Journal of Food Engineering 64 (2004) 179–186

www.elsevier.com/locate/jfoodeng

Viscoelastic properties of microbial alginate gelsby oscillatory dynamic tests

Mauro Moresi a,*, Maria Bruno a, Eugenio Parente b

a Department of Food Science and Technology, Via San Camillo de Lellis, University of Tuscia, Viterbo, 01100 Italyb Department of Biology DBAF, University of Basilicata, Potenza, Italy

Received 27 April 2003; accepted 22 September 2003

Abstract

The jellifying capacity of some alginate gels excreted by Azotobacter vinelandii DSM 576 was studied by oscillatory tests at

alginate concentrations (c) of 1.0% and 1.5% w/w. Frequency dependence of the complex shear modulus (G�) permitted these gels to

be discriminated in terms of their different orders of the relaxation function (a) and ‘‘gel stiffness’’ (Aa) values using Friedrich and

Heymann theory [Journal of Rheology, 32 (1988) 235]. Of the samples tested that characterised by a guluronate content of 25% and

a number-average molecular mass (Mn) of 263.4 kDa exhibited a gel stiffness at c ¼ 1% w/w of the same order of magnitude of that

pertaining to alginates extracted from Macrocystis pyrifera or Lessonia nigrescens with greater guluronate contents (35–40%) but

smaller number-average molecular masses (70–110 kDa) with about half of a.Whereas a was found to depend exponentially on the ratio of guluronate to mannuronate monad frequencies (NG=M) only, Aa was

expressed as a power function of c, Mn, NG=M, and average G-block length (NG). The limited data available, as well as incomplete

understanding of the real controlling factors for ionotropic gelation of alginates, made such empirical correlations just useful for a

preliminary screening of alginates independently of their weed or microbial origin.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: Alginate; Azotobacter vinelandii; Frequency sweep; Storage and loss moduli; Strain sweep; Viscoelastic properties

1. Introduction

Alginates are linear heteropolysaccharides, composed

of residues of b-DD-mannuronic acid and its epimer a-LL-guluronic acid, that are currently extracted from brownalgae (Onsøyen, 1992; Moe, Draget, Skj�ak-Bræk, &

Smidsrød, 1995). They can also be produced by mem-

bers of the genera Azotobacter and Pseudomonas

(Clementi, 1997; Rehm & Valla, 1997; Sabra, Zeng, &

Deckwer, 2001). About 30,000 metric tons/yr of sodium

alginates are currently used in the food, cosmetic,

pharmaceutical, textile and paper industries as thicken-

ing, stabilising, and jellifying agents.The decreasing availability of seaweed (traditional

sources not only of alginates, but also of agar and car-

rageen), the safety concern for using extracts that are

potential accumulators of the heavy metal cations

*Corresponding author. Tel.: +39-0761-357494; fax: +39-0761-

357498.

E-mail address: mmoresi@unitus.it (M. Moresi).

0260-8774/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jfoodeng.2003.09.030

(Hgþþ, Cdþþ, Crþ3, Cuþþ, etc.) present in polluted

seawaters, and increasing costs for their extraction–

purification processes are the main reasons for the

present interest towards the microbial production of

exopolysaccharides (EPS). Their peculiar chemical–physical properties would make them useful not only for

the traditional agro-industrial applications, but also for

other more specific applications in biotechnology and

biomedicine, even if the potential of microbial EPS as

industrial polymers is still a controversial matter.

Moreover, information on either their production costs

or rheological behaviour as thickening or jellifying

agents is yet limited. However, the possibility of usingraw materials free from seasonal and/or geographical

variations, selected strains under closely controlled

operating conditions might be sufficient to compensate

for the relatively low yields and high acetylation degrees

of bacterial biopolymers.

Alginate production from glucose-based media by

Azotobacter vinelandii DSM 576 was previously opti-

mised in the shaken-flask scale (Clementi, Fantozzi,

180 M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186

Mancini, & Moresi, 1995) and then scaled-up in labo-ratory fermenters (Clementi, Crudele, Parente, Mancini,

& Moresi, 1999; Parente, Crudele, Ricciardi, & Cle-

menti, 2000).

The aqueous dispersions of such alginates were found

to behave as pseudo-plastic liquids (Clementi, Mancini,

& Moresi, 1998), similarly to those containing algal

alginates (Mancini, Moresi, & Sappino, 1996). It was

also observed that at constant alginate concentrationand temperature the thickening properties of several

bacterial and algal alginates depended only on the

molecular mass, regardless of their origin (Clementi

et al., 1998).

Formation of alginate gels is quite a complex process,

depending on the type of alginate used (i.e. high-gulu-

ronic ‘‘G’’ or high-mannuronic ‘‘M’’ alginates), degree

of conversion into calcium alginate, source of calciumions (viz. calcium chloride, phosphate, lactate or ace-

tate) and methods of preparation (Moe et al., 1995;

Sime, 1990).

The viscoelastic properties of algal alginate gels have

been assessed so far using either static (Mancini, Moresi,

& Rancini, 1999a, 1999b; Mitchell & Blanshard, 1976;

Nussinovitch, Peleg, & Normand, 1989) or dynamic

(Doublier, Launay, & Cuvelier, 1992; Segeren, Bosk-amp, & Van Den Tempel, 1974) tests. The results of

stress-relaxation exhibited a solid-like behaviour that

was described by a mechanical model consisting of two

Maxwell elements in parallel with one spring (Mancini

et al., 1999a, 1999b; Nussinovitch et al., 1989). On the

contrary, the results of creep experiments showed a

liquid-like viscoelastic behaviour that was described by

a mechanical model consisting of one Maxwell elementin series with two Kelvin–Voigt elements (Mitchell &

Blanshard, 1976). In dynamic tests, the mechanical

spectra of these gels were found to be characterised by a

flat dependency of the shear storage modulus (G0) and

loss modulus (G00) on frequency (f ) for f ranging from

about 10�2 to 102 s�1 with G0 one or two orders of

magnitude greater than G00 (Moresi, Mancini, Bruno, &

Rancini, 2001; Segeren et al., 1974).By using Friedrich and Heymann theory (1988), the

viscoelastic properties of several algal alginates of the

high-mannuronic (M) and -guluronic (G) types, as de-

termined by small amplitude oscillatory dynamic tests,

were discriminated in terms of their different orders of

the relaxation function (aÞ and ‘‘gel stiffness’’ (Aa) val-

ues. The latter was approximately 4–5 times greater in

high-G alginate gels than high-M ones, while the formerwas practically constant and equal to about one twelfth

or one eighteenth for high-G or -M alginates, respec-

tively (Moresi et al., 2001).

The aim of this work was to assess the viscoelastic

properties of a few high-M microbial alginates obtained

from A. vinelandii DMS 576 using oscillatory dynamic

tests, so as to compare their jellifying capacity to that of

a series of high-M and high-G alginate gels previouslytested.

2. Materials and methods

2.1. Production of microbial alginates

Alginate production with A. vinelandii DSM 576 was

carried out in the following medium: glucose, 20 kgm�3;(NH4)2SO4, 0.6 kgm�3; Na2HPO4, 2 kgm�3; MgSO4 Æ7H2O, 0.3 kgm�3; yeast extract (Oxoid), 6 kgm�3. The

inoculum preparation and fermentation conditions were

those described previously (Parente et al., 2000). To

obtain alginates with different molecular weight and

different compositions, agitation speed, medium com-

position, and recovery time were varied as follows. For

the production of sample A, agitation speed was kept at400 revmin�1; recovery was carried out at 48 h. For the

production of sample B, agitation speed was kept at 400

revmin�1 throughout the fermentation, but bentonite (1

kgm�3) was added after 30 h to reduce degradation of

alginate by alginate lyases; recovery was carried out at

72 h. For the production of sample C, agitation speed

was kept at 400 revmin�1 for the first 24 h and increased

at 600 revmin�1 thereafter; recovery was carried outafter 48 h. For the production of sample D, a greater

concentration of (NH4)2SO4 (i.e., 0.9 kgm�3) was used,

agitation speed was kept at 600 revmin�1 throughout

the fermentation and bentonite (1 kgm�3) and glucose

(20 kgm�3) were added after 30 h; alginate was recov-

ered after 142 h. Alginate was recovered from the fer-

mentation broths and finished as white freeze-dried

powders, as previously described (Clementi et al., 1999;Parente et al., 2000).

2.2. Chemico-physical characterization of microbial

alginates

The percentages of mannuronic (M) and guluronic

(G) residues and sequences of MM-, MG- and GG-

blocks for the four samples tested were determined by

NMR spectroscopy at the Polytech S.coop.r.l. (Trieste,Italy) and are listed in Table 1. By using two different

Mark–Houwink relationships correlating the number-

(Mn) and weight- (Mw) average molecular masses of

several weed [Fucus vesicularus (Mackie, Noy, & Sellen,

1980), Laminaria digitata (Smidsrød, 1970), L. hyper-

borea (Mackie et al., 1980; Martinsen, Skj�ak-Braek,Smidsrød, Zanetti, & Poletti, 1991), L. cloustoni (Don-

nan & Rose, 1950), Macrocystis pyrifera (Martinsenet al., 1991)] or bacterial [Azotobacter vinelandii (Mackie

et al., 1980)] alginates to the corresponding intrinsic

viscosities ([g]) in 0.1 kmolm�3 NaCl at 25 �C (Clementi

et al., 1998), it was possible to estimate the Mn and

Mw values of all microbial alginates tested and, conse-

Table 1

Main chemical (percentage of overall guluronic-, FG, and mannuronic-, FM, blocks and of MM-, FMM, MG-, FMG, and GG-, FGG, blocks) and physical

properties {intrinsic viscosity ½g� at 0.1 M ionic strength, number-, Mn, and weight-, Mw, average molecular masses; number-average degree of

polymerization, dp} of the microbial and algal alginates examined in this work

Alginate from Type No. FG(%)

FM(%)

FMM

(%)

FMG

(%)

FGG

(%)

[g](dL/g)

Mn

(kDa)

Mw

(kDa)

dp (–) Ref.

Azotobacter vinelandiia High-M A j 25 75 50.5 24.5 0.5 9.6 120.6 460.4 609 b

B m 24 76 52.5 23.5 0.5 20.4 263.4 990.5 1330 b

C d 25 75 50.5 24.5 0.5 26.6 347.4 1297.1 1755 b

D r 17.5 82.5 65.5 17 0.5 15.1 192.4 729.6 972 b

Laminaria hyperborea High-G BDH 63 37 29 8 55 5.9 73.1 280.7 369 c

LF10/60 57 43 33 10 47 6.8 84.6 324.3 427 c

SF60 63 37 24 13 50 17.0 217.7 822.9 1099 c

Macrocystis pyrifera High-M LV 38 62 44 18 20 5.9 72.7 280.7 367 c

MV 35 65 46 19 16 9.6 119.9 460.4 606 c

CE 35 65 53 12 23 9.3 116.2 445.8 587 c

Laminaria hyperborea SF120RB 45 55 36 19 26 12.7 161.8 611.9 817 c

Lessonia nigrescens LF10/60-L 40 60 43 17 23 5.6 68.9 266.2 348 c

a The chemical composition of microbial alginates was determined by Polytech S.coop.r.l. (Trieste, Italy).b This work.cAs extracted from Moresi et al. (2001).

M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186 181

quently, their number-average degree of polymerization

(dp ¼ Mn=198), as shown in Table 1. These samples were

found to be more or less easily dispersible in water de-

pending on Mn.

2.3. Gel preparation

Calcium alginate gels were made according to the

internal setting method (Onsøyen, 1992) using two dif-

ferent alginate concentrations (c), that is 1.0% and 1.5%

(w/w). Once chosen the alginate concentration (c), it waspossible to calculate: (i) the molar mass of sodium al-

ginate (nNaAlg) to be dissolved in demineralised water; (ii)

the g-equivalents (nCaþþ ¼ 1=2nNaAlgdp) of calcium ionsto be added (to saturate theoretically all the carboxylic

groups present in any alginate molecule, this involving a

Naþ:Caþþ ratio of 0.5); (iii) the mass of calcium acid

phosphate dihydrate (CaHPO4 Æ 2H2O)as nCaþþ times the

molecular mass of the calcium source (i.e. 172.09 Da);

(iv) the mass of glucono-d-lactone being equal to that of

the calcium source used in accordance with Mitchell &

Blanshard (1976) and Nussinovitch et al. (1989).As an example, to prepare 100 g of a gel consisting of

sodium alginate no. A (Table 1) at c ¼ 1% w/w, 1 g of

the above sodium alginate and 0.43 g of the calcium salt

were thoroughly dispersed in 98.13 g of demineralised

water at ambient temperature under vacuum to mini-

mise entrapment of air bubbles. By adding 0.43 g of

glucono-d-lactone under vigorous mixing, pH reduced

from 7.8 ± 0.1 to 6.3 ± 0.1 after about 30 min, thus libe-rating the Caþþ ions. The resulting dispersion was

quickly poured over a 15-cm square glass tray and

stored at room temperature for about 24 h, thus ob-

taining a gel slab with a thickness of ca. 3 mm. Such

a slab was then segmented into 25-mm disks using

a stainless steel sharp-edged cork borer (inside dia-

meter¼ 25 mm and thickness¼ 0.5 mm).

2.4. Rheometrical tests

All rheometrical determinations were performed at 20

�C by using a dynamic stress rheometer (RS200, Rheo-

metric Scientific Inc., Piscataway, NJ, USA) equipped

with a 25-mm parallel plate geometry with smooth

surfaces (type LS-PELT-IP25). During all tests 25-mm

photocopy paper disks were stuck on both plates to avoid

the specimens slipping away; afterwards, the new zeroposition of the upper plate was automatically assessed.

All bases of any gel disk were gently drained off using a

photocopy paper sheet, the specimen axis was then

aligned with the instrument one by letting it adhere to

the paper disk stuck to the upper plate sensor, which

was finally lowered by 2% of the initial height of the gel

disk. In accordance with Mancini et al. (1999b) a 5-min

delay was assumed to be enough to let practically allshear history be relaxed away and to reconfigure the

unperturbed state of any specimen before testing.

The temperature was controlled within 0.1 �C by a

Peltier element in the lower plate. Evaporation of the

sample was prevented using a water-lock.

Dynamic strain sweep tests were performed at a fre-

quency of 1 s�1 and dynamic frequency sweep tests at a

strain amplitude of 0.5%.All tests were replicated at least three times, and mean

values and corresponding standard deviations calcu-

lated.

182 M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186

3. Results and discussion

In agreement with previous work (Moresi et al.,

2001), once assessed the range of linear viscoelasticity

for the microbial alginates tested, a series of frequency

sweep tests were carried out to study their viscoelastic

properties as a function of alginate concentration and

type.

3.1. Rheometrical determinations under strain sweep tests

By performing a series of strain sweep tests at lowfrequency (f ¼ 1 s�1), it was possible to confirm that the

four microbial alginate gels tested behaved as solid-like

materials (G0 > G00). Fig. 1 shows the effect of alginate

type on the complex dynamic shear modulus (G�) and

loss tangent (tan d) for 1.5% (w/w) alginate gels.

It was possible to discriminate two different regions,

namely a linear viscoelastic region in which G� and tan dwere practically constant, and a non-linear one in whichG� started to decrease with increasing strain while tan d

Table 2

Effect of the alginate type (see Table 1) and concentration (c) on the critical st

(G�) and loss tangent (tan d) values, as determined via strain sweep tests at

Alginate type c (% w/w) c0c (%)

A 1.00 2.2

1.50 2.2

B 1.00 0.9

1.50 1.6

C 1.00 2.8

1.50 1.4

D 1.00 3.6

1.50 2.8

1000

10000

0.1 1 10 100

Strain (%)

G*

(Pa

)

0.01

0.1

1

10

tan

δ

Fig. 1. Strain sweep tests for a few microbial alginate gels at 1.5% (w/

w) alginate concentration (c), 20 �C and frequency 1 s�1: complex shear

modulus (G�: closed symbols) and loss tangent (tan d: open symbols)

vs. strain amplitude (c0c). For alginate types same symbols as in

Table 1.

began to increase. As the shear strain amplitude (c0)increased from 0.2% to 10%, tan d was generally far

smaller than 0.1 except for sample C, thus revealing no

real crossing of the G0ðc0Þ and G00ðc0Þ curves (data not

shown). However, the slipping away of gel disks from

the plates made this region inaccessible for all gels

tested.

Despite practically constant the loss tangent tended

to reduce firstly up to reach a minimum value and thento increase. The strain amplitude associated with such a

minimum value was regarded as the upper strain limit

(c0c) of the linear viscoelastic region for these gels. Table

2 shows the effect of the alginate type and concentration

(c) on the critical strain (c0c) together with the corre-

sponding G� and tan d values.

Of the samples labelled A, B, and C in Table 1 pre-

senting almost the same mannuronate content (FM) anddiad sequence with Mn increasing from 121 to 347 kDa,

sample B exhibited the highest gel strength (expressed as

G�) of circa 3.0–4.0 kPa for c increasing from 1.0% to

1.5% w/w with the more restricted range of linear vis-

coelasticity (Table 2). On the contrary, sample D gave

gels with about half G� values (1.6–2.3 kPa) within the

same concentration range, probably because of its

greater mannuronate content (FM ¼ 82:5%) and smallermolecular mass (Mn ¼ 192 kDa).

It can finally be noted that the critical strain ampli-

tude (c0c) for these bacterial alginates did not exceed

3.6%, while it was found to vary up to 6.3% in the case

of the high-M algal alginates previously tested (Moresi

et al., 2001).

3.2. Rheometrical determinations under frequency sweep

tests

The microbial alginate gels showed approximatelyflat mechanical spectra in small amplitude oscillatory

experiments with G0 one or two order of magnitude

greater than G00 (Fig. 2), this being also in agreement

with Segeren et al. (1974) and Moresi et al. (2001).

rain value (c0c) together with the corresponding complex shear modulus

20 �C and frequency of 1 s�1

G� (kPa) tan d (–)

1.60± 0.03 0.039± 0.003

2.3 ± 0.1 0.040± 0.004

3.0 ± 0.3 0.074± 0.002

4.0 ± 0.5 0.078± 0.004

2.6 ± 0.2 0.049± 0.002

3.2 ± 0.2 0.078± 0.004

1.55± 0.05 0.020± 0.001

2.3 ± 0.1 0.036± 0.004

1000

10000

0.01 0.1 1 10 100Frequency (s-1)

G' (

Pa)

1

10

100

1000

G''

(Pa)

Fig. 2. Frequency sweep tests for a few microbial alginate gels at 1.5%

(w/w) alginate concentration (c), 20 �C and strain amplitude 0.5%:

storage modulus (G0: closed symbols) and loss modulus (G00: open

symbols) vs. frequency. For alginate types same symbols as in Table 1.

1000

10000

0.01 0.1 1 10 100Frequency (s-1)

G*

(Pa)

0.001

0.01

0.1

tan

δ

Fig. 3. Frequency sweep tests for a few microbial alginate gels at 1.5%

(w/w) alginate concentration (c), 20 �C and strain amplitude 0.5%:

complex shear modulus (G�: closed symbols) and loss tangent (tan d:open symbols) vs. frequency. The continuous and horizontal lines were

respectively calculated using Eqs. (4) and (3) with the empirical para-

meters listed in Table 3. For alginate types same symbols as in Table 1.

Table 3

Mean values and standard deviations of the gel stiffness parameter (Aa)

as a function of alginate concentration (c) and type, together with the

optimal values of the order of the relaxation function (a)

Alginate Type c (% w/w) Aa (kPa rad�a sa) a (–)

A 1.00 1.68± 0.01 0.022

1.50 2.24± 0.01

B 1.00 2.70± 0.03 0.036

1.50 3.70± 0.01

C 1.00 2.02± 0.04 0.037

1.50 2.90± 0.03

D 1.00 1.54± 0.01 0.015

1.50 2.10± 0.01

M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186 183

In previous work (Moresi et al., 2001), the four-

parameter relaxation function used by Friedrich &

Heymann (1988), to describe the phase transition of

cross-linking networks near the gel point or after the

transition sol–gel, was found to be able to reconstructthe evolution of shear storage (G0) and loss (G00) moduli

in the frequency domain as follows:

G0ðxÞ ¼ G1;a þffiffiffi2

p

rS�a cos

p2a

� �xa ð1Þ

G00ðxÞ ¼ffiffiffi2

p

rS�a sin

p2a

� �xa ð2Þ

where xð¼ 2pf Þ is the angular rate, a is the order of the

relaxation function; while G1;a and S�a are respectively

the equilibrium modulus and material parameter all

pertaining to a. Provided that the equilibrium modulus

(G1;a) were equal to zero (this holding for the sol state

and at the gel point) or negligible (this holding at the gel

state in a limited frequency range only), the tangent ofphase shift would be dependent on a only, while the

complex dynamic shear modulus G� would be function

of a and S�a :

tan d ¼ G00

G0 ¼ tanp2a

� �ð3Þ

and

G�ðxÞ ffiffiffiffi2

p

rS�ax

a ¼ Aaxa ð4Þ

In this way, based upon the model by Friedrich &

Heymann (1988), the three-dimensional structure cha-

racterising a gel was described in terms of the order of

the relaxation function (a), while Aa measured the

‘‘strength’’ of the cross-linking polymer network.

In this specific case, for f ranging from 0.2 to 20 s�1,G0ðxÞ and G00ðxÞ were found to be respectively linear

functions of ½cosðp=2aÞxa� and ½sinðp=2aÞxa�, while the

estimated values of the equilibrium modulus (G1;a) re-

sulted to be negative (that is devoid of any physicalmeaning) or statistically negligible at the confidence

level of 0.05 for all gels tested. Therefore, in the above

limited frequency window for all gels examined G� was

expressed using Eq. (4). Once assumed an arbitrary avalue for any alginate type, the experimental G�ðxÞ datawere divided by xa, thus yielding a series of Aa values,

which were averaged. By minimising the corresponding

sums of squares with respect to a via a non-linear esti-mation method, it was possible to determine the mean

values and standard deviations of Aa together with the

optimal value of the order of the relaxation function a,as listed in Table 3. The continuous lines in Fig. 3 show

a remarkable agreement with the experimental G� traces,

while the horizontal lines in Fig. 3 differ from the

experimental tan d data from as small as )7% to as great

as +55%.

1

10

100

1 10 100Aα

α

,exp

A,c

alc +2.5%

−2.5%

Fig. 4. Comparison between the experimental values of the gel stiffness

parameter Aa of bacterial (d) and algal high-M (�) and high-G (�)

alginates and those calculated using Eq. (6) together with the optimal

set of empirical regression coefficients ai reported in the text.

184 M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186

For the microbial alginates under study the averageorder of the relaxation function (a) was 0.03 ± 0.01, thus

being two or two and a half times smaller than that

pertaining the high-M (0.06 ± 0.01) or high-G (0.079±

0.001) alginate gels previously tested (Moresi et al.,

2001).

By referring to the classical theory of rubber elasti-

city, Segeren et al. (1974), as well as Mitchell & Blans-

hard (1976), were able to estimate the number-averagemolecular mass (Mc) of the portion of the chain between

two consecutive cross-links by using the well-known

relation valid for a network of Gaussian chains:

G0 ¼ cRTMc

¼ cRTðMn=ncÞ

ð5Þ

where c is the concentration of the polymer in the net-

work (in g m�3), T the absolute temperature, R(¼ 8.31

Jmol�1 K�1) the ideal gas-law constant, and nc the

number of cross-links per molecule. By confusing G0

with G� and referring to Table 1, Eq. (5) allowed esti-

mation of 7 up to 37 cross-links per each molecule of thebacterial alginates studied. These cross-links corre-

sponded to sequential arrangements of 80 to 48 mono-

mer units, quite in agreement with those (62) estimated

by Segeren et al. (1974) in the case of a high-M alginate

with Mn of 130 kDa. Despite the number of cross-links

calculated from Eq. (5) appears of the correct order of

magnitude, the deviations from the ideal rubber elas-

ticity observed at high strains and in swollen gels bySegeren et al. (1974) made such a simplified approach

useful just for a preliminary check of data consistency.

As concerning the strength of calcium alginate gels, it

is generally recognised that the proportion of G-blocks

is the main structural feature contributing to gel for-

mation (Moe et al., 1995). In fact, Skj�ak-Braek,Smidsrød, & Larsen (1986) succeeded in correlating the

compressive strength (r) of homogeneous alginate gelswith calcium ions to the average G-block length (NG),

defined as the ratio between the guluronate monad fre-

quency (FG) and mannuronate–guluronate diad fre-

quency (FMG), or to the length of guluronate blocks

larger than 1 (NG>1), defined as the difference between

the guluronate monad (FG) and mannuronate–guluro-

nate–mannuronate triad (FMGM) frequencies divided by

the mannuronate–guluronate–guluronate triad (FMGG)frequency. As pointed out by these authors themselves,

such a relationship accounting for the content and dis-

tribution of G-units only was unable to explain the

relatively high strength of gels made of the epimerised

Laminaria digitata alginate and should be no way re-

garded as fully comprehensive. Moreover, in previous

work (Moresi et al., 2001) the gel stiffness parameter Aa

was found to be a power function of alginate concen-tration and type (intrinsic viscosity ½g�, GG block diad

frequency, FGG, and average G-block length, NG), its

sensitivity with respect to c and NG being greater than

that to ½g� and FGG. However, such a correlation wasunable to predict the gel stiffness of the bacterial algi-

nates tested here.

To fit all the Aa data collected for the algal and

bacterial alginates listed in Table 1, the empirical cor-

relation previously developed (Moresi et al., 2001) was

modified as follows:

lnAa ¼ a0 þ a1 ln cþ a2lnMn þ a3 lnNG=M

þ a4 lnNG þ a5 ln FGG ð6Þ

where the empirical coefficients (ai) were estimated by

using the least-squares method upon bilogarithmic

transformation of the dependent (Aa) and independent

[c, Mn, NG=M, NG and FGG] variables, thus yielding:

a0 ¼ �1:1 � 0:8; a1 ¼ 1:6 � 0:2;

a2 ¼ 0:3 � 0:1; a3 ¼ 0:7 � 0:2; a4 ¼ 0:8 � 0:2;

a5 ¼ �0:07 � 0:06 ðr2 ¼ 0:93Þ

Further minimization of the mean percentage error(e) between the experimental and calculated Aa values

with respect to the only statistically significant parame-

ters ai by using a non-linear estimation method allowed

the following optimal set of the unknown empirical

parameters to be assessed:

a0 ¼ 1:24; a1 ¼ 1:87; a2 ¼ 0:11; a3 ¼ 1:28;

a4 ¼ 0:53; a5 ¼ 0

these being associated with a minimum e value of 23.4%.Fig. 4 shows the calculated against experimental Aa

values: more than two-thirds of the data fall within a

25% deviation band.

It can be noted that for the alginate concentrations

studied Aa was dependent on c1:87, this being in agree-

ment with c1:73� 0:11 dependence previously assessed for

algal alginates only (Moresi et al., 2001) and with c2 onereported by Smidsrød & Haug (1972). Moreover, theeffect of GG diad frequency on Aa was statistically

negligible, at least within the data considered in this

0

20

40

60

80

0 0.5 1 1.5 2NG/M

1/α

Fig. 5. Effect of the ratio between guluronate and mannuronate monad

frequencies (NG=M) on the reciprocal of the experimental order of the

relaxation function for bacterial (d) and algal high-M (r) and high-G

(�) alginates. The continuous line was calculated using Eq. (7).

M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186 185

work. However, this result is apparently in contradiction

with the general statement that contiguous guluronicacid residues exert a prevailing role in the jellifying

process of alginates in presence of bivalent cations, such

as Caþ2, the presence of which being regarded as es-

sential for forming binding sites for calcium ions and

giving rise to junctions in the gel network of the ‘‘egg-

box’’ type (Moe et al., 1995; Sime, 1990; Stokker et al.,

2000). Despite such an empirical correlation accounted

for the presence of G-units in the alginate molecules viatwo distinct parameters, i.e. NG=M and NG, it disregarded

several factors controlling lateral association of chain

segments, as well as the formation of precursor struc-

tures, structural interruptions of regular residue se-

quences limiting the length of the junction zones, etc.

(Stokker et al., 2000). Moreover, the data represented

here are still too limited to allow to elaborate more on

this point.Finally, by plotting the reciprocal of the order (a) of

the relaxation function vs. NG=M the following empirical

hyperbolic function was obtained by the least squares

method:

1=a ¼ exp½ð2:36 � 0:03Þ þ ð0:28 � 0:01Þ=ðNG=M � 0:06Þ� ðr2 ¼ 0:93Þ ð7Þ

thus allowing the experimental a values to be recon-

structed with a mean percentage error of about 10%

(Fig. 5).

The parameter (1/a) tended to decrease from 18± 1 to

12.6 ± 0.1 when dealing with high-M or high-G algalalginates, while it increased from about 27 to 67 for the

bacterial alginates tested here, these being characterised

by a high content of M-blocks (FM > 0:75) and a very

limited content of GG-blocks (Table 1).

4. Conclusions

A series of sequential oscillatory dynamic tests were

able to characterise the different gelling ability of bac-

terial and algal alginates by determining their range ofapparent linear viscoelasticity and frequency depen-

dence of G� in terms of their different order of the re-

laxation function (a) and gel stiffness (Aa) values using

Friedrich & Heymann theory (1988).

Whereas a was found to depend exponentially on the

ratio of guluronate to mannuronate monad frequencies

(NG=MÞ, Aa was expressed as a power function of alginate

concentration (c) and type [number-average molecularmass Mn, NG=M, and average G-block length (NG)], its

sensitivity with respect to c and NG=M being greater than

that to Mn and NG. However, the limited data available,

as well as incomplete understanding of the real con-

trolling factors for ionotropic gelation of alginates,

made the prediction capability of such correlations just

useful for a preliminary screening of alginates indepen-

dently of their weed or microbial origin.Under constant alginate concentration and number-

average molecular mass, as well as temperature, the

viscosifying properties of the bacterial alginates in

aqueous dispersions were found to be comparable to

those of the algal counterparts (Clementi et al., 1998).

However, this cannot be extrapolated to the gelling

properties. In fact, of the samples tested that charac-

terised by a guluronate content of 25% and a number-average molecular mass of 263.4 kDa exhibited a

gel stiffness at c ¼ 1% w/w of the same order of

magnitude of that pertaining to alginates extracted from

M. pyrifera or Lessonia nigrescens with greater guluro-

nate contents (35–40%), but smaller number-average

molecular masses (70–110 kDa). Provided that the

greater molecular mass of such a bacterial alginate

compensated for the corresponding lower guluronatecontent, should the limited range of linear viscoelasticity

observed for such a calcium gel be a signal of a shear

sensitivity typical of a physical rather than a chemical

gel?

Further studies might be needed to increase the gu-

luronate content of the bacterial alginates, by optimising

the culture media formulation (i.e. by adding Caþþ ions)

and fermentation conditions (pO2 levels and oxidativestress), as suggested by Rehm & Valla (1997) and Sabra

et al. (2001), or resorting to sequential treatments con-

sisting of a preliminary deacetylation step followed by

enzymatic processes, such as those used by Skj�ak-Braeket al. (1986) to epimerise for instance DD-mannuronan

from Pseudomonas aeruginosa into a polymer containing

40% G.

Acknowledgements

This research was supported by a special grant

(COFIN2000) from the Italian Ministry of Research

and University.

186 M. Moresi et al. / Journal of Food Engineering 64 (2004) 179–186

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