -
O
Prodem,%
FIGURE 12. Phase diagrams: (A) for gelatidextran-water, (B and D) legu-
min-NaCI-water and (C) gelatin-pectin-water. (From Refs. 11,124,160.)
126
pH 8.0 and 5OoC, which, after separation from
starch and other insoluble ingredients, is di-
luted to 0.3 M and 0.15 M NaCl and cooled
down to 25°C and 5°C to separate legumin and
vicilin, respectively.1LJ22
6. Effect of Shearing
Figure 12B shows that on the right-hand
side of the rectilinear diameter, W/W emul-
sions have a concentrated continuous phase
and a diluted dispersed phase. Such phase con-
centration ratios are typical of many food sys-
tems, such as doughs and extruded products.
Both co-existing phases of the system (Figures
12B and D) contain the same individual protein
but in different concentrations. It may be as-
sumed that this reflects an incompatibility of
associated and nonassociated forms of the same
seed storage globulin. Accordingly, Figure 12D
shows that with the mechanical destruction of
protein associates at a sufficiently high shear
rate gradient in a rotational rheometer, the two-
phase system undergoes a reversible transition
into a single-phase state.11Jz.89JM This transi-
tion is presumably due to shear-induced dis-
ruption of protein associates at temperatures
below the critical temperature of phase separa-
Gelatin + pectin (MW 23 kD, DE=57%)
pH 6.0,0.5 M NaCI
o
 I Broad Bean Flour
Extraction of proteins I

l
I

u 4 ’ I
I
I
O 1

1
e
b
I
I

40

25
i p i m n of 7s fracrim (viaiia)
5

Protein. %wt
FIGURE 13. Phase diagrams for the broad bean (pea) globulins-NaCl-water system at
different salt concentrations and temperatures. General scheme of the 1IS and 7s seed
globulin isolation.

tion. Accordingly, stopping shear flow rein-
duces the self-association of protein molecules
and phase separation. This means that shear
conditions during extrusion affect both the stnic-
tural homogeneity and the anisotropic structure
of the extrudates. Because shear forces can
counteract the aggregation of proteins, the
“shear dissolving” effect is important for pre-
cipitating protein from solution. The deforma-
tion of such W/W emulsions at relatively low
shear rates could lead to anisotropic systems
with two continuous phases104J12-114,200
(the next
section). Thus, the two most common food
processing operations, that is, thermal and
mechanical treatments, affect phase state and
structural properties of foods.’04.200

127
B. Low Interfacial Tension: Spinneret-
less Spinning of the Dispersed
Particles
A low interfacial tension varying from
0.0001 to O. 1 dyn/cm is typical of W/W emul-
sions.201For instance, the maximum value of
the interfacial tension for gelatin-dextran-wa-
ter systems equals 0.027 dyn/cm and decreases
when the system approaches the boundary con-
ditions.’16To measure interfacial tension in W/
W emulsions, because of the low values and
opaqueness of the emulsions, two techniques
have usually been used: the horizontal rotating
tube methodzo2and the capillary method.203The
low interfacial tension in W/W emulsions re-
flects the close compositions of the coexisting
aqueous phases that are composed mainly of
water and contain the same limitedly co-solu-
bility biopolymers but in different proportions.
The low interfacial tension contributes to the
easy deformability of the dispersed particles
and a highly developed interfacial surface of
W/W emulsions in flow. Figure 14 illustrates
how the deformability of the dispersed par-
ticles affect the morphology and texture of many
foods. The stresses developed in a dispersion
medium of a flowing W/w emulsion can de-
form and orient the liquid dispersed particles.
This occurs if the viscosity of the dispersed
particles is lower than that of the dispersion
medium. Under such conditions in a flowing
emulsion, dispersed particles take the shape of
liquid fibrils, can then disrupt into smaller
spherical droplets, and coalesce in larger spheri-
cal drops and liquid filaments. The formation
mechanism of fibers and anisotropic materials
in the processing of such two-phase liquid sys-
tems has been called spinneretless spin-
ning.33,34”,*9This mechanism affects the mi-
crostructure and quality of thermoplastic
extrusion products,LL2-114
and protein isolateszm
in particular. In the latter case, filamentous
particles of a precipitated protein can be sepa-
rated easily from the supernatant, dewatered
under pressure, and dried. The capillarity of
dry fibrous instant foods affects their rehydra-
128
tion behavior, for example, splitting and lump-
ing of the dispersed particles, their swelling,
and dissolving rateszw
C. Gelation: Elasto-osmometry
An increase in the concentration (Figure
9H) of one of the system phases can result in its
gelation. A gel-dispersed particle formed in the
bulk of the coexistent phase behaves as an
osmometric cell with elastic walls (Figures 10
and 11). Its swelling-deswelling behavior de-
pends on the osmotic pressure of the surround-
ing biopolymer solution and the elastic proper-
ties of the gel. The latter therefore can be used
to measure the osmotic pressure of the sur-
rounding solution. The pressure applied to the
gel to stop its swelling, that is. the passage of
the solvent into the gel, equals the osmotic
pressure. Figure 11 illustrates the principle of
the method called elasto-osmometry that was
developed by Hermanc and Pnns with co-work-
ers to determine polymer molecular
The authors used an elasto-osmometer con-
sisted essentially of a glass cell filled with poly-
mer solutions of different concentration. In the
cell a gel strip was fixed between two clamps
in which the upper was connected to a balance
arm and the lower clamp was attached to the
base plate by a micrometer moving this lower
clamp in order to adjust and keep the length of
the strip constant. The balance-type device was
to measure the retractive force resulted from
the deswelling of the gel at a constant length.
Figure 11 shows that the change in retractive
force of the gel with an increase in concentra-
tion of the surrounding polymer solution is
proportional to the activity of this solution and
inversely proportional to the number average
molecular weight of the polymer, which cannot
penetrate into the gel. The instrument (elasto-
osmometer) was calibrated for both the solvent
used and the dissolved polymer of known
molecular ~ e i g h t . ~ OThe
5 strips of poly-
dimethylsiloxane or polycisisoprene gel were
used to determine the molecular weight of poly-
0 W
a
r,
æ
5 0
u.

1
129
styrenes and polyvinylacetates.The results were
in good agreement with the independently de-
termined molecular weights. The close corre-
spondence between the values of molecular
weight obtained by the elasto-osmometer and
an independent method means that there is no
penetration of the solute into the gel. Elasto-
osmometry effects therefore are of interest for
many processed food systems and composite
foods with liquid and solid aqueous phases. For
instance, it would be useful for assessing the
quality of a barrier separating the dough and
the sauce and preventing the penetration of
low- and/or high-molecular-weight substances
into gel-like doughs. Such food barrier films
are usually responsible for both the mechanical
properties of the gel and the stability and rhe-
ology of the sauce.
D. Incompatibility and Surface Activity
Control of Biopolymers
Lipids are the third (after proteins and
polysaccharides) structure forming food com-
ponents and macronutrients. Stabilization of oil-
in-water (Om)emulsions and the structure-
forming behavior of lipid-dispersed particles in
W/W emulsions therefore are of great impor-
tance for structure-property relationships in
foods.18-21 There is a great difference in the
emulsion stabilizing mechanism between low-
and high-molecular-weight s ~ r f a c t a n t s . ~ ~ - ~ *incompatible
Chemical molecular design is the main tool for
designing low-molecular-weight surfactants. In
contrast,for biopolymers the most adequatetech-
nique is to design the
and physically modify the biopolymers, includ-
ing changing their conformation and promoting
interbiopolymer ~ o m p l e x i n gFor
. ~ ~instance,
~ ~ ~ it
is well known that both acetic acid and ethanol
are miscible with water in any proportions. Their
miscibility with water, however, decreases with
an increase in the length of the hydrocarbon
chain of the molecules. This increase in the
length of the hydrocarbon chain leads to surfac-
tants, fatty acids, and fatty alcohols, which mol-
130
ecules have an affinity for both polar and non-
polar phases of O/W emulsions.
Protein molecules contain hydrophilic and
hydrophobic groups and have an affinity for
both water and oil phases. The accessibility of
hydrophobic side groups to the medium prob-
ably increases within the series of albumin,
globulins, glutelines, and prolamines. Unlike
low-molecular-weight surfactants, the emulsi-
fying properties of proteins can be improved
without chemical modifications. Several tech-
niques that are usually used for controlling
surface activity of proteins are based on changes
either in conformation (to decrease the mim-
icry) or in the medium (to increase the ex-
cluded volume). These techniques include:
(1) the formation of interbiopolymer complexes
and conjugates, ( 2 )the conformational changes,
for example, due to thermal denaturation and
aggregation of the protein, and (3) complexing
with lipophilic molecules. The latter method
changes the chemical composition of a protein,
for example, by the adsorption of the oil phase
components, to improve its hydrophobicby-
drophilic balance. Lipids form complexes with
both proteins and polysaccharides, usually re-
ducing their solubility, decreasing the confor-
mational stability of proteins and provoking
phase separation.11J2However, unlike low-
molecular-weight surfactants, the efficiency of
proteins as food emulsion stabilizers can be
controlled more simply by the addition of an
J~ biopolymer, that is, without any
chemical or enzymatic modifications whatso-
ever. 10-12.14,18.21,53
It has been proposedI1J2J4that the incom-
patibility of the native and denatured, that is,
structurally dissimilar forms of the same pro-
tein, is the reason for an extended plateau on
the adsorption isotherm of a globular protein
on oiywater interfaces. This plateau corresponds
to the formation of a monolayer around dis-
persed particles and covers a large range of
concentrations of the protein where the amount
adsorbed per unit area is not changed. It was
argued that this is a result of the incompatibil-
ity between adsorbed and dissolved protein
 molecules. Adsorption of protein molecules
usually leads to their concentration, orienta-
tion, and partial unfolding. The protein mol-
ecules dissolved in the dispersion medium and
adsorbed on the dispersed particles cannot, pre-
sumably, recognize each other as being the
same. Adsorbed protein molecules forming a
monomolecular adsorption layer therefore can
inhibit further adsorption.”J2J4 It has been
shown that adding dextran to a protein solution
(1 1s globulin from Vicia juba) increases the
surface activity of the protein at the non-polar
phase/water interfaces. When the amount of a
polysaccharide added to a protein solution ex-
ceeds the phase separation threshold, phase
separation of the aqueous dispersion media
results in the encapsulation of the dispersed
particles of the O/W emulsion. Thus, demixing
probably causes the formation of protein
multilayers at the interface.11,12,14,100,101
Due to
the excluded volume effect the addition of one
biopolymer to a solution of another reduces the
protein concentration required for its multi-
layer adsorption at the O/W interface. Compar-
ing the phase diagram with the protein adsorp-
tion isotherm shows that the formation of protein
multilayers is due to phase separation in the
aqueous emulsion phase.11J2.14The formation
of protein multilayers can be regarded as a
transition from the O/W emulsion to the W/W/
O emulsion with very low interfacial tension.
Additional factors improving the activity of
biopolymers as O/W emulsion stabilizers is
biopolymer-lipid complexing. Complexing of
linear anionic polysaccharides with oppositely
charged micelle-forming lipids could lead to
intramacromolecular lipid-microphasemicelles,
which is similar to synthetic polymer-colloidal
com plexe^.^^^^^^^ Interbiopolymer casein-Ca-
pectin complexes were used to stabilize a thixo-
tropic oil-in-water emulsion applied as meat
extender for the manufacture of “cooked sau-
sages”.21 Owing to complexing, an anionic
polysaccharide can act either as a flocculating
agent increasing the particle size (bridging floc-
culation) or as a protective colloid increasing
the surface hydrophilicity of the dispersed par-
ticles, that is, their mimicry. Thus, the two
universal techniques for improving the effi-
ciency of biopolymers as stabilizers of oil-in-
water emulsions are based on incompatibility
and complexing of biopolymers. Both methods
achieve the encapsulation of lipid droplets by
highly viscous or gel-like biopolymer lay-
ers.11,I2,14
E. Interfacial Layer Between
Immiscible Aqueous Phases:
Depletion Flocculation of Liquid and
Solid Dispersed Particles
Figure 15 shows that phase separation re-
sults in the formation of a thick, low-viscosity
interfacial (depletion) layer between the two
immiscible phases of W/W emulsions. This
layer is the direct result of mutual depletion of
biopolymers from the interfacial area. The
depletion layers around dispersed particles are
responsible for an association of dispersed par-
ticles. The self-association (or depletion floc-
culationj of dispersed particles, that is, destabi-
lization of a W/W emulsion, is counteracted by
an adsorption within the interfacial layer (i.e.,
between the aqueous phases) of dispersed par-
ticles of different mainly hydrophobic species
(e.g., lipids, denatured proteins, and
Two large-scale industrial applications illus-
trate the importance of the latter phenomenon.
The adsorption of hydrophobic particles within
the interfacial layers between immiscible aque-
ous phases are used for: (I) manufacturing low-
and (2) fractionating cells,
fat spreads10-12.2’~200
cell organelles, and macromolecules using W/
W emulsions based on synthetic water-soluble
polymers.20’~209-212
The basic difference between the phenom-
ena of depletion flocculation and limited ther-
modynamic incompatibility is that the former
is of a nonequilibrium nature. Unlike molecu-
lar dispersions (biopolymer solutions), colloi-
dal dispersions are thermodynamicallyunstable.
Therefore, the transition from molecular to
colloidal biopolymer dispersions makes, the
131
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132
phase separation even more pronounced. Deple-
tion flocculation can also be treated as a par-
ticular case of flocculation and coagulation of
colloidal particles in a nonwettable medium.
Depletion flocculation causes a rapid coales-
cence of water-in-water emulsion droplets. Fig-
ures 9B and 9G show phase diagrams for mix-
tures of casein and sodium caseinate with pectin
and sodium alginate, respectively. The phase
diagrams illustrate the similarity between phase
separation of biopolymers (Figure 9B) and
depletion flocculation (Figures 9G and 9H) of
large-ize casein particles. The dissociation of
casein micelles increases both the phase sepa-
ration threshold and the co-solubility of the
biopolymers, and also involves the fraction-
ation of the casein chains. An excess of Ca-
ions in milk is likely to prevent the incompat-
ibility of casein chains with each other. Unlike
phase separation of a protein-polysaccharide
FIGURE 16. Schematic representation of interfacial layers around dispersed
particles filling the gel. Gelatin gel network disrupted by granules of maltodextrin
droplets. (From Ref. 117.)
mixed solution, which usually occurs at a total
biopolymer concentration exceeding 4%, deple-
tion flocculationof larger dispersed particles of
m i k casein or chloroplasts can occur at con-
centrations of less than 1%. An example of
depletion flocculation coinciding with the phase
separation of an infant formula containing casein
and maltodextrin occurs when the product is
sterili~ed.~'~ Because a low co-solubility and
its reduction with temperature are typical of
mixtures of polysaccharides with unordered
structure proteins, such as casein, adding
polysaccharides (maltodextrin) reduces the heat
stability of this infant formula as a casein-sta-
bilized oil-in-water emulsion. This effect is due
to depletion flocculation.2w~2'l
The important rheological role of interfa-
cial layers between the two immiscible (liquid
or solid) phases is preserved during geletion of
one or both of the p h a s e ~ . ' ~ Figure
. ~ ~ J ~16
~
133
illustrates the result of electron microscopic foods. The spherical gel granules separated by
investigations of gelatin gels filled with dis- thin lipid films (honeycomb-like structure) have
a low adhesion to each other and can rotate in
persed particles of d e ~ t r a n . ”It~has shown that
the fibrils of the gelatin gel trend to minimizeshear flow. It was also proposed that the rota-
contact with the dispersed particles filling thetion of solid granules results in so-called “ball-
gel. Within the area around the dispersed par- bearing” e f f e ~ t , ’which
~ . ~ ~ is probably respon-
sible for the fluidity of many food systems and
ticles the fibrils of the gei network are oriented
by their free ends normally to the surface of fat mimetic behavior of non-fat material, cos-
these particles. The ruptures and imperfections metics, and foods (Figure 21). Examples in-
of the gel network within the interfacial layer clude ice-cream mixtures, wheat flour doughs,
decrease the elastic modulus of the gel with low-fat spreads, fat replacers, and even extend
increasing volume fraction of the dispersed to cosmetics based on starch and talc powders,
phase. which have been used since time immemo-
fia1.17,18,20
To produce low-fat butter replacers, W/W
F. Lipids as Component of Water-in- emulsions based on gelatin-maltodextrin and
Water Emulsions: Fat Replacement gelatin-amylopectin mixtures are the most
widely applied.214-220 Other biphasic biopoly-
Figure 15 shows the two types of behavior mer mixtures can also be used for producing
of dispersed lipid droplets added to a W/W small gel granules of proteins, polysaccharides
emulsion. Normally, lipid droplets would be and low-fat spreads.221-228 For instance, to re-
either encapsulated by the protein-rich phase place a part or all of the fat in such foods as ice
and/or adsorbed within the interfacial layer cream, salt dressing, spreads, and sauce^,*^^-*^^
between immiscible aqueous phases. Adsorbed small gel granules of amylose,196geiatin,229-231
lipid droplets can coalesce within the interfa- thermosetting proteins,232gelling polysaccha-
cial layer and form thin lipid layers. These rides, and Ca-alginate (containing gum-arabic
layers can also coalescence (in their turn) and as surface active p o l y s a c ~ h a r i d e )and
~~~
form a three-dimensional lipid network, so- microparticulated protein and protein-polysac-
called “honeycomb-like” structure.18-21J18.200 charide complex gels have been proposed.
The formation of this new lipid continuous
phase is favored by surfactants stabilizing wa-
ter-in-oil emulsions. This phenomenon of ad- VI. CONCEPTUAL APPROACH TO
sorption and coalescence of lipids around aque- SKIMMED MILK PROCESSING. ICE
ous dispersed particles can be applied to CREAM MIXES
significant advantage in processed food mate-
rials, for example, less than 30% wt of added Figures 9G, 1 1 , and Figure 15 show that a
lipids form the continuous phase encapsulating mixture C of skimmed milk with 2% high-ester
more than 70% aqueous phase. This principle pectin solution separates into two phases: D
underlies a number of low-fat spreads (butter containing from 20% to 30% casein and a small
replacers) based on different biopolymer mix- amount of whey proteins and E containing about
tures.1s.21Gelation of both aqueous phases and 1 % pectin and most of the milk whey proteins.
solidification of the lipid continuous phase on Figure 17 shows that the two operations: (1)
cooling yields a solid composite with abutter- the addition of a polysaccharide to skimmed
like texture. This composite material formed milk and (2) the heating (pasteurization) of the
by a lipid phase filled with thermoreversible mixture result in two consecutive phase separa-
gel globules is a good model for obtaining tions. Both yield small spherical gel granules
insights into the fat-like mouthfeel of many consisting of Ca-caseinate and of whey pro-

134
 Jmim
Dispesed Phase:
1540% Casein
Phase
I
Milk Curd
Separation-1
0 0
Fat Replacer,
Protein gel granules
FIGURE 17. Schematic representation of flow diagram for processing skimmed milk into “full-fat”
milk foods. (From Ref. 21 .)
teins, respectively. Mixing these two fractions
underlies the manufacturing of “full-fat”-type
m i k products based on skimmed m i k z 1The
process is based on the difference in both phase
behavior and gelation conditions between
casein-polysaccharide and whey protein-
polysaccharide mixtures. The first phase sepa-
ration of skimmed milk-polysaccharide mix-
tures occurs at room temperature and gives the
dispersed phase (Figures 9G and 9H, point G),
which is Ca-caseinate gel granules. The Ca/
protein ratio depends on partitioning the Ca-
ions between the casein-rich and the polysac-
charide-rich phase and is controlled by the
degree of esterification of the pectin. The latter
can be separated, purified, and reused. The use
of polysaccharides for clotting milk casein pro-
vides a new generation of cheese-making with-
out rennin and equally simplify the production
of dry cheese p0wders.2~~ The heat pasteuriza-
tion of the phase L (point L in Figure 9H)
containing pectin and milk whey proteins re-
sults in the denaturation and a decreased co-
solubility of the proteins with pectin and phase
ispersion Medium:
Whey proteins,
Lactose
4
~ 9-- Whey
Dispesed Phase:
protein gei
Ik
o Q- Dispersion Medium: USE
separation. The separated mass of small gel
granules (Figure 15) has a fat-like texture. Mix-
tures of the whey protein gel granules with the
casein-rich phase produced by the fust phase
separation leads to a “full-fat”-milk curd pro-
duced from skimmed milk. The pectin-rich liq-
uid continuous phase can be separated, puri-
fied, concentrated, and reused.240These
principles can be further expanded for addi-
tional processing advantages. For example, an
anionic polysaccharide used in this process acts
as liquid ion-exchangers binding heavy metal
cations.239This selective binding provides the
means to extract possible pollutants from the
milk into the polysaccharide-rich phase L. The
phase behavior of skimmed milk-polysaccha-
ride mixtures presented on Figure 17 is a viable
model for a wide variety of foods, from ice
cream mixes to bread doughs.
Figures 9G and 15 illustrate the possible
activity mechanism of structural stabilizers of
ice c r e a r n ~ ,which
~ ~ . ~are
~ usually polysaccha-
rides or gelatin. The amount of polysaccharide
stabilizers in commercial ice cream varies from
135
0.25% (for sodium alginate and carboxymethyl
cellulose) to 0.1% and below (for locust bean
gum and carrageenan). These values are pro-
portional to the excluded volume of the polysac-
charide macromolecules and below the phase
separation threshold of their mixtures with
skimmed milk. Due to the pronounced phase
diagram asymmetry of casein-polysaccharide
mixtures,20.200 the first phase separation of ice
cream mixes (Figure 17) rcsults in a strong
concentration of the phase rich in casein (Fig-
ure 11). Freezing of about 50% of the water
leads to an additional concentration, coales-
cence, and gelation of the dispersed Ca-casein-
ate phase. The melting characteristics of this
gel-network determine the heat shock stability
of ice creams. For instance, Figure 9G shows
that less than 0.5% pectin or alginate added to
skimmed milk provokes phase separation. The
homogenization and pasteurization conditions
of the ice cream mixes correspond to those of
both types of phase separation mentioned above.
The first phase separation occurs during the
homogenization of the ice cream mixes and
yields the casein-rich phase and the biopoly-
mer-stabilizer-rich phase containing milk whey
proteins. The latter liquid phase can be phase
separated during pasteurization of the ice cream
mixes. These two phase separations result in
the two specific structures (Figure 15):protein
gel granules and thin lipid layers separating
aqueous phases, both contributing to fat-like
texture. Lipid layers forming honeycomb-like
structures would also be responsible for a de-
crease in ice crystal size. Air bubbles covered
by lipids are also a possible factor, reducing ice
crystal size. One more mechanism of reducing
ice crystal size appears to be a nonspecific
adsorption of hydrated macromolecules to the
surface of the growing crystal, that is, an
encapsulation of ice crystals by food
hydrocolloids.21Unlike low-molecular-weight
cryoprotectants such as salts, sugars, glycerin,
and ethanol, the activity of hydrocolloids
(polysaccharidesand proteins of unfolded struc-
ture) depends on the excluded volume of mac-
romolecules and is not proportional to their
136
concentration. During ice formation the long
hydrated chains and their aggregates filling the
solution space, can rapidly be bound (coopera-
tively adsorbed by many H-bonds) onto, and
modify the growing faces of small ice crystals.
Cooperative adsorption of hydrophilic side
groups of an amphiphilic macromolecule cov-
ers the ice surface by hydrophobic side groups.
This “antifreezing” activity obviously rises with
the degree of water crystallizationthat increases
the concentration of the macromolecular stabi-
lizer.
Figure 18 shows the thermodynamic simi-
larity between low-fat spreads and ice cream
mixes. Their similar composition, phase be-
havior, and possible formation of honeycomb-
like structure should be noted. Because ice
cream mixes contain less lipids, which are partly
adsorbed by gas bubbles, a continuous lipid
phase cannot be formed. It should also be noted
that both types of phase separation (due to
interbiopolymer complexing and incompatibil-
ity) encapsulate the food ingredients dispersed
in the lipid phase?41-245 This technique using
lipids as a dispersion medium for encapsulated
nutrients and flavours decreases the rate of many
chemical reactions when compared with the
rate in aqueous media. This principle is impor-
tant for controlling nutrient delivery, flavor
release, and shelf-life stability of foods con-
taining biologically active and chemically un-
stable compounds. The phase separation tech-
nique has been used widely for encapsulating
nutrients, flavors, and for producing granular
functional and decorative foods, such as ana-
logues of caviar, grains, berries, and dry
rehydratable berries, drinks, fat-replacers, etc.
and for controlling food texture.11-19s22.233.246,247
VII. PHASE STATE OF DOUGHS: A
CONCEPTUAL MODEL
Recently, a model was proposed to under-
stand the functional properties of wheat flour
doughs is based on the assumed thermody-
namic similarity between milk protein-polysac-

systems
FIGURE 18. Schematic representationof the composition areas of low-fat spreads and ice cream mixes. (From Ref.
36.1
charide mixtures and wheat flour doughs.20
There are several reasons for this assump-
tion.''J0 It is proposed that the distinct struc-
tural properties of these biopolymers are not
coincidental but relate to a common basis of
physiological function arising from biological
selection. There is a common physiological
function of milk and wheat proteins. Both glu-
ten and milk casein are storage proteins, that is,
sources of amino acids, short peptides, and
nitrogen for use during germination and the
early stages of growth. This common physi-
ological function underlies the similar thermo-
dynamic and structural features of these pro-
teins. Actually, both gluten and casein:
-:h -phase
otein-ric
D
b
Protein, Yo wt.
(1) consist of associated heterogeneous sub-
units; (2) contain polypeptide chains of un-
folded structure; (3) are relatively poorly soluble
at physiological pH, (4) are insensitive to heat-
ing; and (5) form very large particles that are
incompatible with polysaccharides. Covalent
disulfide bonds and coordination bonds (via
Ca-ions) and hydrophobic interactions contrib-
ute to the formation of the large-size particles
and prevent the incompatibility of the polypep-
tide chains of both casein and gluten. The next
thermodynamically important issue related to
their common physiological function is that
mik and wheat flour dough contain nearly the
same proportion of the main protein classes
137
defined according to Osborne's classification.
Wheat flour contains about 85% seed storage
glutenins and gliadins and about 15% albumins
and globulins. Milk proteins comprise about
80% of casein (i.e., glutelin) and about 20%
milk whey proteins, albumins, and globulins.
The next common feature is that gel networks
of both casein and gluten are formed using
covalent (gluten), coordination (with Ca-ions),
H-bonds, and hydrophobic interactions. Both
are notable for high critical concentrations of
gelation, the syneresis and structural changes
of the gels during mixing (the peak time, the
mixing peak, the maximum consistency of
doughs, and the ripening of rennet coagulated
milk curd in cheese making). The chain mobil-
ity and high excluded volume of gluten and
milk casein gels would make the hydrolysis of
these proteins slower, matching the rate of the
biosynthetic reuse of the amino acids.
Because wheat flour always contains some
amount of soluble pentosans and starchy
polysaccharides (from granules damaged dur-
ing milling), the model system proposed is a
milk protein-polysaccharide mixture.20Figures
9G and 11 illustrate the phase behavior of
skimmed milk protein-pectin m i x t u r e ~ , ' ~ ~ J ~ ~
where the pectin content corresponds to the
concentration range of soluble polysaccharides
typical of wheat doughs. Figure 19 and Table 2
show that the concentration of both the gluten
and casein-phases reaches 20 to 30%, at equi-
librium with a solution containing about 1%
polysaccharides, soluble pentosans, or pectin,
respectively. According to this model, on mix-
ing wheat flour with water two immiscible
continuous aqueous phases must be formed.
The first is a viscous continuous phase contain-
ing soluble polysaccharides and most of the
soluble proteins, that is, albumins and globu-
lins. This liquid phase as an unwettable me-
dium causes an aggregation of the dispersed
phase particles containing the gliadin and glu-
telin fractions. The aggregation of gluten-dis-
persed particles minimizes contacts with the
unwettable medium and leads to the second
continuous phase. Both continuous dough
phases are filled with several types of dispersed
particles: intact and damaged starch granules,
insoluble pentosanes, other food fibers, lipid

6 12 18 24 30
Proteins,%

FIGURE 19. Schematic phase diagram of wheat flour dough

based on the data (Table 2) about the compositions of the
dough phases separated by ultracentrifugation. (From Ref. 20.)

138
TABLE 2
Experimental Studies on Separation of Wheat Flour Dough Phases

Mixing, Baker, Parker, Mauritzen, Stewart, MacRitchie, Larsson, Eliasson,

:entrifugation Mize, 1946, 1965 1976 1996

:onditions and ~481 WJI 12501 [251,252]

:omposition of a

iough and ih 3 Layers: 5-7 Layers: 3 Layers: 4 Layers:

Fractions, wt % (i) (i) lipids, (0 (i) liquid phase,

viscous liquor; (ii) liquid phase, viscous (ii) pentosan gel,

(ii) (iii) pentosan gei, liquor; (iii) giuten gel,

giuten+starch (iv) gluten gel, [ii) (iv) starch

intermediate and (v) starch and gluten+cta Cchemical

(iii) starch (vi) and (vii) two rch analysis of the

intermediate layers phases did noi

containing damage made

starch granules

Dough composition 73% H20; 2% 63.4% H20; (52.0- 46% H20, 38 - 50% H2O;

NaCl; 69.5% 0.5M NaCl 7.Y? 6 flour:8.8+16.7?4

25 flours: 9 . 8 ~solution) at 30"C, protein, proteins,

14.9 % protein, 7 flo~n:8.2+ 11.4% 2% NaCl, 76.6+84.7%

average 12.2 %; protein flOW starch anc

fermented and 13.4% 2.3+8.6%

non-fermented protein damaged starch.

Mixing and 65 rpm, > 1.5 7+210 min at 30°C; 25 OOO+ 60 rpm, 5 min, a

:enîrifugation min., 40 O00 g, 105 O00 g for 70 407 O00 g, 30°C; 100 O00 j

139
TABLE 2 (continued)
:onditions 20 min, 29.4 min. at 30°C 50 min or 1 hour.

*IT

iquid Quantity 23.8 (8.8 G37.5) 9.5 + 20.6; 19.0 50th overmixing

>hase ûvermixing: 20.60 results in a loss

11.3; if water binding

19.2 010.7 :apacity) and an

Solids 13.1 (10.1+18.0) 14.5+15.5 13.7 :xcess water

Proteins 2.61 (1.99+2.4) Aibuminíglobulin 3.4 uided to dough

ratio: 3 . 4 4 . 0 ncreases the

Pentosans 1.53 (1.09+2.4) 7.0 iquid phase

sugars 6.6 (4.1û+10.98) iolume and

increases due to iough stickiness

enzymatic

hydrolysis of the

starch

H20 87 84.5i85.5 86.0 38

Fu~c- High foaming A highly viscous Good

tionai properties, liquid. The viscosity foaming

features increases the is greatly reduced properties

dough over during 36 h. The (defetted

spring and reducing sugar doughs)

improves color content rises during

of the crust 6 h.

140
TABLE 2 (continued)

Solid byer- 1, Ouantity 1.6+9.7, &antity U% H20;

(Pentosangel) ovennixing reduces: 31%H20; 4 large amount of

4.5 00; 3.9 3 o. Protein imaged starch

Solids 2e22.8; 3.9%; )y microscopy)

1.9+3.8% protein. Pentosans IS at the bottom of

+ starch rhe gel phase

Solid Layer-3, Ouantity 9.8i36.1, 55%; 50~55% H20;

(Gluten gel) overmixing NaCl Addition of

increases: 9.8022.3; 3.8%; lecithin leads to

11.8+2 1.9; H20 an increase in the

Solids 52~54; 34.5%. water content of

17+22.3% protein;. the gluten phase

(Starch Layer) Ouantity 42+65, about 30% H2O.

overmixing

increases: 62065;

65066;

Solids:7Ck72;

O.l+l.49%N

Lipid (upper) Layer 1-2 d450 g dough A thin oil film is

on the top of the

liquid phase

141
droplets and air bubbles. Because in the solvent
medium a gel-like dispersed particle behaves
similarly to an osmometric cell with elastic
walls (Figures 9H, 10, and 1 I), the degree of
swelling at equilibrium of gluten gel particle in
a polysaccharide solution under standard con-
ditions (including mixing) could be used to
assess the bread-making quality of wheat flour.
Because starch is more hydrophilic than gluten
proteins, its gelatinization on baking provokes
dewatering of the continuous dough phases.
Other factors contributing to the gelation of the
gluten phase are (I) a decrease in water binding
capacity due to protein denaturation, (2) water
evaporation from an exterior loaf layer; (3) the
Maillard reaction and other cross-linking reac-
tions of the macromolecules; and (4) glass tran-
sitions in the gel networks and within the exte-
rior layer of the loaf that fix its shape, structure,
and retarding staling.20J86
A. Ultracetrifugational Separation of
Dough Phases
There is considerable experimental evi-
dence supporting the proposed thermodynamic
model and its pre di ct ion^.'^.^^ The direct ex-
perimental data248-252
on the separation of wheat
Ultracentrifugational fractionation of
wheat flour dough
FIGURE 20. Wheat flour dough fractions separated by ultracentrifugation.
142
dough phases using high-speed centrifugation
are presented in Table 2. Figure 20 illustrates
the result of dough ultracetrifugation. Baker
et al. first demonstrated the heterophasic na-
ture of Their results were then con-
firmed and developed by Mauritzen and Stew-
ard,249Ma~Ritchie,*~O and Larsson and
Elia~son~~~,~ a number
~ ~ u s i of
n gdifferent wheat
cultivars and dough compositions. Table 2
shows a varying number of the phases ob-
tained by the authors, as the separation effi-
ciency of dough phases increases with water
content and depends on ultracentrifugation
conditions. The detailed chemical analysis of
dough fractions camed out by Mauritzen and
Steward249 and MacR~tchie~~O (Table 2) showed
that the composition of dough phases is quite
consistent for different wheat flours.
B. Thixotropy of Dough and Its
Foaming and Gas-Holding Properties
Thixotropy (i.e., a reversible liquid-solid
transformation under loading-unloading condi-
tions, respectively) seems to be a specific fea-
ture of the gluten phase of doughs that is of key
importance for dough processing.I7Thixotropy
(during kneading when dough phases becom-
ing liquid) removes the “gel sieving” effect of
the gel network and ensures mixing of colloi-
dal and coarser dough ingredients. Also, the
effect of washing starch granules out of mixing
dough reflects the thixotropy of the gluten phase.
Finally, the thixotropic solidification of the
gluten phase at rest contributes to the high gas-
holding capacity and structural stability of the
dough as a solid foam and emulsion.
The liquid aqueous phase containing globu-
lar proteins, soluble pentosans, and starch is
presumably responsible for the high foaming
properties of doughs. The bulk biopolymer
concentration of this phase is close to the phase
separation threshold (4% and higher) typical of
protein-polysaccharide r n i ~ t u r e s . ~ ~ , ’ ~ ~ .precipitation
Phase separation could be caused by additional
polysaccharides dissolved during kneading and
their interactions with the lipids and other dough
components. Owing to phase separation,denser
membranes around gas bubbles and oil drop-
lets can be formed.9-12~18~21~46~52~53Thixotrophy
solidifies the gluten phase of the dough foam
and emulsion structures. In other words, thixot-
ropy provides dough foaming and prevents the
separation of the phases at rest. Thus, two
membranes are formed around gas bubbles in
doughs. The first is formed by the precipitation
of the liquid phase components and the second
by the gluten phase due to its thixotropic gela-
tion.17
The proposed responsibility of water-
soluble fractions of wheat flour for the high
foaming capacity and foam stability of doughs20
is in agreement with experimentai data.248-2M,253-259intensity of mechanical dough treatment? Why
Baker et Mauritzen, and
Ma~Ritchie~~O showed good foaming proper-
ties of the water-soluble fraction of wheat flour.
Therefore, this flour fraction could find various
applications in foods and biomaterial products.
These extracts could be used as “natural” low-
price dough improvers,20and for encapsulating
nutrients and flavors. Aqueous extracts of flour
of winter wheat and rye cultivars could be
sources of biological antifreeze, and could im-
prove the structural stability of frozen doughs
and ice cream An alkaline pro-
teinaceous fraction of rye was proposed to
improve loaf volume and crumb q ~ a l i t y . ~ ~ ~ - ~ ~
It was also that a small addition of
pumpkin powder markedly increases the vol-
ume of wheat bread loaves. The authors be-
lieved that this unexpected effect was due to
the surface activity of the highly acetylated
pectin as a major component (about 30% of the
total dry-matter content) of pumpkin tissue. It
is important, however, that the effect of addi-
tives on the specific volume of the bread went
through a maximum at about 5 to 10%concen-
tration of pumpkin powder (g/kg flour). This
would reflect the phase separation threshold of
the mixtures. Phase separation results in the
‘ ~ ~ , ~ ~ ~ of interbiopolymer protein-pectin
and/or protein-pentosan complexes at the gas-
water interfaces and stabilizes foam and emul-
sion structures.18-z1 The proposed role of the
liquid fraction in the stickiness of dough was
also recently shown e~perimentally.’~~
C. Thermodynamic and
Microrheological Aspects of Dough
Functionality
Thermodynamic and microrheological ap-
proaches are important in practice to interpret
the relationships of dough processing function-
ality.17,20For instance, such approached help to
understand why mechanical treatment is so
important to dough functionality? Why does
the extractibility of lipids decrease with the
can pasta be dried without cracks? Why are
doughs and cooked pasta products opaque, but
dry pasta and breadcrumb are transparent?
Mechanical handling greatly affects dough
structure at the molecular, supermolecular, and
colloidal levels. The stress gradient arising
during dough mixing and sheeting can deform
and orient flexible molecular chains, macro-
molecular aggregates, gas bubbles, and dis-
persed liquid drop let^.'^.^^ An increase in the
degree of orientation of protein chains can in-
tensify hydrophobic interactions and H-bond-
143
 ing between oriented polypeptide chains. En-
hanced hydrophobic interchain interactions may
form gluten aggregates with a hydrophobic
central core and hydrophilic side groups on the
surface. Because the hydrophobic core acts as
a solvent for lipids, mechanical treatment usu-
ally decreases the amount of lipids that are
extractable from the dough. This effect is simi-
lar to that of globular protein denaturation,
which enhances protein-lipid interactions and
encapsulated lipids. Lipids and flavors are usu-
ally bound more strongly by denatured globu-
lar proteins than by native ones. Mechanical
treatment also contributes to the chemical sta-
bility of lipids, due to the antioxidative activity
of gluten. The molecular encapsulation of lip-
ids within the hydrophobic core of the gluten
aggregates and crumb encapsulationby the crust
of the loaf seem to be responsible for the less
pronounced rancidity of baked products than
that of most other foods. Taking into account
the previously mentioned ability of lipids to
form honeycomb-like structures between im-
miscible aqueous phases (Figure 15), it can be
assumed that similar layered lipid structures
form between the phases of chemically differ-
ent insoluble food fibers. This adsorption of
lipids within the bulk of the insoluble fibers
favor the reduced mobility, dispersibility, and
absorption of lipids.18J9This effect could un-
derlie the well-documented effect of food fi-
bers on the digestion, absorption, and subse-
quent metabolism of lipids and protein^.^^"'^^
Deformation and orientation of dispersed
particles can form an anisotropiccapillary dough
structure, presumably by the mechanism of
spinneretless spinning (Figure 14).33,7L-73,84.*9,L'2-
116,260-266 The continuous gluten phase between
oriented capillaries therefore could consist of
noncylindrical strips. Shear conditions affect the
size of these gluten network elements. Because
the liquid particles deformed in flowing dough
are not stable, a dynamic equilibrium between
droplet deformation,break down of liquid cylin-
ders, and droplet coalescence may establish it-
self (Figure 14). The dynamic equilibrium can
be established during kneading, and fixed by
144
thixotropic gluten gelation at rest. Slow dough
mixing can shift the equilibrium toward coales-
cence of droplets and increase the heterogeneity
of porous structures. The liquid phase can be
squeezed from the capillary structures onto the
surface of the dough during mixing and sheeting
and affect dough stickiness. High-energy mix-
ing can shift the equilibrium toward smaller
dispersed particles and more homogeneous pore
structures. The reinforcement of gluten strips
due to orientation of gluten macromolecules in
an intensive shear flow normally leads to a de-
crease in gluten network thixotropy and reduces
loaf volume. An elastic deformation of the cap-
illary dough structures oriented in flow can cause
its anisotropic shrinking. Destabilization of glu-
ten networks may be due to the hydration of
non-polar groups. An intensive mixing and heat-
ing can also cause the collapse of overmixed
~~~g~s~20,112-114,117
The liquid phase particles and starch gran-
ules act as a lubricant in doughs (Figures 16
and 21). This lubrication effect is due to the
previously mentioned ball bearing effect.
Starchy polysaccharide-proteinincompatibility
corresponds to a negative mutual affinity be-
tween starch granules and gluten protein phase.
However, because of biopolymer incompatibil-
ity, the interfacial layers around starch gran-
ules can adsorb other components of flour or
dough (e.g., specific proteins and/or lipids de-
pending on cultivars) and stick to the gluten
phase. This adsorption increases the interac-
tion between immiscible aqueous phases (Fig-
ure 14). The mechanism of lipid adsorption
seems to be similar to the formation of honey-
comb-like structures (Figure 15). Weak inter-
actions between the granules and the gluten
phase are ruptured when the shear stress in-
creases and exceeds the friction force. The
dough becomes fluidized above a certain criti-
cal shear rate. When the shear stress decreases
sufficiently, sticking is restored again. Figure
21 illustrates the principle underlying the ball
bearing effect. The revolving of starch gran-
ules, which creates the ball bearing effect, de-
creases the friction between adjacent gluten
 Thixotropy and high fluidity of a dough
Ball-bearing effect am’
Homogeneous structure and thixotropy of the bulk of a dough
Rolling-pin effect
! Gluten strips i
“Starch-empty’’ surface layers and a “starch-full” central layer
FIGURE 21. Schematic representationof microrheoiogical effects governing structural and physical
features of wheat flour doughs. (From Refs. 17 and 20.)
layers flowing at different rates. The ball bear-
ing effe~t’~<*Owould explain why dough fluid-
ity is so unexpectedly high for such highly
concentrated dispersions. The ball bearing ef-
fect may, as a consequence, prevent the forma-
tion and propagation of cracks in the gluten
matrix during bread and pasta drying. Thus,
starch granule dynamics, and the stick-slip ra-
tio, are changed by both shearing and adsorp-
tion of lipids and/or proteins on the surface of
the granules. The slipping-sticking behavior of
starch granules affects the thixotropy of wheat
flour doughs and the fat-like texture of starch
based fat replacers and cosmetic powders.
In a flowing dough the difference in rela-
tive velocities between gluten layers on both
sides of each starch granule corresponds to a
difference in pressure. This pressure varies
according to Bemouilli’sprinciple, which states
that when the flow velocity is greatest, the
pressure is least. This principle results in the
migration of revolving starch granules toward
the central layers of a flowing d o ~ g h . ‘ An
~,~~
Revolution of starch granules
decreases the friction between
adjacent giuted layers
undergoing a shear flow
Revolving starch granules are
“roliing-pins“ for r o l l i out the
structural elements of the gluten
network
R towards faster layers, e.g. a faster
increase in the concentration of starch granules
implies a decrease in water content in the cen-
tral layer of extruded dough and can be inter-
preted as an “extrusion drying” Be-
cause of the relatively large size and volume
fraction of starch granules compared with those
of gluten strips, starch granules roll out the
latter. This “rolling-pin effect” is important to
the formation of three-dimensional homoge-
neous dough structures during mechanical treat-
ment.” The rolling-pin effect also underlies the
homogeneous aeration, gas retention, and thixo-
tropic solidification of the dough. Thus, me-
chanical handling is an important aspect of the
design and overall relationship of the structure
properties of doughs and fimal baked products.
An important rheological feature of many
baked foods is caused by their composite na-
ture. The attractiveness of many layered com-
posite foods, such as pizza, cakes, waffle-based
chocolate bars, baked and fried potatoes, pre-
sumably results from the spreading of flavored
layers during chewing in the mouth. The spread-
145
ing increases the surface area of the contacts
between flavored layers and the taste buds on
the tongue surface. The spreading reflects the
difference in mechanical properties between
the layers, where the stronger layer acts as a
“knife”. When the effort created by this knife
exceeds the yield stress (or the elastic limit,
beyond which the solid is no longer elastic),
the coverage layer should change into the liq-
uid state, that is, melts under pressure. As two
kinds of plastic deformation are typical of
spreading: (1) the elongation flow (within the
spread layer along the knife) and (2) shear flow
(within the spread layer between the knife
and the surface), the yield stress and thermo-
mechanical properties are the main textural
characteristics of baked and fried composite
foods. Flavor encapsulation between extended
rigid layers (nonpenetrable to flavor), their
gradual destruction and flavor release during
mastication are key factors contributing to the
palatability of layered composite foods.
D. Some Aspects of Dough
Formulation
According to Le Chatelier’s principle, as
mentioned previously, an increase in the con-
centration of food macromolecules stimulates
their aggregation, gelation, crystallization and
adsorption to interfaces. The rate of these pro-
cesses can, however, be reduced because an
increase in the concentration of macromolecules
usually increases the viscosity of the system.
The relative role of the thermodynamic and
kinetic factors depends on the dough composi-
tion and the time-temperature and shearing
conditions of the dough making. For instance,
several factors underlie the slow hydrolysis of
starch in pasta products, which are considered
as a source of slow-released carbohydrates. The
first factor is the increased crystallization rate
of starch (an increased amount of resistant
starch) in cooked pasta due both to its higher
concentration compared with that of bread and
to a memory effect of gluten networks. The
146
second factor is the partitioning feature of amy-
lolytic enzymes in pasta products because the
continuous gluten phase, which encapsulated
starch granules, is a good solvent for proteases
but a poor solvent for amylases. The third fac-
tor is the high excluded volume in the gluten
gel phase due to the chemical nature of gluten
gels. The latter may be a reason for the slow
hydrolysis rates of unfolded structure proteins,
such as gluten and casein. However, the first
factor, that is, an increase in concentration of
incompatible macromolecules under conditions
favoring the crystallization of some of them, is
the most important because it explains many
other experimental observations. For instance,
it was found that the content of the enzyme-
resistant starch increases in pasta dried at high
and ultrahigh temperatures compared with that
of products dried under low tern pera tu re^.^^^
According to excluded volume effects, both
the soluble and insoluble polysaccharides added
to dough increase the efficient concentration of
the soluble proteins and thus improve crumb
texture, porosity, oven spring, crust color, bread
break, and shred properties and loaf vol-
A reduction in the rate of
ume.249~250~255~256~267-270
metabolism of starch (i.e., an increase in the
amount of resistant starch) could be achieved
by adding an incompatible polysaccharide, for
example, guar gum, cereal brans and dough
heating. These effects are well d0c~mented.l~~-
199,265-275 Guar gum and other food fiber are used
for producing wheat bread for d i a b e t i ~ s .For
~~~-~~
instance, it was shown that in guar-containing
wheat bread, guar gum acts as a physical bar-
rier to starch digestion.278It was also shown
that guar gum is thermodynamically incompat-
ible with amylopectin in aqueous media.198J99
Based on these observations, it was possible to
conclude21 that the addition of guar gum to
wheat flour dough provokes phase separation
in the liquid dough phase containing soluble
starch from damaged starch granules. Heating
concentrated aqueous starch-guar gum mixtures
during baking results in the gelatinization of
starch granules, phase separation, and encapsu-
lation of starchy phase by the guar gum-en-
riched phase. This can favor crystallization of
the starch and the formation of resistant starch
particles encapsulated by dense layers of the
guar gum-enrichedphase. The net result of these
modifications explains therapeutic efficiency of
guar gum supplement, that is, a reduction in the
digestion rate of the wheat bread starch to a level
required for people with diabetes.l9Z1
The idea of the thermodynamic similarity
between milk protein-polysaccharide mixtures
and wheat flour doughs helps to understand the
wide application of milk ingredients as the
means to improve many dough functional prop-
Whey and skimmed milk have been
used in bread making since antiq~ity.**~ An
opposite approach, that is, applications of dough
ingredients to improve dairy food functional-
ity, has perhaps a greater potentiaL20Aqueous
extracts of many seeds have been processed
into milk analogues (vegetable milk products)
from time immemorial. Aqueous extracts of
wheat flour, which have good foaming, emul-
sifying, gel-forming properties, and are com-
patible with milk proteins, would find a num-
ber of applications. For instance, such
components would be thickening and structure
stabilizing agents for ice cream, frozen yogurt,
and “light food” formulations,replacers of egg-
white in whipped foods, including cakes and
biscuits, for edible films, etc. It should be noted
that similar textural properties of doughs and
ice cream mixes reflect their structural similar-
ity. These are simultaneously thixotropic emul-
sions, suspensions, and foams. As discussed
previously, the structural features of these com-
posite materials are the consequence of altering
thermodynamic conditions during processing.
Products and processes that may at first appear
to be entirely different are driven by the same
underlying principle. The increase in concen-
tration promotes the gelation of the continuous
protein phase in the final products. Dewatering
the wheat gluten phase is the result of starch
gelatinization during baking, while the separa-
tion of water is achieved by freezing, which
leads to the formation of the Ca-caseinate gel
of ice creams.
Another application of the thermody-
namic similarity between milk protein-
polysaccharide mixtures and dough is that
exopolysaccharides and cell wall polysac-
charides of lactic acid bacteria control the
texture of yogurt and other dairy products
and may also contribute to the texture of
non-yeast sour doughs and breads. These
functions of polysaccharides as dough im-
provers could be due to their ability to com-
plex with grain proteins at acid pH. It should
be stressed that the structure and rheologi-
cal properties of yogurt obviously do not
only depend on its pH, but also on the con-
centration of exopolysaccharides, their in-
teractions (incompatibility and complexing)
with milk proteins, and the degree of pro-
teolysis. For instance, it was shown that the
exopolysaccharide xanthan (0.05 to O. 1%)
decreases the solubility of gluten in acid
media and has a strengthening effect on both
gluten and the dough. This aspect is prob-
ably due to complexing with the proteins.275
It can be assumed that antifreezing activity
would be biologically necessary for some
exopolysaccharides and exocellular
protein-polysaccharide complexes, thus con-
trolling the cellular microenvironment. Such
antifreezing defense of the cell could be
based on ice nucleating and antifreezing
agents. These exocellular biopolymers would
be of interest for controlling the texture of
ice creams, frozen yogurts, and some other
frozen foods.253,254.286-289
Other applications of the milk protein-
polysaccharide-water system would be model-
ing of: (1) the structure of chyme, ( 2 )the mecha-
nisms of food digestion, and (3) cereal-milk
food compositions. Much as the hydrophobic
aggregation of casein particles within an
exopolysaccharide-rich phase forms thixotro-
pic gel networks in yogurt and the hydrophobic
aggregation of gluten phase particles forms
dough networks, the dispersed particles of de-
natured proteins, and protein-polysaccharideco-
precipitates tend to form the gel-like properties
of intestinal chyme.
147
E. Rye Flour Dough: Sour Dough
Rye sourdough produces bread with a rela-
tively low loaf volume, but is important for
quality improvement of crust, shelf life and fla-
vor of mixed rye-wheat breads, fabrication of
edible films, and containers. The content of
nonstarch polysaccharides is the main differ-
ence between wheat flour and rye flour. De-
pending on cultivars and the harvest year, the
content of rye pentosan varies from 6.5 to 10.5%,
of which 16 to 25% is water soluble. In contrast
to wheat flour, rye flour contains from 6 to 11%
proteins, 35% of which are water dispersible.
Soured rye dough contains up to 85% proteins
dispersible in The information about
rye polysaccharides and proteins,268,z90-295 is sig-
nificantly richer than that about their interac-
tions in sour dough. For instance, it was shown
that the digestibility of rye proteins is increased
considerably by pretreating grains with
p e n t o ~ a n a s eIt. ~was
~ ~ also shown that rye pro-
teins are precipitated from alkaline extracts after
the addition of a polyanion and an acid to adjust
the pH to 3-5.”6 It was found by Schneider et
al.296-298 that rye pentosans are incompatible with
some globular proteins (bovine serum albumin,
ß-lactoglobulin, and ovalbumin) at their IEP,
while below the IEP rye pentosans form electro-
static complexes with the same individual pro-
teins and with wheat gliadin. It was also found
that electrostatic protein-polysaccharide
complexing can greatly decrease the conforma-
tional stability of globular proteins, makes their
denaturation irreversible, and presumably con-
verts globular proteins into unfolded (gluten-
l i e ) proteins.11,13,36.44.53 Protein-polysaccharide
complexes can form gels under conditions where
their macromolecular components taken indi-
vidually do not ge1.35,48-51
Based on these observations, it is possible
to assume that the mechanism of rye dough
making is based on the formation and gelation
of protein-polysaccharide complexes. These
complexes are formed in the acid medium and
are probably incompatible with other macro-
molecular dough components. The presence of
148
the latter reduces the solubility and the gelation
threshold of the interbiopolymer complexes and
the degree of swelling of their gel-like phase.
These trends are due to the excluded volume
effects of macromolecules and elastoosmotic
interactions between the structural elements of
gel network and the dispersion medium. In
other words, the mechanisms underlying the
formation of rye dough seem to be similar to
those of wheat flour dough. Actually, as is true
for the gluten phase network, the formation of
a gel network in rye dough could be due to the
hydrophobic aggregation of individual particles
of insoluble interbiopolymer complex within
the unwettable liquid phase. This aggregation
and the gelation of insoluble complex particles
minimize contacts with the unwettable medium
and lead to the second continuous phase of
protein-polysaccharide complex in the rye
dough. However, unlike protein continuous
phases of wheat dough, protein-polysaccharide
complexes in rye sourdoughs can have a higher
affinity for insoluble polysaccharides. The gel
network of rye dough therefore may be filled
and reinforced by several types of dispersed
particles, such as starch granules, insoluble
pentosanes, and other food fibers. This means
that an additional difference between wheat
and rye doughs is that the gluten phase contains
protein-proteincomplexes and co-soluble mac-
romolecules whereas the rye gel network is
formed by protein-polysaccharide complexes,
co-soluble macromolecules and dispersed par-
ticles as active (reinforcing) filler. Both doughs
have sufficiently high gelation thresholds, be-
low which their heterogeneous networks are
converted into liquid dispersions, for example,
pancake doughs or batters. It should also be
noted that high-molecular-weight,good hydra-
tion properties and oxidative gelation of
polysaccharide components could also be im-
portant for composition-property relationships
in rye doughs. Exopolysaccharides of lactic
acid bacteria in traditional sour doughs pre-
sumably reduce the solubility and enhance the
gelation of interbiopolymer protein-polysaccha-
ride complexes in acid pHs. Because there is,
however, no information on thermodynamic
compatibility between exopolysaccharides with
soluble non-starchy flour polysaccharides or
on exopolysaccharide-rye protein complexing,
any discussion of the exopolysaccharide func-
tions in the formation of dough structures must
be speculative. However, the assumptions men-
tioned agree with the experimental data on rye
dough making. The assumed thermodynamic
and structural similarity of wheat and rye doughs
is proposed to underly the functional efficiency
of rye flour and soluble rye polysaccharides as
improvers of wheat d o ~ g h s . ~ The ~ ~ -high
*~~
dispersibility of interbiopolymer complexes in
aqueous media?15.34,37,38,53 presumably deter-
mines the remarkably higher dispersibility of
rye flour and rye dough components compared
with those of wheat flour doughs. It has been
shown that modified highly expanded starch
granules together with dispersed particles of
endosperm walls participate in the formation of
the continuous (protein-polysaccharide) phase
of the rye dough n e t w ~ r k . ~ ~ - ~ O ~
F. Glassy Foods and Their
Rehydration: Thermoplastic
Extrudates, Edible Films
Generally, if two synthetic polymers are
not mixible in dilute solution, they are also
incompatible in bulk.77,304 Biopolymers, espe-
cially globular proteins, usually exhibit higher
co-solubility than polymers. 19,24,30,31,105.106,I10
However, with an increase in their concentra-
tion in common solvents the phase behaviors
of biopolymer mixtures come closer to those of
synthetic polymers. In other words, as poly-
mers biopolymer incompatibility in solutions
presumably implies the incompatibility of a
given biopolymer pair in concentrated systems
at low moistures.11,33J13J14 Normally, two (or
muitiple) glass transitions of a macromolecular
mixture can be regarded as a criterion of their
in~ompatibility.3~J~~-~**
Due to the high excluded volume effects of
anionic polysaccharides, their small addition to
pasta dough provides an additional thermostable
gel network.15.20 For instance, it was shown that
a small amount of an anionic polysaccharide
added to pasta dough based on wheat flour
remarkably improved the product q ~ a l i t y . ~ ~ ~ - ~ ~
Both demixing of biopolymers and the sub-
sequent partitioning of other components be-
tween the biopolymer phases decrease the prob-
ability that they will interact chemical during
heating, for example, the Maillard reaction.
The intensive mechanical mixing of the macro-
molecular components and their mechano-
chemical destruction during thermoplastic ex-
trusion provoke the Maillard reaction and
produce the colored extrudates. On the con-
trary, in the absence of mechanical mixing, the
incompatibility of these biopolymers (i.e., the
absence of miscibility on a molecular scale)
was presumably the reason that heating bovine
serum albumin-starch and gluten-starch model
mixtures at 23% water level did not show (DSC
within the temperature range from 20°C to
180°C) any evidence of interactions between
components.308Accordingly, a so-called “dry
heating” method is used to synthesize protein-
polysaccharide c o n j ~ g a t e s . ~ O
An~ -optimum
~~~
temperature of heating the biopolymer mix-
tures prepared by lyophilization of their dilute
mixed solutions, providing protein-polysaccha-
ride conjugates,presumably corresponds to the
interval between the denaturation and the glass
transition temperatures of the protein.l3J4JI8
Both the denaturation and the glass transition
temperatures of a protein are constant within a
wide concentration range of its dilute and
semiconcentrated systems. Both temperatures
rise with the decrease in water content below
30%. The latter value is usually regarded as the
level of complete primary hydration of the pro-
tein. An increase in the stability of proteins in
low-moisture systems is possibly due to the
glassy state of the polypeptide segments and a
decrease in the amount of piacticizer, water.19J8G’88
As a result, the dry heating method used to
synthesize protein-polysaccharide conjugates
provides the molecular dynamics and preserves
the conformational stability of reagents.I3
149
 An interesting aspect of phase separation in
low-moisture foods is the thermodynamic com-
patibility of different polypeptide chains of the
same protein. Most food proteins, for example,
the 1IS and the 7s storage globulins of legume
seeds and oil seeds, are oligomeric heteroge-
neous-type proteins. Their molecules consist of
several (usually 12 and 3) polypeptide chains
differing in the composition and molecular
weight. The polypeptide chains constituting a
protein molecule form structuraldomains of ther-
modynamically equivalent stability. This means
that they are characterized by the same values of
the temperature, enthalpy, increment of specific
heat, and free energy of d e n a t u r a t i ~ n . ~ ~ , ~processing
However,after thermal denaturationthese chains
are not compatible in solution and plasticized
melt.'1,33J14Their incompatibility and self-asso-
ciation are important for both the critical con-
centration of the protein gelation and the forma-
tion of anisotropic fibrous structures of
thermoplastic extrudates." Unlike polypeptide
chains of oligomeric seed storage proteins, those
of gluten seem to be compatible due to lower
hydrophilicity and contributions of disulfide and
coordinative interchain bonds.
Both molecular and supermolecular struc-
tures affect the overall properties of extrudates.
Deformation of dispersed phase particles in
flow leads to fibrous (fibrilla) and lamella struc-
tures due to the mechanism of spinneretless
spinning (Figure 14). Phase diagram asymme-
try is important for phase inversion and the
structure-property relationships in foods pro-
duced by thermoplastic extrusion, drying, and
solidification. It was shown that the expansion
volume of a cereal starch jet leaving the extru-
sion die (which depends on the barrel and the
nozzle temperatures, extrusion rate, other ex-
trusion conditions, moisture level, and amylose
content) goes through a maximum in the vicin-
ity of 50% amylose content.318These results
could reflect the incompatibility of amylose
and amylopectin178and the phase inversion of
the extruded system.33 In low-moisture food
systems, separation of the phases is counteract-
ing by their high viscosity. Phase diagram asym-
150
metry and the phase volume ratio govern the
porosity of thermoplastic extrudates and dry
foods in particular. Generally, a higher water
content of the dispersed phase results in porous
dry products. On the contrary, a lower water
content of dispersed phase leads to dry materi-
ais with increased density.
In the shear and elongation flow (e.g., on
taking up the extrudate) long polysaccharide
chains can be stretched and oriented. Presum-
ably, unlike polysaccharides, globular proteins
cannot be unfolded by shear forces under the
flow conditions of thermoplastic extrusion,
which are likely to be the most severe of food
~ ~ J ~ ~ technologies. Globular proteins can
unfold and align in flow only in a special sol-
vent, such as aqueous urea and sodium
dode~ylsulfate.~~~ Unfolding and orientation of
polysaccharide chains and low-rate relaxation
processes in highly viscous polysaccharide-
enriched phases contribute to the nonequilib-
rium nature of extruded foods.11.33.112-1L4,259-262
The deformation/relaxationand the solidifica-
tiodrelaxation rate ratios of the extrudatephases
during extrusion affect the internal stresses,
propagation of cracks, porosity, and densities
of the products. The ratio between the extru-
sion temperature and the temperature of the
glass transition of the phases, the rate of drying
and cooling, influence the bulk structure of
extruded and dried food materials.33J12-114,259-262
It should be noted that glassy biopolymer
components are not only limited by low mois-
ture and frozen foods. The high concentration of
the chain segments in native and denatured pro-
tein molecules and the macromolecular associ-
ates forming biopolymer gels determine the
glassy state of structural elements of most
foods.186The glassy state of biopolymer gel
networks, low moisture, and frozen foods deter-
mine their memory effects relative the history of
preparation con dit ion^.'^.^^.^^ For instance, the
memory effects include accumulation of me-
chanical energy by drying under loading and
during extrusion depending on the extrusion-
cooling-drying conditions (Figure 22) and swell-
ing behavior of the dried gels.Is6Memory of dry
 \ll
Sha
elas%c
inFood
avisco-
material
Tg < T < Tm
FIGURE 22. Schematic representation of memory effects of low-moisture foods.
Accumulation of mechanical energy and self-stirring effects due to viscoelastic recov-
ery during rehydration. (From Ref. 186.)
gels can be illustrated by the fact that the de-
gree of swelling of dry gelatin powders pre-
pared by drying the gels of different initial
concentrations is inversely proportional to the
initial concentration of the gel before drying. In
other words, the maximum degree of swelling
is shown by the powder obtained from the gel
with the lowest concentration. Memory effects
can also be illustrated by the dependence of
wetting behavior of gels on the medium in
which the gel was formed.I9 Figure 22 shows
that glassy structural elements of foods pre-
serve the mechanical energy accumulated dur-
ing deformation and its release above the glass
transition temperature. For instance, cooling a
loaded gel or liquid viscoelastic food system
results in its vitrification and an accumulation
of mechanical energy, which can be released
(elastic recovery) due to heating and/or addi-
tion of a plasticizer, water. Memory effects are
important for rehydration of instant foods and
drinks, the swelling capacity (swelling behav-
ior) of dry particles, agglomeration, and the
preservation or changing the initial shape, in-
cluding self-stimng effects during food rehy-
dration and The phase state, the
composition, and the glass transition tempera-
ture of the phases are the factors affecting the
rehydration of dry foods and macromolecular
Viscoelastic Recover
Rehydration
(hot water)
T g < T < Tm
food ingredients. The diffusion rates of water,
air, and other gases through a film depend on
the composition, the volume fraction ratio, and
the difference in properties between its con-
tinuous and dispersed phases. Therefore, the
biphasic nature of food films (e.g., casing ma-
terials and moisture barriers), packaging mate-
rials, and three-dimensional foods opens the
way to controlling permeability, mechanical
properties, and rehydration behavior of low-
moisture and dried foods.lI~33~~12-~14.186-188
VIII. PHASE SEPARATION IN
BEVERAGES
Foods and beverages differ greatly in their
composition and the phase behavior of their
macromolecular components. The main rea-
sons underlying this diversity are the low vis-
cosity of beverages and the water dispersibility
of macromolecular components, their associ-
ates, aggregates, and complexes. Generally,
macromolecular components of drinks cover
proteins, polypeptides, polysaccharides,
polyphenols, complexes of these macromol-
ecules and products of their cross-linking,ther-
mai decomposition, and chemical modification.
For instance, polysaccharides and phenolic
151
compounds affect the rheological properties,
“body” and mouthfeel of many beverages.320 tion of ions, surfactants, lipids, and flavors by
These macromolecules contribute to the astrin-
gency of coffee, tea (green and black), and to
the clarity and color of red wines and l i q u o ~ s ? ~ ~ ~and
Besides processing conditions (e.g., drying,
roasting, infusion, and extraction), some en-
zymes are important to the formation of phe-
nolic and other polymers. The composition and
concentration of macromolecules are impor-
tant for the phase state, colloidal stability, vis-
cosity, turbidity, and flavor of most beverages
and liquid foods. Phase separation can occur
both during processing and storage. Phase sepa-
ration induced by interpolymer complexing,
for example, the limited co-solubility of pro-
tein-polyphenol complexes, is the most fre-
quent cause of haze in beer, wine, and clear
fruit juices.323
Phase separation techniques based
on denaturation and coagulation of proteins,
interbiopolymer complexing, and incompatibil-
ity (bridging and depletion flocculation) are
widely used to reduce the cloudiness and im-
Formation of Tea Cream
Hot (%O°C)
Homogeneous
Infusion
Cooling
I
I
Phase separatiolfjj
Heatin g
FIGURE 23. Schematic representation of tea cream formation.
152
prove the stability of many drinks. An adsorp-
dispersed particles within the interfacial layers
results in a local increase in their concentration
* ~ enhances their perception. Flavor binding
by macromolecular components, partitioning
of individual flavors between co-existing phases
and interfacial layers, and their volatility from
the continuous phase are important to the qual-
ity of many be vera ge^.^^,'^,^^.^^
A. Tea
Formation of so-called “tea cream” is one
of the most studied examples of phase separa-
tion in beverages (Figure 23). A strong aque-
ous infusion of black tea leaf becomes turbid
on cooling. Phase separation of tea with the
formation of a dispersed phase, called tea cream,
results in a loss of transparency, and, typically,
changes in color and flavor. The formation of
tea cream is also a technologically negative
Cold (<40°C)
Biphasic Infusion
Water-in-Water Emulsion
Partitioning Flavours
between the Phases
 phenomenon hindering the application of many
techniques that could concentrate tea extracts,
for example, low-temperature freeze-drying and
membrane filtration. Factors influencing the
formation of tea cream include the concentra-
tion, pH, and time-temperature history of the
tea i n f ~ s i o n .The
~ ~majority
~ - ~ ~ ~of the tea cream
phase formed in a normal brew (the total solids
content 30 g.1-I) dissolves on heating above
40"C, whereas complete dissolving occurs only
at 90°C. Tea cream is not dissolved in clarified
tea Supernatant, but can be dissolved in water
or dilute tea infusion on heating.330The mor-
phology of tea cream depends on the concen-
tration of tea solids. Spherical liquid droplets
are formed in sufficiently concentrated (above
5% wt) infusions. An increase in tea concentra-
tion raises both the amount of the tea cream
and the rate of its formation. At a concentration
above 40%, phase inversion occurs. Both the
spherical form of the dispersed phase particles
and the phase inversion provide evidence of
the liquid-liquid phase separation,which makes
an equilibrium between the phases possible.
Black tea infusions have an upper critical tem-
perature, about 60°C, on cooling below which
phase separation The phase dia-
gram for the tea cream formation has been
determined by light scattering3;;
The creaming properties of teas differ
greatly depending on the content of the macro-
molecular components, first of all proteins and
phenolic polymers. Black tea can contain more
than 100 polyphenols formed mainly due to
oxidation and condensation processes.;% The
first data describing high-molecular-weight(up
to 40,000) polymer phenols were obtained by
Vuataz and B r a n d e n b ~ g e rMolecular
. ~ ~ ~ weight
and solubility of phenolic polymers are changed
due to the formation of complexes with other
components, for example, proteins, polysac-
charides, lipids, and metal ati ion s.^^^,^^^ The
kinetics of infusion of tea leaf is dependent on
the tea leaf/water ratio, the temperature, and
hydrodynamic ~ o n d i t i o n s . Caffeine
~ ~ ~ , ~ ~and
~ mainly polyphenol-protein complexes, while
epigallocatechin gallate are preferentially in the
cream phase.334The tea cream composition,
including the partitioning of polyphenols and
multivalent cations335between the tea phases,
is in agreement with the assumed dominant
role of the tea proteins in cream f ~ r m a t i o n . ~ ~ ~ . ~ ~ ~
The detailed chemical analysis of tea infu-
sion phases has been carried out by Chao and
Chia11g.3~'They showed that the semifermented
tea infusion (prepared at a 10% tea leaf/water
ratio, at 90°C and 20 min extraction) containing
3.81 g total soiids/100 ml breaks down on cool-
ing to 30°C into two phases: tea cream and
clarified infusion, with concentrations 0.57 and
3.24 g total solids/100 ml, respectively. The tea
cream phase contains 30% catechins, 20% caf-
feine, 16% protein, 2% pectin, and 4.98 ppm Ca
cations. The clarified infusion contains 40%
catechins, 2.8% pectin, 0.154% protein, 8.3%
caffeine, 5.14 ppm Fe, and 0.92 ppm Zn cations.
Aging leads to solidification of the separated tea
cream phase. Chao and Chiang337reported that
an increase in the tea leaf/water ratio from 1.25
to 15% increases the total solids in the infusions
and the amount of tea cream, but does not change
the concentration of each component in the tea
cream. These data are in good agreement with
the principle that tea creaming is a phase sepa-
ration caused by both interpolymer complexing
and limited co-solubility of these heterogeneous
complexes with one another and other macro-
molecules?' Pectin is the main polysaccharide
of leaves, while the most important green leaf
protein (about 25% of water-soluble proteins) is
ribulosediphosphate carboxylase, an oligomeric
protein, containing 16 subunits of two types.
Complexing with pectin decreases their confor-
mational stability.' l s 4 A different affinity of
nbulosediphosphate carboxylase and pectin to
polyphenols, lipids, and multivalent cations in-
creases the chemical difference between them
due to the formation of pectin-polyphenol and
protein-polyphenol complexes. The latter com-
plexes are presumably limitedly co-soluble and
concentrated in the different phases. The dis-
persed phase, that is, the tea cream, contains
pectin-polyphenol complexes are in the clarified
tea infusion phase. Both complexes are prob-
153
ably highly heterogeneous triple complexes:
pectin-Fe-polyphenols and protein-ca-polyphe-
nols stabilized by coordinative interactions with
cations, hydrogen bonds, and hydrophobic in-
teractions. Tea lipids presumably also form com-
plexes with proteins and polyphenolic compo-
nents of tea. A significantchemical difference of
the complexes determines the remarkable asym-
metry of the phase diagram of teas. The pectin-
Fe-polyphenolcomplexeshave a higher excluded
volume, hydrophilicity, and are more competi-
tive for water than the protein-Ca-polyphenol
complexes forming the dispersed tea cream
phase. In the latter, protein molecules can be
cross-linked by polyphenols by hydrogen bonds
and hydrophobic interactions that presumably
determine the reversibility of the phase separa-
tion on heating. Owing to the higher hydropho-
bicity of its various components, the tea cream
phase is a better solvent for flavors and caffeine.
These assumptions are in agreement with
experimental results, particularly those that
concern the applications of tea creams requir-
ing preconcentration of tea components338and
the methods proposed for overcoming cream-
ing in manufacturing soluble These
methods include: (1) separation of the phases
under cooling, their separate drying, and dry
mixing; (2) tea cream dissolving in the second
aqueous extract of leaves on heating;339
(3) drying at above 75°C; (4) separation and
oxidation of the tea cream phase to increase
solubility;340and (5) the addition of catechin
extracted from green tea leaves to improve the
solubility of the tea components in cold wa-
ter;341(6) the addition of O. l % beta-cyclodextrin
(which is a cream inhibitory agent) to tea infu-
; ~ (7)
s i o n ~and ~ an application of tannase, which
can be (8) used to treat green tea before fer-
~ ~(9)
m e n t a t i ~ nand ~ .immobilized
~~ to treat an
aqueous tea extract at 50 to 65°C.345Manufac-
turing stable tea extracts and instant teas with-
out forming tea cream on cooling and freeze
concentration is also possible when a sufficient
amount of milk or a caseinate solution is added
before and/or after cooling and tea creaming.349-351
The effect of casein is interpreted to be to
154
increase the compatibility of tea macromolecu-
lar components. It can be assumed that the
initial incompatibility reflects the great chemi-
cai difference between tea proteins, polysac-
charides, and phenolic polymers. Casein cov-
erage would make complex particles chemically
similar, that is, achieving molecular mimicry at
the colloidal level, presumably contributing to
an increase in co-solubility of the c~mplexes.~'
B. Coffee
Unlike macromolecular components of
green leaves and grasses, which usually have a
quite similar chemical composition because of
the same fundamental functions: photosynthe-
sis and transpiration, seeds differ markedly in
their chemical composition because germina-
tion yields greatly different plants. Neverthe-
less, the macromolecular ingredients of black
tea and coffee extracts, their content, heteroge-
neity, and phase behavior seem to have many
common features. Dry coffee beans contain ca.
40 to 65.5% carbohydrates, including 3 to 4%
soluble polysaccharides, 6.7 to 12.1% chloro-
genic acids, and 1 to 3% lignin, 7.9 to 12%
proteins, 25 to 40%of which are water-soluble
proteins, and 8 to 18% lipids.352A specific
feature of roasted cafe is a pronounced hetero-
geneity of macromolecular components. Cof-
fee proteins with the IEPs in the ranges 4.4 to
4.7 and 5.7 to 6.3 have a high proportion of
storage proteins, about 45% of which are legu-
min-like 11s g l o b ~ l i n . 3The
~ ~ -macroscopic
~~~
structure of coffee beans is the result of com-
plex processes and interactions that occur post-
harvest. Water vapor, carbon dioxide, and car-
bon monoxide formed at the roasting due to
pyrolysis and condensation are mainly respon-
sible for the puffing and porous structure of
roasted coffee beans. The glassy state of the
roasted porous beans encapsulatesand preserves
coffee aroma during storage. Grinding roasted
beans releases the aroma compounds from the
porous glassy matrix. Accordingly, coffee fla-
vor enhancers are produced by grinding fresh
roasted coffee beans heated at a temperature of
85 to 105”C, adsorbing the flavors on solid
adsorbents and extracting them into organic
solvent/water mixtures. The coffee lipids, for
example, produced from pressed roasted cof-
fee, are also used as a good solvent for coffee
aroma compounds. Volatile aroma compounds
(collected from grinding gas, steaming of
ground roasted coffee before extraction) can be
dispersed subsequently in the coffee oil and
~ ~ ~ , ~ ~60%
added to the final coffee p o ~ d e r .Depend-
ing on the roasting and extraction conditions,
soluble coffee contains variable amounts of
melanoidins, polysaccharides, phenolic poly-
mers, and their complexes. Macromolecular
components of soluble cafe include: about 15
to 35% melanoidins, 1 to 21%“proteins”, and
18 to 50% polysaccharide^.^^^-^^^
The chemistry of coffee bean roasting re-
mains poorly understood. The roasting results in
chemical modification of macromolecular com-
ponents due to pyrolysis, oxidation, depolymer-
ization, cross-linking, polymerization, polycon-
densation,the Maillard reaction, etc. The Maillard
reaction, with participation of free amino acids,
peptides, proteins, monosaccharides, sucrose,
polysaccharides, chlorogenic acid, and lipids, is
responsible for color, flavor, cross-linking of
proteins, and the formation of partially soluble
(acid-precipitated) me la no id in^.^^^,^^^ The mac-
romolecular products of the Maillard reaction
aiso include protein-polysaccharide conjugates
and modified polysaccharide~.3~~-~~
A iimited number of publications are devoted
to the transformations of coffee proteins, polysac-
charides, and phenolic polymers. Heterogeneous
high-molecular-weight galactomannans and
arabinogalactansare the main polysaccharides in
coffee extracts?“ Arabinogaiactomannans be-
tween 5000 to 50,000 molecular weight were
found in coffee brews.367In the hot water extracts
of dark roasted Coffea arabica,two fractions with
10,900Da and 270,000 Da were An
important role of the high-molecular-weight
polysaccharide fractions (about 70,000 Da and
2000 kDa) was also shown in the foam stability
of espresso coffeem Phenolic polymers can be
formed by thermal decompositionof chlorogenic,
caffeic, and other polyphenolic a ~ i d s .Phenolic
3~~
polymers form complexes and condensationprod-
ucts with polysaccharides, proteins, and with
multivalent cations. The hydrophobicity and sur-
face activity of modified polysaccharides are most
responsible for the foaming of both coffee and
tea.
The extract used for manufacturing instant
coffee is typically concentrated up to 35 to
~ wt solids by either water evaporation or
freezing and separation of ice crystals.362These
highly concentrated coffee liquors containing
chemically and physically heterogeneous mix-
ture of macromolecular components obviously
cannot be thermodynamically stable. However,
unlike in tea cream formation, the phase sepa-
ration of coffee extracts and coffee liquors re-
mains practically unstudied. Phase separation
in concentrated coffee extracts on cooling was
only considered in specific patents, whose ob-
jectives were (1) freeze concentration of coffee
extracts; (2) manufacturing coffee concentrates,
which can be stored at room temperature; and
(3) fortificationof coffee with bioavailable iron.
Freeze concentration of a coffee extract re-
quires that it is stable on cooling. Therefore, it
was proposed to cool a Concentrated coffee
extract up to 20 to 45°C to precipitate tars and
waxes and remove them by filtration prior to
freeze concentration, to be able to separate the
concentrate from the ice by c e n t r i f ~ g a t i o n . 3 ~ ~ ~ ~ ~ ~
A similar method was proposed for producing
liquid coffee concentrates that can be stored.
The dispersed particles and bacteria are re-
moved by filtering the concentrate before bot-
tling.373
The fist unique patent on the phase separa-
tion of coffee liquor was aimed at the problem of
iron fortificationof c0ffee.3’~Because iron-defi-
cient anemia takes place in some developing
countries, such as Costa Rica, where coffee is
widely consumed, iron fortification of coffee
was of practical importance. The direct addition
of iron compounds to coffee is impossible be-
cause phenolic polymers and polyphenolic acids
as multidentate ligands form insoluble chelate
155
complexes with iron ions. Actually, a more re-
cent study showed that polymeric phenols are
responsible for formating metal (iron and zinc)
coordination complexes in instant coffee.375
Therefore, phase separation of coffee liquor on
cooling was used to separate phenolcontaining
fractions374and to avoid precipitation of added
iron. The authors found that polyhydroxyphenols
and polyhydroxyphenol-polysaccharide com-
plexes are precipitated when liquid coffee ex-
tracts with concentrations above 15 wt.% are
cooled below 35°C. After separating the pre-
cipitated material, an assimilable Fe compound
can be added to the extract before drying. The
efficiency of this technique374in isolating phe-
nolic compounds proves that coffee extracts
break down into two phases on cooling and that
macromolecular components are concentrated
in the different phases and have a very low co-
solubility. Like black tea infusions, a coffee
extract has an upper critical point. Figure 24
illustrates the phase separation of coffee extracts
on cooling. Polysaccharides, including polysac-
charides modified by the Maillard reaction, can
form the continuous phase of coffee extracts due
to the higher co-solubility with each other than
with polymeric phenols. The stability of coffee
extracts after adding iron means a complete sepa-
ration of phenolic ingredients into the dispersed
phase. Phenolic polymers and polysaccharides
chemically modified by polyphenolic acids (of
low and high molecular weights) can be concen-
trated in the dispersed phase of coffee brew. A
higher concentration, density, and viscosity of
the dispersed phase and a relatively lower vis-
cous dispersion medium favor the separation of
the coffee extract phases by centrifugation. Like
in the case of tea, the content of phenolic acids,
iron, and other polyvalent cations, which may
change with the coffee cultivar and growing
conditions, thus could affect the phase behavior
and viscosity of coffee liquor.
Phase separation of coffee extract on cool-
ing is more important than previously be-
l i e ~ e to ~ ~quality of soluble coffee. An
d ~the
important role of phase separation in coffee

Coffee Extract
as Biphasic Polymer System

Phase separation Encapsulation

Water-in-Water Emulsion

Hot ( > 600C) BIPHASIC

Soluble Coffee
Coffee Extract Coffee Extract
Powder
(>lo% wt) Cold (< 4OOC)
FIGURE 24. Phase separation of coffee liquor. Schematic representation of soluble coffee powder as a biphasic
system.

156
 manufacturing is evidenced by the well-known
difference in flavor quality and intensity of
flavor between a soluble coffee and a classic
freshly prepared expresso or a roasted coffee
during milling. This great difference in flavor
is preserved even when the original quality of
coffee beans, their roasting, and storage condi-
tions are changed. This shows (Figure 24) that
the flavor of instant coffee is not a chemical but
physicochemical problem. Actually, the dis-
persed phase containing phenolic compounds
could be a better solvent for coffee flavors. The
drying of concentrated phase separated liquors
is accompanied by nonequivalent losses of fla-
vors from both phases. The phase separation
and nonuniform partitioning of flavor compo-
nents between the dispersed and the continuous
coffee phases can decrease the flavor intensity
and change the flavor profile. Because poly-
mers, which are incompatible in solution, are
also incompatible in a melt, dry coffee powders
are biphasic in nature (Figure 24). This phase
separated nonequilibrium nature and glassy state
of dry foods and drinks are important for rehy-
dration, swelling, and dissolving of dehydrated
par tic le^.'^,^^ A marked difference between the
chemical compositions of the phases corre-
sponds to a different rate of their swelling,
dissolution in water, and their different affinity
for flavors. The phase separationtechnique used
for isolating the phenolic components is impor-
tant for improving coffee drying technology
and the quality Characteristics of soluble coffees.
For instance, this can include a decrease in
viscosity and an increase in concentration and
stability of coffee liquors before drying. The
separation of phenolic coffee components would
control pH, which is an important parameter of
As the functions of polyphe-
the final drink.376*377
nolic acids in seeds presumably include de-
fense against digestive enzymes, viruses, and
infections, the phenol-rich phase isolated from
coffee liquors could find various novel applica-
tions. This phenolic material and its fractions
could be used for extracting coffee flavors, for
coating packaging materials used for perish-
able foods, for food designed for weight con-
trol, for new cross-linking agents for food
macromolecules to control the permeability and
texture of food membranes, and finally for
encapsulating nutrients for functional foods,
drinks, etc.
IX. CONCLUSION
Although pursued for many thousands of
years, the search and development of formulated
foods is still in the bloom of its youth. Several
thousand food products and beverages are devel-
oped every year. Comparable dynamism is only
typical of some other areas of the arts, in music
and Ijterature. There is an increasing interest in
functional and instant palatable foods with con-
trollable compositions. Because phase state, stnic-
mal, and physical properties of conventional and
novel foods are determined mainly by the protein
and polysaccharide components, food formula-
tion and processing require attention to the physi-
cal chemistry of food macromolecules in order to
control their functionality in biological systems,
in food systems, and in chyme.
Due to molecular mimicry, native globular
proteins as food components (e.g., milk whey,
egg white, seed storage proteins) are signifi-
cantly co-soluble with other ingredients, add-
ing minimal contributions to the viscosity of
food systems and maximum reproducibility in
their properties. Physical and chemical modifi-
cations of proteins and polysaccharides during
food processing usually reduce molecular mim-
icry and the co-solubility of macromolecules.
Phase separation therefore is more typical of
foods than biological systems and are impor-
tant for designing specific food structures. The
main advantages of long-chain charged polysac-
charides are the low-phase separation thresh-
old with proteins, low critical concentration
needed for gelation, high viscosity, and
complexing ability with proteins. Symbiosis in
mixtures of globular proteins, polysaccharides,
and protein-polysaccharide mixtures, which is
usually called functional “synergy”, is widely
used in the food indusiry.
157
 The main conclusion of this review is that
nonspecific macromolecular interactions con-
tribute greatly to their functionality and the
processes of structure formation in biological,
food systems, and beverages. Information on
the phase behavior of biopolymer mixtures,
however, remains limited. With greater under-
standing of these relationships, the rational
design of formulated foods will accelerate.
ACKNOWLEDGMENTS
The author is indebted to Professor Dr.
Bruce German and Dr. Robert Redgwell for all
their help in preparing the manuscript.
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