Properties of Xanthan Obtained From Agricultural Wasted Acid Hydrolysates

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Properties of xanthan obtained from agriculturalwastes acid hydrolysates

M.J. Loopez *, M.C. Vargas-Garca, F. Suarez-Estrella, J. Moreno

Area de Microbiologa, Departamento de Biologa Aplicada, Universidad de Almera, CITE II-B, La Ca~nnada de San Urbano, 04120 Almera, Spain

Received 27 February 2003; accepted 21 July 2003

Abstract

The properties of xanthan produced by Xanthomonas campestris from agricultural wastes acid hydrolysates (AHW-xanthan) and

standard xanthan were compared. Both polymers had similar acetyl/pyruvyl ratio but had different amounts of other compounds,and therefore conditioned wide differences in viscosity of solutions. AHW-xanthan was less pseudoplastic and gave solutions of

lower apparent viscosity than standard xanthan. In contrast, AHW-xanthan solutions were more stable to changes in temperature,

pH and ionic strength than standard xanthan. These results suggest that xanthan obtained from agricultural wastes acid hydro-

lysates, low-cost and abundant substrates, may found additional applications to standard xanthan because of its higher stability and

lower production costs.

2003 Elsevier Ltd. All rights reserved.

Keywords: Xanthan; Agricultural wastes hydrolysates; Xanthomonas campestris; Rheology

1. Introduction

Xanthan is an extracellular heteropolysaccharide

produced by the bacterium Xanthomonas campestris.

This polymer is one of the major microbial polysac-

charides actually employed in many industrial processes

because of its unique rheological behaviour. Solutions of

xanthan are highly pseudoplastic and show very good

suspending properties. This makes xanthan very useful

as suspending, stabilizing, thickening and emulsifying

agent for food, cosmetics, pharmaceuticals and oil re-

covery among other applications (Sutherland, 1996).

The primary structure of xanthan is a cellulose-like

main chain with trisaccharide side chains composed of

DD-mannose/DD-glucuronic acid/DD-mannose linked to al-

ternate glucose residues (Jansson, Kenne, & Lindberg,1975). This molecule can reach weight ranges from 0.9

to 1.6 106 daltons (Shatwell, Sutherland, & Ross-

Murphy, 1990). Side chains usually carry an O-acetyl

group ester linked attached to the internal mannose,

while terminal mannose may contain a ketal-linked

pyruvate group. Composition of xanthan is affected by

several factors such as X. campestris strain, batch, cul-

ture media and downstream processing. The changes

in composition, mainly the extent of acetylation and

pyruvylation, affect properties of xanthan solutions

(Sutherland, 1994). Consequently, a wide range of dif-

ferent behaviour may be obtained and it is advisable to

test each polymer produced under specific conditions.

Main applications need a stable biopolymer and flow

behaviour of aqueous solutions under specific environ-

ment. The effects of temperature, ionic strength, coun-

terion valency and pH on viscosity of xanthan solutions

are of great importance (Moorhouse, Walkinshaw, &

Arnott, 1977).

In a previous report, we demonstrated that xanthan

could be obtained from agricultural plant wastes hy-

drolysates (Moreno, Loopez, Vargas-Garca, & Vaazquez,

1998). In this work, the properties of xanthan obtainedfrom these substrates were tested and compared with

standard xanthan. The viscosity of solutions was ana-

lysed as well as the effect of different chemical and

physical parameters in solution.

2. Materials and methods

2.1. Microorganism

Xanthomonas campestris NRRL B-1459 S4-LII

was obtained from the USDA National Center for

Journal of Food Engineering 63 (2004) 111115

www.elsevier.com/locate/jfoodeng

*Corresponding author. Tel.: +34-950-015-890; fax: +34-950-015-

476.

E-mail address: [email protected] (M.J. Loopez).

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

doi:10.1016/S0260-8774(03)00289-9
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Agricultural Utilization Research (Peoria, IL, USA) and

used throughout this study. The strain was maintained

on yeast malt (YM) agar (Difco, MI, USA) slants stored

at 4 C and subcultured at weekly intervals.

2.2. Media and culture conditions

Acid hydrolysates of sun-dried plant (AHW) of

melon (Cucumis melo) were used as substrates. AHW

were prepared as follows. A 10% (w/v) of chopped plant

wastes were mixed with 1.5% (v/v) sulphuric acid.

Mixtures were autoclaved at 121 C for 2 h. Waste hy-

drolysates were paper filtered and pH of the filtrates was

adjusted to 6.67 with Ca(OH)2 and filtered again. Total

carbohydrate content was determined in the hydroly-

sates as shown in analytical determinations.

Media for xanthan production was composed of

AHW added to a basal mineral solution (in g/l of dis-

tilled water: NH4Cl, 1; KH2PO4, 5.0; Na2CO3, 0.5;

Na2SO4, 0.114; MgCl2

6H2O, 0.163; ZnCl2, 0.0067;CaCl2 2H2O, 0.012; FeCl3 6H2O, 0.0014; H3BO3,

0.006) to provide a final carbohydrate concentration of

1 g/l.

Inocula were prepared in YM broth (Difco, MI,

USA). X. campestris cells were incubated in this medium

at 30 C under continuous shaking (120 rpm) for 18 h.

Then, 1 ml of this culture was inoculated into 250 ml

Erlenmeyer flasks containing 50 ml of mineral basal

medium supplemented with AHW. After incubation at

30 C on a shaker at 120 rpm for 5 d, xanthan was ex-

tracted.

2.3. Polymer extraction

Cells from 5 d cultures were centrifuged at 16,873 g

for 20 min. The product was recovered from the su-

pernatant by precipitation with two volumes of cold

isopropyl alcohol, using 1% (w/w) KCl as an electrolyte.

After washing with a 70% (v/v) isopropyl alcohol, the

precipitate was freeze-dried. The product was milled to

get a homogeneous powder.

Xanthan, obtained from AHW (AHW-xanthan), was

dissolved in deionised water (0.1% w/v) and centrifuged

at 16,873 g for 10 min. Supernatant was dialyzed against

deionised water for 72 h and solutions were freeze-dried.

2.4. Solutions preparation

Standard xanthan gum (Sigma, MO, USA) and

xanthan from AHW (AHW-xanthan), obtained as de-

scribed above, were dispersed in deionised water and

mixed using a magnetic stirrer for 812 h. The solutions

were gently stirred to remove bubbles and foam, and

used as prepared.

In order to study the effect of different parameters on

polymer viscosity, polymer concentration, temperature,

pH and salts concentration of solutions were modified

according to values summarized in Table 1.

For pH adjustment, 1 N NaOH or HCl was added to

provide the desired values.

To study the effect of monovalent and divalent salt

concentration on viscosity, 2%, 5% and 10% (w/v) of

either KCl or MgCl2 was added to xanthan solutions.

2.5. Viscosity measurements

Viscosity measurements were performed on a Brook-field LVT concentric cylinder viscometer (Brookfield,

MA, USA) at 25 C, unless otherwise indicated. Tem-

perature was controlled using a circulating wash bath.

Viscosity readings were taken after solutions reached

temperature. Regression analyses were performed to

describe the relationship between each tested parameter

and viscosity.

2.6. Analytical determinations

Carbohydrate content of acid hydrolysates was mea-

sured using the anthrone method. 1 ml of sample wasplaced into thin-walled glass tubes, cooled in ice-water,

and 5 ml cold anthrone reagent (0.2% of anthrone

(Sigma, MO, USA) in sulphuric acid 75% (v/v)) were

added by swirling the tube in the ice-water. After al-

lowed to stand a few minutes, the mixture was trans-

ferred to a boiling water-bath for exactly 10 min. After

cooled in an ice-bath, green colour was measured in a

Shimadzu UV-200 spectrophotometer at a wavelength

of 625 nm.

Polymer Analysis. Powered samples of AHW-xan-

than and standard xanthan, were chemically analysed

for their content in glucuronic acid, pyruvate and acetyl

content.Pyruvate content was determined enzymatically,

using lactate dehydrogenase (type II, Sigma), after hy-

drolysis of 0.20.4 g dried xanthan in 50 ml of 1 M HCl

for 3 h, and neutralization with BaCO3 (Sloneker &

Orentas, 1962).

Acetyl content was determined according to the

method of McComb and McCready (1957). A 200 ll

sample of xanthan solution (1% w/v) was added to 400

ll of a 1:1 mixture of 2 M hydroxylamine HCl and 3.5

M NaOH. After standing for 2 min at room tempera-

ture, 200 ll of 5.65 M HCl and 200 ll of 0.37 M

Table 1

Range of parameters analysed

Parameter Range

Polymer concentration (%) 0.52

Monovalent salt (KCl) (%) 010

Divalent salt (MgCl2) (%) 010

pH 210

Temperature (C) 25100

112 M.J. Loopez et al. / Journal of Food Engineering 63 (2004) 111115


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FeCl3 6H2O in 0.1 M HCl, were added. Brown reddish

colour was measured at 540 nm in a Shimadzu UV-200

spectrophotometer. Solutions of 0.04 M acetyl choline

HCl in 0.001 M sodium acetate (pH 4.5) ranging 1080

lg/ml were used as standard.

Uronic acids were determined using the method of

Blumenkratz and Asboe-Hansen (1973). 0.2 ml of 0.1%

xanthan solutions were added of 1.2 ml of 0.0125 M

sodium tetraborate in concentrated H2SO4. The mixture

was chilled in an ice bath for 5 min, homogenized, heated

at 100 C in a boiling bath and chilled again. Then, 20 ll

of 8.8 mM 3-phenylphenol in 125 mM NaOH, were

added and optical density was measured at 520 nm in a

Shimadzu UV-200 spectrophotometer. Pure glucuronic

acid solutions (100400 lg/ml) were used as standard.

3. Results and discussion

3.1. Polymers composition

Main components known to affect xanthan properties

in solution were analysed in standard and AHW-xan-

than (Table 2). AHW-xanthan had slightly lower con-

tent of each chemical tested but both xanthans had

similar acetyl/pyruvic ratio.

The pyruvic acid content has been suggested as an

indicator of some rheological properties (Rochefort &

Middleman, 1987). Our results agree with these findings

as noted by the lower apparent viscosity of AHW-xan-

than than standard xanthan solutions (Fig. 1), which

may be a consequence of the lower pyruvic content inthe former. This factor alone would not explain the wide

difference in the values of viscosity observed, whose

values in standard xanthan can reach more than fivefold

of viscosity obtained in AHW-xanthan. Other chemical

properties such as tridimensional conformation and

probably impurities and the formation of aggregates in

AHW-xanthan solutions may contribute to this broad

difference.

3.2. The effect of polymer concentration and shear rate on

viscosity

The concentration of polysaccharide in solution is

known to affect directly the viscosity and the degree of

pseudoplasticity (Sutherland, 1994). This factor also

influences the significance of solution physicochemical

parameter such as ionic strength (Torres, Sanchez,

Galindo, & Nienow, 1993). The viscosity of polysac-

charide solutions at different concentrations (0.52%) is

shown in Fig. 1. Standard xanthan gave higher viscosity

values at all concentrations. According to regression

analyses, the viscosity exponentially increased with

xanthan concentration in both polysaccharides

(r2 0:99). This effect was more marked in standardxanthan solution as indicated by the higher constant

values in the regression equation. Other researchers have

found similar relationships between concentration and

viscosity of xanthan solutions (Vanhooren & Vand-amme, 1998; Xuewu et al., 1996).

The measurement of viscosity at different shear rates

showed that the two polymers have a similar pseudo-

plastic rheological behaviour (Fig. 2). Relationship

between viscosity and shear rate is described by the

Ostwald de Weale model :

g Kcn1

where g is the viscosity, K is the consistency index, c is

the shear rate and n is the flow behaviour index.

A flow behaviour index lower than unity, indicates

the fluid has a pseudoplastic behaviour. Samples testedfollowed the model described, with a correlation coeffi-

cient r2 of 0.99. The values of consistency (K) and flow

behaviour (n) were obtained from the regression ana-

lyses. The n value was, in all cases, lower than one

(Table 3). This flow parameter for AHW-xanthan so-

lutions increased from 0.1 to 0.48 when polymer con-

centration was increased, while in standard xanthan it

remained almost constant, about 0.13 (Table 3). This

fact may reflect differences in molecular weight. Van-

hooren and Vandamme (1998) reported this relationship

in solutions of dextrans with different molecular weights.

Table 2

Chemical characteristics of xanthan samplesa

Component Standard xanthan (%) AHW-xanthan (%)

Uronic acids 14.16 1.02 10.60 2.06

Acetic acid 4.83 0.80 3.33 0.50

Pyruvic acid 3.74 0.50 2.81 0.43

Acetyl/pyruvic ratio 1 .29 0 .15 1.19 0 .21

aValues are the average of five determinations. Average standard

deviation.

y = 0.3752e0.9487x

y = 0.023e1.6478x

0

1

2

3

0 1 2 3

Xanthan concentration (g/l)

Viscosity(Pa.s

)

Fig. 1. The effect of concentration on viscosity of standard xanthan

() and AHW-xanthan (d) solutions at a shear rate of 20 s1.

M.J. Loopez et al. / Journal of Food Engineering 63 (2004) 111115 113


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The consistence index (K) tended to increase with

xanthan concentration (Table 2). This effect has been

also reported by Xuewu et al. (1996). Values for this

parameter were always higher in standard xanthan than

in AHW-xanthan.

3.3. The effect of temperature on viscosity

Solutions subjected to increases in temperature

showed reduced viscosity (Fig. 3). This effect was

marked in standard xanthan, whereas AHW-xanthan

was more stable to changes in temperature.

Temperature is known to affect mainly the confor-

mational structure of polysaccharide in solution. Xan-

than solutions undergo a conformational transition

from a rigid ordered structure to a disordered coil upon

heating above the melting point (Milas & Rinaudo,

1986). Acyl and pyruvate contents of polymer have a

noticeable influence on those conformational changes,

acetyl groups stabilize the ordered form, whereas pyru-

vate groups cause the opposite effect (Sutherland, 1994).

Hence, differences in polysaccharides used throughout

this study as effect of temperature may be the con-

sequence of the slight different content in acetyl andpyruvyl groups (Table 2).

3.4. The effect of pH

The viscosity of AHW-xanthan solutions was less

affected by changes in pH than standard xanthan solu-

tions (Fig. 4). In both solutions, viscosity decreased at

pH different from neutrality. The lowest values were

obtained at extreme pH values. Miladinov and Hanna

(1996) reported similar response. Conformational

changes as consequence of structure modification by

ionic interactions (Moorhouse et al., 1977) or compo-sition alteration by alkalis (Nasr-el-Din & Noy, 1992)

have been reported to be the effect of variations in pH.

In the case of polymers under study, the lower ionic

charge of AHW-xanthan may explain its higher stability

with changes in pH.

3.5. Effect of cation type and concentration (or simple

salts) on viscosity

The effect of ionic strength on viscosity is of primary

importance in the characterization of polyelectrolytes.

0

2

4

6

8

1 10 100 1000

shear rate (s-1

)

Viscosity(Pa.s

)

0

0.5

1

1.5

2

1 10 100 1000

shear rate (s-1

)

Viscosity(Pa.s

)

(a)

(b)

Fig. 2. The effect of concentration and shear rate on viscosity of (a)

standard xanthan and (b) AHW-xanthan solutions: (j) 2%; (M) 1.5%;

(d) 1%; (}) 0.5% (w/v).

0

1

2

3

0 20 40 60 80 100 120

Temperature (C)

Visco

sity(Pa.s

)

Fig. 3. The effect of temperature on viscosity of standard xanthan ()

and AHW-xanthan (d) solutions at a concentration of 2% (w/v) and

a shear rate of 20 s1.

Table 3

Effect of polymer concentration on rheological behaviour (flow be-

haviour (n) and consistency (K) indexes) of standard xanthan and

AHW-xanthan solutions

Concentra-

tion (%)

n K (Pas)

Standard

xanthan

AHW-

xanthan

Standard

xanthan

AHW-

xanthan

0.5 0.13 0.11 7.58 0.52

1.0 0.14 0.23 13.66 0.99

1.5 0.13 0.38 21.36 1.65

2.0 0.14 0.48 31.32 3.52

114 M.J. Loopez et al. / Journal of Food Engineering 63 (2004) 111115


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The increase of salt (MgCl2) concentration led to slightdecrease of viscosity in xanthan standard solutions (Fig.

5). This variation was more marked when a divalent

cation was present (MgCl2). AHW-xanthan viscosity

was independent of type or concentration of salt. The

lower stability of standard xanthan solutions to salts in

comparison with AHW-xanthan may be ascribed to its

more ionic character. Nasr-el-Din and Noy (1992)

reported similar results in solutions of xanthan with

different ionic charge. Uronic and pyruvyl contents

influence the behaviour of the polysaccharide solutions

in the presence of cations. Their contributions to the

total charge of polymer influence the interaction with

cations and hence the tertiary structure (Sutherland,

1994). This interaction is different depending on cation

valency. Selectivity of xanthan for divalent cation is

higher than that for monovalent (Rinaudo & Milas,

1978). Indeed, we found lower variation in the beha-

viour of solutions with monovalent cation.

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0

1

2

3

0 2 4 6 8 10 12 14

pH

Visco

sity

(Pa.s

)

Fig. 4. The effect of pH on viscosity of standard xanthan () and

AHW-xanthan (d) solutions at a concentration of 2% (w/v) and a

shear rate of 20 s1.

0.1

1

10

0

Standard Xanthan

AHW-Xanthan

4 6 8 10 12

Salt concentration (%)

V

iscosity

(Pa.s

)

Fig. 5. The effect of cation type KCl (j) or MgCl2 (d) on viscosity of

standard xanthan () and AHW-xanthan (- - -) solutions at a con-

centration of 2% (w/v) and a shear rate of 20 s1.

M.J. Loopez et al. / Journal of Food Engineering 63 (2004) 111115 115

Properties of Xanthan Obtained From Agricultural Wasted Acid Hydrolysates

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