Characterization of the Effect of Solutes on the Water-Binding
and Gel Strength Properties of Carrageenan
D. K. REY and T. P. LABUZA
-ABSTRACT
The water activity of carrageenan gels with incorporated solutes
was determined by Inyoscopic osmometry. Kappa (K), kappa-
lambda (Kh) and iota (!) carrageenan
were utilized at concentrations
of from O.l-2% solids producinga sol, viscous sol, andgel. The sol-
utes used were sucrose, NaCl, Na2S04, KCl, NH&l and urea at
concentrations ranging from 0.05-1.0 kinetic units per kg H,O.
AU the solutes were fcund to give a, values as measured cryoscop-
ically within 0.0005 u:rits of literature values.The water activity of
pure carrageenans at (1.1-2s solids was greater than 0.999. Addi-
tion of scme solutesg creasedthe water binding of the carrageenan-
solute-water system as was found by a lower a, than expected. This
interaction effect incn:ased with increasing concentration of solute
and carrageenanand was greater for solutes with lower activity
coefficients.The intenlctidn effect increasedin the following order:
Na#04, NaCl, KCl, a Id NH4 Cl. The force required to rupture the
gel was measured using: the Instron Universal Testing Machine. Sol-
utes were found to inlluence the gel strength of K and oh carragee-
nans,but not L carragcenan.The solutesincreasedoh carrageenan’s
gel strength in incmtsing order: urea, sucrose, NazS04, NaCl,
NH&l, and KC1 and K carrageenan’sgel strength in increasing order:
sucrose,Na,S04 and :gaCl. There appearsto be no simplerelation-
ship betweengel &en :th and water-bindingwhen solutesare added
to carrageenan.
INTRODUCTION
CARRAGEENAN is a polysaccharide derived from several
varieties of seaweetl. The ability of carrageenan to imbibe
large amounts of lvater, thus yielding a solid-like elastic
gel, has found many applications in today’s food technol-
ogy,such as in the tlairy, dessert, and confection industries
(Stoloff, 1959; Towle, 1973). Carrageenan’s primary use
deals with its uniqc e water-holding and viscosity-increasing
abilities, which mainly affect the textural and stabilizing
properties of food:;. The water-holding capacity of a gel
refers to the amount of water a given system is able to re-
tain within its boutldaries, and is therefore, directly related
to the moisture con tent.’
The term water-binding should not be confused with
the water-holding capacity (WHC) which is the amount of
water held in the g:l after a physical stress such as centri-
fugation, a methocl developed by Wierbicki et al. (1957)
to determine the lvater-binding properties of meats. This
stress-related phenomenon measures a property that is
somewhat between true water-holding and water-binding.
True water-binding reflects a physico-chemical alteration
in water structure which affects the colligative properties
of water and is gelterally referred to in terms of water ac-
tivity (a,) lowering. Microbial growth, enzymatic, and
chemical reactions have been shown to be a function of
water activity (Labuza, 1970). Thus, water can be physic-
ally held within the gel matrix at a concentration of less
than 1% solids whi .e the gel’s water activity remains almost
indistinguishable fi.om that of pure water (Labuza and
Busk, 1979).
Authors R8y and Labuze are affiliated with the Dept. of Food
Science & Nutrition, 1 lniv. of Minnesota, St. Paul, MN 55108.
786-JOURNAL 0 F FOOD SCIENCE-Volume 46 (19811
According to a review by Labuza et al. (1976), the com-
mon water activity techniques, such as the vapor pressure
manometer, electric hygrometer, and dew point device, are
inaccurate in the very high water-activity region. The cryo-
scopic osmometer, however, theoretically can measure water
activity in a high dilution range (> 0.96); therefore, cryoscop-
ic osmometry is well suited for the study of high water ac-
tivity systems as typified by most gel concentrations. Cryo-
scopic osmometers have generally been used in the health
field to monitor osmolality changes in selected body fluids
(Boyd and Baker, 1971; Boyd et al., 197 1). They have also
been used to determine adulteration in milk by added water
(AOAC, 1965). Recently vapor pressure osmometry has
been used to assessmicro-macro-molecular interactions in
pharmaceutical hydrogels (Etter and Rey-Bellet, 1980).
Carrageenans are large polymers ideally consistmg of al-
ternating a-1,3 and pl,4 glycosidic linkages of galactose
with various amounts of 3,6-anhydro-D-galactose and sul-
fate half-esters. The three major types of carrageenan-
kappa (K), lambda (h), and iota ($--exhibit different gell-
ing properties due to the degree of substitution on the poly-
galactosyl chain (Percival, 1978). Rees and his co-workers
have published many papers elucidating the structure and
conformation of carrageenan gels. The sol to gel transfor-
mation was found to be dependent on the shifting from
random coil to double helix conformation (McKinnon et
al., 1969; Anderson et al., 196,9). The presence of the
hydrophobic 3,6-anhydro ring causes the galactose ring
structure to be so constrained as to allow for winding and
unwinding of the double helix (Percival, 1978). This con-
straining effect caused by the 3,6-anhydro group was found
to increase gel strength proportionally with the amount of
3,6-anhydro-D-galactose present in the carrageenan (Ains-
worth and Blanshard, 1978). The 2,6disulfate and 6 sul-
fate groups cause kinking in the helical chain resulting in
inter-chain association, thus setting up the three-dimensional
network which is characteristic of gels (Rees, 1972; Percival,
1978).
Solutes have been known to interact with carrageenan to
influence the gel strength. Several researchers found that
the order of effectiveness in increasing gel strength of car-
rageenan is potassium salts > ammonium salts > sodium
salts. This enhancement of the gel strength is considered to
be an ionic attraction between electronegative sulfate es-
ters and specific cations (Smith et al., 1954; Zabik and Al-
drich, 1968; Glicksman, 1969; Towle, 1973). The charge
to mass ratio of the cation is considered to play an impor-
tant role in the gelation phenomenon. Since the effective
ionic radius of sodium is larger than the radii of potassium
or ammonium, it is thought to make sodium a less effective
gelling agent (Smith et al., 1954). When selected anions of
potassium were added to carrageenan, the resulting gel
strength of Cl- was greater than SO47 (Zabik and Aldrich,
1965).
Solutes not only affect the gel strength, but also the
water binding in carrageenan systems. A procedure devel-
oped by Lewicki et al. (1978) utilized capillary suction
potential to determine the water activity of gel systems.
Carrageenan, by this method, was found to bind more
water than starch or gelatin gels. The theoretical water
 CARRAGEENAN-WATER BINDING/GEL STRENGTH.. .

activity as a function of carrageenan solids in these gels, known standard NaCl solutions to bracket the unknown
was very close to 1.0 as measured by the VPM technique samples. The conversion from mOsm to a, (Eg. 1) is simply
(Labuza and Busk, 1979). It should be noted that they a rearrangement of Raoult’s Law;
stated the measured values were probably in error because nl
of the problem of measuring a, close to 1.O. a,= 71nl+n2 (2)
When solutes are added to carrageenan gels, the observed
water activity may be significantly different from what is y1 = activity coefficient of the solvent
expected. If no interaction between the solute and polymer
occurs, the solute and polymer would bind the same n2 = mku solute
amount of water with or without the presence of the other where n2 is a function of y1 and nr2.
components. Competitive binding of water by micro and Comparisons of the water activity of various solutes as
macromolecules can affect the water activity of the three measured cryoscopically vs literature values are seen in
component system. The solute can (1) displace water from Table 1. Literature water-activity values were calculated
the polymer hydration shell, (2) bring new water into the from osmotic coefficient data where:
immediate vicinity via ion hydration. (3) alter the macro-
molecule’s conformation, and (4) alter the bulk structure a, = e(-O.O18 mu@)
of water (Kuntz and Kauzman, 1974). These effects could m = molality (3)
either increase or decreasethe water activity.
V = number of kinetic units generated by
The purpose of this research was to evaluate a new pro-
cedure using cryoscopic osmometry, which can be used to one molecule
determine the water activity of carrageenan gel systems. 4 = osmotic coefficient
This procedure should also have the potential to assessthe
extent of the interaction effects with respect to water bind- For all the solutes studied, osmometric results predicted the
ing when solutes are added to carrageenan-water systems. water activity to within 0.0005 a, units of reported litera-
Finally, an attempt was made to relate the force reuquired ture values, which is as good as or better than typical water-
to rupture the gel, using the Instron, with the water bind- activity measurement devices. Labuza et al. (1976) deter-
ing as measured by cryoscopic osmometry. mined, in a collaborative study, that the accuracy of water
activity measurements was no better than kO.005 a, for
MATERIALS&METHODS most equipment. Thus, the cryoscope is considered to be
an accurate measure of high water-activity solute systems.
Materials A question arises as to whether this cryoscope can also
Kappa, high kappa-lambdaand iota carrageenansolutions and measure the water activity of a biopolymer solution. Cryo-
gels (lot numbersRe-7997,Re-7998,Re-8005,respectively,Marine scopic osmometry intrinsically measures the water binding
Colloids, Inc.), were prepared similarly to the method outlined
by Persidskyand Luyet (1955). The appropriateweight of carra-
geenanpowder to include the sol, viscoussol, and gel range was
dispersedin cold deionizedand distilled water by rapid stirring with Table l-Water binding of selected solutes es measured by the cryo-
a magneticmixer for 10 min. The samplewas heatedin an 80°C scope
water bath with occasionalstirring until the carrageenan was com-
pletely in solution.The carrageenan solution wasquickly transferred Solute cont.
into 20.0-g aliquots and returned to the water bath so as not to
allow the gel to set. The solutesNaCl, Na2S04, NH&l and urea Solute
type ( Ki~~z~its) “5 Calw$tad Literrre
were eachrapidly stirred into an aliquot at concentrationsof 0.10,
0.25, and 1.0 kinetic units (ku) per kg H,O, while sucroseand KC1
were addedat 0.05, 0.10, and 0.25 ku. NaCla 0.10 95 0.9983 0.9983d
1.0 906 0.9839 0.9836
Methods KCla 0.05 48 0.9991 0.99924
Water activity measurement. Water activity was determined 0.25 228 0.9959 0.9959
using the AdvancedDigimatic Osmometer3DII (AdvancedInstru- NH&la 0.10 94 0.9983 0.9986
ments, Inc.) where 0.25 ml of a bubble-free sampleis Pasteur 0.25 195 0.9965 0.9964
pipetted into a samplecuvette and held coveredat 24°C for l-2 NapSOeb 0.10 84 0.9985 0.9982’
hr prior to measurement.Water activity was calculatedusing a 1.0 672 0.9880 0.9872’
modification of Raoult’sLaw where: SucroseC 0.05 50 0.9991 0.9991d
0.25 268 0.9954 0.99544
“1 UreaC 0.10 99 0.9982 0.9982
aw=- (1)
n1 + n’z 1.0 949 0.9832 0.9828

a, = water activity E Hamer

Stokes
and Wu (1972)
(1948)
n1 = nmolesof solventper kg Hz0 z Scarchard et al. (1938)
Found by interpolation
n’z = effectivemku (“mosm” the machinereadout)
Gel strength measurement. Gel strength was determinedas the Table 2-Evalaution of the water activity la,) of carrageenan sys-
force required to rupture the gel surfaceusing a 5/16” cylindrical tems
probe monitoredby the Instron UniversalTestingMachine.
RESULTS&DISCUSSION Carrageenan cone a, of Carrageenan types
9 Theoretical aw
CRYOSCOPIC OSMOMETRY has not been used to any de-
100 ml H20 kappa kappa-lambda iota predictionsa
gree to determine the water activity of high moisture sys-
tems. The cryoscopic osmometer does not measure the 0.1 0.99996 0.99996 0.99995 1 .ooooo
water activity directly, but measures the colligative proper- 0.5 0.99986 0.99982 0.99978 0.99998
ty of freezing point, which is internally converted to an 2.0 0.99961 0.99939 0.99923 0.99993
effective osmotic concentration (nz), given as milliosmoles
a Interpolation of values using the Flow-Huggins equation (Labuza
per kg Hz0 (mOsm). The cryoscope is calibrated with and Busk, 1979)

Volume 46 (1981)-JOURNAL OF FOOD SCIENCE-787

 within the sample; therefore, it should not matter what centrations up to 2-3% gel.
type of solute is binding the water to depress the freezing When solutes are added to carrageenan, the observed
point. Carrageenan is considered to bind considerably more water activity in many cases is significantly lower than
water than predicted by Raoult’s Law (Kawai, 1958; Craig expected. Six common solutes were chosen for their par-
and Trivisonno, 1963). Flory (1942) and Huggins (1942) ticular characteristics; NaCl, NazS04, KCl, NH4CI, and
developed theories to explain the water activity of polymer urea exhibit a positive Raoult’s Law deviation, while
solutions. Labuza and Busk (1979) used the Flory-Huggins sucrose binds more water than predicted by Raoult’s Law.
equation to predict the water activity of carrageenan- Sucrose and urea are nonionic solutesl as contrasted with
Ha0 systems. The theoretical calculation of water activity the remaining solutes which are essentially fully ionizable.
indicates that a strong self-standing gel of 2% carrageenan NaCl was also selected because it is commonly used in
is almost equiva1er.t in water activity to pure water. The foods to control water activity and the sodium ion is
aw’s measured by :he cryoscope for the three carrageenan considered to be a net water structure former (Fennema,
types at 2% solids were found to be greater than 0.999, 1976). NaCl has also been found to produce weak gels in
as shown in Tab:e 2. The slight differences in water K carrageenan (Zabik and Aldrich, 1968).
binding between tile predicted and measured carrageenan Sucrose is commonly used in foods to control water
systems, are proba3ly due to the fact that the theoretical activity and induce pectin gels, as in jelly. Sucrose fits
calculation of Flo:y and Huggins does not take into ac- well into the hydrogen bonded structure of water and is
count the direct binding of water by the strongly electro- considered to behave as a net water structure former
negative sulfate esler groups. An increase in water-binding (Fennema, 1976). Although NazS04 is not allowable in
is seen in the carral:eenans as ordered K < Ki < 1, which can foods, carrageenan naturally contains various levels of sul-
be attributed to all increased sulfate ester content. Cryo- fate esters; therefore, the addition of NaaS04 to car-
scopic measuremert of gel ,concentrations of greater than rageenan was of interest.
2-3% gel was inhibited by lack of nucleation and subse- KC1 is commonly used as a food ingredient, especially
quent crystal grojvth. The cryoscope can be, from this in sodium modified diets. The potassium ion was found to
evidence, a reasonable tool for the measurement of the increase gel strength of carrageenans (Stoloff, 1954; Mar-
water activity of n.any gels and especially carrageenan con- shall and Orr, 1954; Zabik and Aldrich, 1968). It is con-
sidered to behave as a weak net water structure breaker
(Fennema, 1976).
Table 3-Effect of eqrlivalent solute levels on gel strength and water NH4Cl is used in the baking industry where carrageenan
binding of 2% KA camlgeanan may have an application. The ammonium ion is considered
to be a net water structure breaker (Fennema, 1976) and
Water Interaction has been found to moderately increase the gel strength of
Gel Strength binding effect carrageenan (Zabik and Aldrich, 1968).
System Type (g Force) (mOsm) (mOsm) Urea has hydrogen-bonding sites geometrically incom-
0.25 ku KCI in 2% oh 1611 280 +18a
patible with the hydrogen-bonding structure of water, thus
0.25 ku NH4CI in 2% KA 147 270 +8 providing disruption in water structure. It has limited use in
0.25 ku NaCl in 2% K;L 18 271 +9 foods although it is approved for use in the wine industry.
0.25 ku Na SO4 in 2!6 KA 6 237 +8 A synergism or interaction effect, with respect to water
0.25 ku SU~rOSS in 2% Kh 4 406 +115a binding, can be assessedin the following way. A, is the de-
0.25 ku Urea in 2% K;. 4 310 +31a viation from Raoult’s Law for the solute: the measured
2% Kh 1 34 -
solute water binding minus Raoult’s predicted solute OS-
a Interaction Is significant at the 0.001 level (> 14.5 mOsm);
motic concentration. A2 is the actual measured osmotic
~it$terattloneffeCt cf > 56 mOsm Is 2 0.001 a, units).r concentration for the carrageenan-solute system minus the
measured plain carrageenan (which is close to 1.0) and that
predicted for the solute from Raoult’s Law. The interac-
tion effect is simply Az - Ar. If the interaction effect
Table 4-Effect of equivalent solute levels on gel strength and water
equals zero, there is no effective change in water binding;
binding of 2% K carra! faenan
however, if the interaction effect is less than or greater than
Gel Water Interaction zero, the system respectively binds less or more water than
strength binding effect expected. A greater value than zero could be because the
System type (g Force) (mOsm) (mOsm) solute may alter the conformation or hydration shell of
carrageenan, may bring ion hydrated water near the poly-
0.25 ku NaCl in 2% K 232 270 +18a mer and may alter the bulk structure of water, causing the
0.25 ku Na2S04 in 2’16 K 156 220 +3
carrageenan structure to change. Of the solute-carrageenan-
0.25 ku Sucrose in 29; K 157 423 +143a
2% K 120 22 - water systems studied, there was either no significant in-
teraction or a positive interaction effect, which lowered the
a Interaction Is sign flcant at the 0.001 level (> 14.5 mOsm); water activity more than expected as can be seen in Table 3.
(Interaction effect of 2 56 mOsm is 2 0.001 a, units).
The 2% oh carrageenan data with equivalent solute
levels ranked in decreasing order of gel strength are also
Table S-Effect of ec ruivalent solute levels on gel strength and water
seen in Table 3. The K+, NH4+, and Na+ ions strengthen
binding of 1% L carra! reenan
the gel significantly, probably by decreasing the sulfate
charge repulsion. The mechanism of gelation with these
Gel Water Interaction ions has been visualized as a zipper arrangement between
strength binding effect polymer strands, where the cation. bridges between two
System type (g Force) (mOsm) (mOsm) sulfate radicals (Bayley, 1955). Sodium’s effective radii
is larger than the K+ or NH4’ ion and may be too bulky
0.25 ku NaCl in 1% L 4 267 +16a to be an effective gelling agent. KC1 was reported to pro-
0.25 ku NazS04 in 1% r 4 219 +4
duce carrageenan gels of higher strength than KaS04
0.25 ku Sucrose in 1’6 L 3 303 +25a
l%l 3 20
(Zabik and Aldrich, 1965). The anion probably reduces
the gel strength via electrostatic repulsion. The mono-
a Interaction Is slgnl;icant at the 0.001 level (2 14.5 mOsm). valent Cl- does not reduce the gel strength as much as the

788~JOURNA L t IF FOOD SCI EKE- Volume 46 ( 198 1)

 CARRAGEENAN-WATER BINDING/GEL STRENGTH.. .

divalent SO4- when associated with sodium. Since sucrose between the polymer and the structure modified water in
and urea can not negate the sulfate ester’s charge, one the solutes’ hydration cloud. This reduced constraint on
would not expect them to be very effective gelling agents, the polymer by the modified water may prove to be great
as is seen. The solute’s effect on gel strength may be con- enough to allow more polymer-polymer hydrogen-bonding,
sidered largely electrostatic; however, a greater selection one form of which, the helical conformation, is very ther-
of solutes is needed to prove this theory. modynamically stable (Rees, 1972). This interaction would
There appears to be no direct relationship between gel then result in very little extra reduction in the a,.
strength and water-binding as measured by the cryoscope A net water structure former is considered to enhance
for 2% K h carrageenan, as seen in Table 3. There does, how- the overall hydrogen-bonding character of water in the sys-
ever, appear to be a relationship between water binding and tem. Sucrose and its structured water shell would promote
the interaction effect. The solute which has a lower activ- hydrogen-bonding with the polymer, and thereby reduce
ity coefficient or binds more water than predicted by the weaker polymer-polymer hydrogen-bonding. The
Raoult’s Law tends to produce a larger interaction effect net effect could be to enhance the overall hydrogen-bond-
with the carrageenan. Thus, the solute that interacts well ing, and therefore water-binding, of the entire system. Of
with water (i.e. greater a, lowering in Table 1)) also the solutes studied, sucrose generally interacted the strong-
tends to interact with carrageenan to bind more water than est to increase water-bonding with carrageenan systems, and
expected, where sucrose > urea > KC1 > NH4Cl> NaCl > thus had the largest-interaction effect.
NaCl > Na#04. The significance of the interaction effects Urea destabilizes most hydrogen-bonding structures and
(A, - A,) were determined by pooling the variances, con- would tend to act as a strong net water structure breaker
ducting an ANOVA, and an F-test at the 0.01 and 0.001 as well as a polymer-polymer hydrogen-bond breaker. If
significance levels. The very conservative confidence inter- polymer interactions are reduced, one would expect an in-
val chosen was necessary due to the large amounts of data crease in water-binding as well as a decreasein gel strength.
pooled (over two to four replications for most points), However, urea, being nonionic, would not be expected to
plus the desire not to report false-positive results. affect the gel strength by electrostatic joining as in the case
It is necessary to note that the interaction in some in- of the ionic solutes.
stances is almost as large as or larger than the water-binding The multi-component system is intrinsically complex to
of the plain carrageenan. For example, 0.25 ku sucrose in analyze. Many points have been raised as to theoretical in-
2% Kh has an interaction effect of 115 mOsm, while 2% teractions between polymer-solute and water. There is,
Kh carrageenan has a water binding of only 34 mOsm. unfortunately, no simple relationship evident to relate gel
This degree of interaction may alter the water-binding strength to water binding. However, there does appear to
conditions in formulated food systems in such a way that be a correlation between a solute that binds well with water
microbial .fermentation, chemical, or enzymatic reactions and a strong interaction effect, with respect to water bind-
can be affected. ing when the solute is added to a carrageenan-Hz0 system.
The 2% K carrageenan data resemble the 2% KX, as seen
in Table 4. The gel strength was in the order of NaCl > REFERENCES
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Na2S04 > sucrose. Water-binding and interaction effects of molecular structure and s&ength of carrageenan and-carragee-
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Anderson, N.S., Campbell, J.W., Harding, M.M.. Rees, D.A., and
appears to be no relationship between gel strength and Samuel. J.W.B. 1969. X-ray diffraction studies of polysaccharide
water binding but the K fraction gels are stronger. This is sulfates: Double helix models for K and r-carrageenans. J. Mol.
because X does not generally gel and thus acts more as a Biol. 45: 85.
AOAC. 1965. “Official Methods of Analysis.” 10th ed. Association
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The 1% L carrageenan data in Table 5 suggest that the Barley, S.T. 1955. X-ray and infrared studies on carrageenan. Bio-
them. Biophys. Acta. 17: 194.
solutes tested do not induce strong gelation as occurs Boyd, D.R., Addis, H.M.. Chllimintris, C., Lowe, R.J.. Folk, F.A.,
in the other carrageenan fractions. The water-binding and and Baker. R.J. 1971. Utilization of osmometrv in criticallv ill
interaction effects again are ordered sucrose > NaCl > surgical patients. Arch. Sum. 102: 363. 1 -
Boyd. D.R. and Baker, R.J. 1971. Osmometry: A new bedside
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Direct comparisons.are limited to the fact that 1 was tested Clinics of North America. 51(l): 241.
Craig, D. and Trivisonno. N.M. 1963. Cryoscopic segment weight
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polymer, one would expect weaker hydrogen-bonding -Continued on page 793

Volume 46 11981)-JOURNAL OF FOOD SCIENCE-789