Journal of Food Engineering 63 (2004) 111115 www.elsevier.

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Properties of xanthan obtained from agricultural wastes acid hydrolysates

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 dierent amounts of other compounds, and therefore conditioned wide dierences 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 hydrolysates, 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 polysaccharides 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 recovery among other applications (Sutherland, 1996). The primary structure of xanthan is a cellulose-like main chain with trisaccharide side chains composed of D -mannose/D -glucuronic acid/D -mannose linked to alternate glucose residues (Jansson, Kenne, & Lindberg, 1975). This molecule can reach weight ranges from 0.9 to 1.6 106 daltons (Shatwell, Sutherland, & RossMurphy, 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 aected by several factors such as X. campestris strain, batch, culCorresponding author. Tel.: +34-950-015-890; fax: +34-950-015476. pez). E-mail address: [email protected] (M.J. Lo 0260-8774/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00289-9
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ture media and downstream processing. The changes in composition, mainly the extent of acetylation and pyruvylation, aect properties of xanthan solutions (Sutherland, 1994). Consequently, a wide range of different behaviour may be obtained and it is advisable to test each polymer produced under specic conditions. Main applications need a stable biopolymer and ow behaviour of aqueous solutions under specic environment. The eects of temperature, ionic strength, counterion 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 hypez, Vargas-Garc drolysates (Moreno, Lo a, & V azquez, 1998). In this work, the properties of xanthan obtained from these substrates were tested and compared with standard xanthan. The viscosity of solutions was analysed as well as the eect of dierent 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

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M.J. L opez et al. / Journal of Food Engineering 63 (2004) 111115 Table 1 Range of parameters analysed Parameter Polymer concentration (%) Monovalent salt (KCl) (%) Divalent salt (MgCl2 ) (%) pH Temperature (C) Range 0.52 010 010 210 25100

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 hydrolysates were paper ltered and pH of the ltrates was adjusted to 6.67 with Ca(OH)2 and ltered again. Total carbohydrate content was determined in the hydrolysates as shown in analytical determinations. Media for xanthan production was composed of AHW added to a basal mineral solution (in g/l of distilled water: NH4 Cl, 1; KH2 PO4 , 5.0; Na2 CO3 , 0.5; Na2 SO4 , 0.114; MgCl2 6H2 O, 0.163; ZnCl2 , 0.0067; CaCl2 2H2 O, 0.012; FeCl3 6H2 O, 0.0014; H3 BO3 , 0.006) to provide a nal 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 asks 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 extracted. 2.3. Polymer extraction Cells from 5 d cultures were centrifuged at 16,873 g for 20 min. The product was recovered from the supernatant 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 described 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 eect of dierent parameters on polymer viscosity, polymer concentration, temperature,

pH and salts concentration of solutions were modied 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 eect 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 Brookeld LVT concentric cylinder viscometer (Brookeld, MA, USA) at 25 C, unless otherwise indicated. Temperature 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 measured using the anthrone method. 1 ml of sample was placed 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 allowed to stand a few minutes, the mixture was transferred 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-xanthan 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 hydrolysis 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 temperature, 200 ll of 5.65 M HCl and 200 ll of 0.37 M

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FeCl3 6H2 O 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 H2 SO4 . 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.

Viscosity (Pa.s)

y = 0.3752e

0.9487x

y = 0.023e

1.6478x

0 0 1 2 3 Xanthan concentration (g/l)

Fig. 1. The eect of concentration on viscosity of standard xanthan () and AHW-xanthan (d) solutions at a shear rate of 20 s1 .

3. Results and discussion 3.1. Polymers composition Main components known to aect xanthan properties in solution were analysed in standard and AHW-xanthan (Table 2). AHW-xanthan had slightly lower content 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 ndings as noted by the lower apparent viscosity of AHW-xanthan than standard xanthan solutions (Fig. 1), which may be a consequence of the lower pyruvic content in the former. This factor alone would not explain the wide dierence in the values of viscosity observed, whose values in standard xanthan can reach more than vefold 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 dierence. 3.2. The eect of polymer concentration and shear rate on viscosity The concentration of polysaccharide in solution is known to aect directly the viscosity and the degree of
Table 2 Chemical characteristics of xanthan samplesa Component Uronic acids Acetic acid Pyruvic acid Acetyl/pyruvic ratio Standard xanthan (%) 14.16 1.02 4.83 0.80 3.74 0.50 1.29 0.15 AHW-xanthan (%) 10.60 2.06 3.33 0.50 2.81 0.43 1.19 0.21

pseudoplasticity (Sutherland, 1994). This factor also inuences the signicance of solution physicochemical parameter such as ionic strength (Torres, Sanchez, Galindo, & Nienow, 1993). The viscosity of polysaccharide solutions at dierent 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 eect was more marked in standard xanthan 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 & Vandamme, 1998; Xuewu et al., 1996). The measurement of viscosity at dierent shear rates showed that the two polymers have a similar pseudoplastic rheological behaviour (Fig. 2). Relationship between viscosity and shear rate is described by the Ostwald de Weale model : g K cn1

a Values are the average of ve determinations. Average standard deviation.

where g is the viscosity, K is the consistency index, c is the shear rate and n is the ow behaviour index. A ow behaviour index lower than unity, indicates the uid has a pseudoplastic behaviour. Samples tested followed the model described, with a correlation coecient r2 of 0.99. The values of consistency (K ) and ow behaviour (n) were obtained from the regression analyses. The n value was, in all cases, lower than one (Table 3). This ow parameter for AHW-xanthan solutions increased from 0.1 to 0.48 when polymer concentration was increased, while in standard xanthan it remained almost constant, about 0.13 (Table 3). This fact may reect dierences in molecular weight. Vanhooren and Vandamme (1998) reported this relationship in solutions of dextrans with dierent molecular weights.

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(a)

Viscosity (Pa.s)

Viscosity (Pa.s)

0 1 10 100 1000 shear rate (s-1 ) 2

0 0 20 40 60 80 100 120 Temperature (C)

(b)

Fig. 3. The eect 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 .

Viscosity (Pa.s)

1.5

0.5

0 1 10 100
-1

1000

shear rate (s )

Fig. 2. The eect 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).

marked in standard xanthan, whereas AHW-xanthan was more stable to changes in temperature. Temperature is known to aect mainly the conformational structure of polysaccharide in solution. Xanthan 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 inuence on those conformational changes, acetyl groups stabilize the ordered form, whereas pyruvate groups cause the opposite eect (Sutherland, 1994). Hence, dierences in polysaccharides used throughout this study as eect of temperature may be the consequence of the slight dierent content in acetyl and pyruvyl groups (Table 2). 3.4. The eect of pH The viscosity of AHW-xanthan solutions was less aected by changes in pH than standard xanthan solutions (Fig. 4). In both solutions, viscosity decreased at pH dierent from neutrality. The lowest values were obtained at extreme pH values. Miladinov and Hanna (1996) reported similar response. Conformational changes as consequence of structure modication by ionic interactions (Moorhouse et al., 1977) or composition alteration by alkalis (Nasr-el-Din & Noy, 1992) have been reported to be the eect 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. Eect of cation type and concentration (or simple salts) on viscosity The eect of ionic strength on viscosity is of primary importance in the characterization of polyelectrolytes.

The consistence index (K ) tended to increase with xanthan concentration (Table 2). This eect 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 eect of temperature on viscosity Solutions subjected to increases in temperature showed reduced viscosity (Fig. 3). This eect was
Table 3 Eect of polymer concentration on rheological behaviour (ow behaviour (n) and consistency (K ) indexes) of standard xanthan and AHW-xanthan solutions Concentration (%) n Standard xanthan 0.13 0.14 0.13 0.14 AHWxanthan 0.11 0.23 0.38 0.48 K (Pa s) Standard xanthan 7.58 13.66 21.36 31.32 AHWxanthan 0.52 0.99 1.65 3.52

0.5 1.0 1.5 2.0

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Viscosity (Pa.s)

valency. Selectivity of xanthan for divalent cation is higher than that for monovalent (Rinaudo & Milas, 1978). Indeed, we found lower variation in the behaviour of solutions with monovalent cation.

References
1

0 0 2 4 6
pH

10

12

14

Fig. 4. The eect 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 .

The increase of salt (MgCl2 ) concentration led to slight decrease 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 dierent ionic charge. Uronic and pyruvyl contents inuence the behaviour of the polysaccharide solutions in the presence of cations. Their contributions to the total charge of polymer inuence the interaction with cations and hence the tertiary structure (Sutherland, 1994). This interaction is dierent depending on cation
10

Standard Xanthan

AHW-Xanthan

0.1 0 4 6 8 10 12

Salt concentration (% )

Fig. 5. The eect of cation type KCl (j) or MgCl2 (d) on viscosity of standard xanthan () and AHW-xanthan (- - -) solutions at a concentration of 2% (w/v) and a shear rate of 20 s1 .

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Viscosity (Pa.s)