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Effects of microfluidization on the functional properties of xanthan gum
Food Hydrocolloids 12 (1998) 365±371
Eects of micro¯uidization on the functional properties of xanthan gum N. Lagoueyte, P. Paquin* Centre de recherche en sciences et technologie du lait (STELA), DeÂpartement de sciences des aliments et de nutrition, Universite Laval, Pavilion Paul-Comtois, QueÂbec, QueÂbec G1K 7P4, Canada Received 5 February 1997; received in revised form 16 May 1997; accepted 19 December 1997
Abstract The eects of micro¯uidization on xanthan gum were studied by ¯ow behaviour, hydration rate, water uptake and molecular weight determinations. The eect of pressure and number of passes on the xanthan solution produced a decrease in all these functional properties. Consequently the thickening and, stabilizing properties were reduced. We argued that the high shear, turbulence forces and cavitation involved in the micro¯uidization process produced ordered±disordered conformational transition (by opening of the molecule) and polymer degradation. The opening of the molecule occurred ®rst, followed by polymer degradation due to mechanical stress. Dierences observed in viscosity between treated xanthan dispersion and their spray-dried powders is associated with reorganisation of xanthan aggregates during heating and cooling, which occurred in spray-drying, but these phenomena do not change the high pressure eect on the biopolymer. # 1998 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Xanthan gum is an hydrocolloid widely used in food industries for its thickening and stabilizing properties, and for its unusual stability towards temperature, ionic strength, pH, enzymatic and chemical reactive (Anon., 1988). It is an exocellular heteropolysaccharide consisting in a linear backbone of 1-4 linked b-d glucose residues having a trisaccharide side-chain attached to alternative d glucosyl residues, and it is produced by a distinct fermentation process of Xanthomonas campestris. Nevertheless the knowledge on its composition and secondary structure is still under discussion. Indeed, in aqueous solutions, xanthan undergoes an ordered±disordered conformational change depending on temperature, ionic strength, counterion valence and pH, as well as acetyl and pyruvyl substituent content (Lambert, Milas, & Rinaudo, 1985; Milas & Rinaudo, 1981; Morris, 1977; Paradossi & Brant, 1982). Many authors suggested that the ordered form is a double-stranded helix, and that the conformation transition induced the complete or * Corresponding author.
partial dissociation of the double helix into single chains (Kitamura, Takeo, Kuge, & Stokke, 1991; Liu, Sato, Norisuye, & Fujita, 1987; Sato, Norisuye, & Fujita, 1987). Other results suggested that the ordered conformation is a single strand (Morris, Rees, Young, Walkinshaw, & Darke, 1977; Norton, Goodall, Frangou, Morris, & Rees, 1984) and that below the melting temperature, ordered and disordered regions co-exist within the same xanthan molecule (Norton et al., 1984). Recent studies (Chazeau, Milas, & Rinaudo, 1995; Milas, Reed, & Printz, 1996) proposed that the native xanthan conformation is single-stranded whereas re-natured xanthan conformation (xanthan obtained by puri®cation including heat treatment) is a doublestranded helix. Its has been postulated (Richardson & Ross-Murphy, 1987) that rheological properties of xanthan solution are consistent with a picture of a tenuous network, which from other evidence involves non-covalent intermolecular forces. The exact nature of these forces still remains to be completely established, and possibly a balance between hydrogen bonding, hydrophilic/hydrophobic interactions and cation-speci®c interactions. However, others workers (Lim, Uhl, & Prud'homme, 1984; Milas & Rinaudo,
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1979; Schorsch, Garnier, & Doublier, 1995) have suggested that the unusual rheological properties of xanthan solutions for concentrations of 1% and above are due to the formation of liquid crystalline domains, these structures being disrupted by shear rates greater than 0.1 sÿ1. Micro¯uidization technology is a very unique type of high pressure homogenization: the reaction chamber is built in such a way that the liquid is divided into two microstreams that are projected against one another at high speed and at an angle of 180 (Cook & Lagace, 1985). This technique is currently used by the pharmaceutical and cosmetics industries to produce ®ne emulsions (Chandonnet, Korstvedt, & Siciliano, 1985). Possible applications in production of dairy-based products have also been reported (Paquin & Giasson, 1989; Paquin, Lebeuf, Richard, & Kalab, 1993; Robin, Blanchot, Vuillemard, & Paquin, 1992). Other workers have also demonstrated that micro¯uidization can aected polymeric structure for small polymers (Silvesti & Gabrielson, 1991). The aim of the present work was to study the in¯uence of the mechanical treatment (micro¯uidization) on the characteristics of xanthan. 2. Materials and methods 2.1. Micro¯uidized xanthan Xanthan gum (Ketrol F, Keleo Division of Merck and Co. Inc., San Diego, CA) was dispersed in distilled water (1.0% w/w) and stirred at room temperature for 1 h. The solution was then kept at 4 C for another 12 h to achieve complete hydration. The micro¯uidization treatment was carried out with a micro¯uidizer (model M-1102, Micro¯uidic Corporation, Newton, USA) at 75 MPa. The solution was recirculated for dierent numbers of passes (0, 2, 4, 8, 12, 16 and 20 passes). The micro¯uidizer is a recent high pressure homogenization technology developed in the early 1980s (Cook & Lagace, 1985). The principle is: the liquid is divided in two jet streams, which enter microchannels where they are subjected to shear forces. After
that, the jet streams are directed in an area where there is liquid/liquid contact, and the liquids are submitted to cavitation and turbulence due to pressure drop when the homogenized liquid passes out of the reaction chamber (Fig. 1; Paquin & Giasson, 1989). During the experience, the temperature is kept constant (23 C) by immersing in ice the reaction chambers and the coiled heat exchanger is connected at the outlet of the micro¯uidizer. The micro¯uidized solutions are spray-dried (Niro Atomizer, Copenhagen, Denmark) at an inlet temperature of 200 C and an outlet temperature of 80± 85 C. The powders are stored in sealed containers and are kept at 4 C until used. The moisture content of the dry material determined by drying at 103 C for 18 h, was 11.00.5% (AOAC, 1990). 2.2. Size exclusion chromatography (SEC) SEC characterizations were carried out on a HP 1050 apparatus (Hewlett±Packard Ltd, Mississauga, Canada) with a TSK gel 600OPWXL column (Hosohaas, Montgomeryville, USA) and a dierential refractometer as the detector (HP 10474). Powder samples were dissolved at 0.5 mg/ml in NH4NO3 (0.1 M), with 0.5 g/l Na3N as preservative, at room temperature, and stirred for 2 h. The solutions were stored at 4 C for 12 h before use. After sonication (50±60 Hz, 30 min), the solutions were ®ltered through 0.45 mm membrane ®lters (Nucleopore MF Canada Inc., Toronto). Typically, 20 ml of xanthan sample was injected for each SEC run. Molecular weights were determined by extrapolation of a calibration curve, obtained with Pullulan standards (American Polymer Standars Corporation, Mentor, USA). All measurements were duplicated and performed at 30 C with an elution rate of 0.5 ml/min. 2.3. Water binding of xanthan by the Baumann apparatus Spontaneous water uptake was determined by a modi®cation of the Baumann method according to Kneifel, Paquin, Albert, & Richard (1991). A small amount of powdered sample, in the range of 2±12 mg,
Fig. 1. Shematic representation of the reaction chamber of the micro¯uid equipment.
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was spread in a thin layer onto a Nucleopore MF membrane (Nucleopore Canada Inc., Toronto), which was then placed on the plastic ®lter of the apparatus. The membranes used were mixed esters of cellulose with an average pore diameter of 0.1 mm. Water uptake, as measured by the calibrated capillary (2.000.01 ml), was recorded as a function of time for 60 min. A value of 0.0130.001 ml/h water uptake was found for the blank value and attributed to evaporation. It was evaluated by measuring the water uptake by three membranes on the Baumann device. This small value was considered negligible when compared to xanthan water uptake and was not considered in further calculations.
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likely by strengthening both polymer±polymer and polymer±water interactions during the atomization process. All the samples (ketrol F, non-micro¯uidized spraydried, and micro¯uidized) showed a typical pseudoplastic ¯ow behavior associated to the formation of a weak, tenuous, gel-like network. We observed an important dierence between the non-micro¯uidized samples (commercial product ketrol F and spray-dried product) and the micro¯uidized samples. The results presented in Fig. 3 demonstrated that the ¯ow behavior of the micro¯uidized samples is changed as the samples are submitted to more severe treatment from 0 to 20 passes through the micro¯uidizer. The dependence of the
2.4. Rheological measurements The dry powders were suspended in distilled water (1% w/w) and stirred at room temperature for 1 h. Subsequently, the solutions were stored at 4 C for 12 h before use. The ¯ow behaviour of each micro¯uidized xanthan gum solution prior to drying and rehydrated micro¯uidized powders was determined with a controlled stress viscometer (Carri-Med CS500, Westech Industrial Ltd, Mississauga, Canada), equipped with cone-and-plate geometry. The stainless steel cone employed was CP 5544 (40 mm diameter and 1.58 C). Three independent tests were carried out with a fresh sample of each solution at 25 C and the shear stress was increased linearly from 0 to 15 Pa in 10 min. 3. Results and discussion 3.1. Flow characterization In order to be able to measure functional properties (molecular weight/HPLC, hydration properties/Bauman), micro¯uidized xanthan solutions were spraydried. We also spray-dried the non-treated xanthan solution to compare it to the commercial xanthan product (ketrol F). The rehydrated non-micro¯uidized, spray-dried solution and the commercial product (ketrol F) solution show small dierences in viscosity pro®le (Fig. 2). The spray-dried xanthan higher viscosity is related to the fact that the spray-dried product gives spherical particles when it is dried, compared to commercial product that is dried by alcohol drying, which gives a ®ber-like product. Heating involved the modi®cation of the state of aggregation and, eventually, reversible conformational change. On subsequent cooling, an alternative pattern of side chain±backbone interactions, presumably dictated by kinetic rather than thermodynamic factors, was established (Milas & Rinaudo, 1986). We argue that the reorganization of the aggregates led to a stronger gel-like formation, most
Fig. 2. Viscosity of commercial and spray-dried xanthan versus shear rate.
Fig. 3. Flow behavior of rehydrated micro¯uidized spray-dried xanthan solutions.
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rheology must be related to the orientation and the deformation of the molecules and the modi®cations of the interchain interactions (Mitchell, 1979). Launay, Doublier, & Cuvelier (1986) have suggested that xanthan molecules may exist at rest in an equilibrium state of aggregation and that the extent of aggregation was reduced on shearing. The eect of shear rate on the viscosity of xanthan gum solutions may be described by the following power-law model (Barnes, Hutton, & Walters, 1989): K _ nÿ1
1
where =apparent viscosity, K=consistency index, _
=shear rate and n=¯ow behavior index. Fig. 4 shows the eect of shear rate on the viscosity of micro¯uidized xanthan gum according to Eq. (1). The viscosity decreased as the shear rate increased, the consistency index tended to decrease with the number of passes, whereas the ¯ow behavior index increased (Table 1). The pseudoplastic behaviour was less and less marked with the increase of the number of passes. With reference to K and n values, the micro¯uidization had no more signi®cant eect on viscosity beyond eight passes for the micro¯uidized xanthan dispersions and beyond 12 passes for the rehydrated micro¯uidized spray-dried solutions. These dierences were possibly associated with the fact that spray-dried samples have a larger aggregation state than the dispersion samples. 3.2. Molecular weight determination The values of average molecular weight obtained were higher than those in the literature (25106). Southwick, Jamieson, & Blackwell (1982) noted that the large discrepancies reported in the studies on molecular weight for xanthan (2106±50106) probably resulted
Table 1 Consistency index and ¯ow behavior index of xanthan gum dispersions Micro¯uidized dispersions Number of passes ketrol F 0 2 4 8 12 16 20 a
n
K
r2
0.218 0.572 0.587 0.641 0.630 0.627 0.614
4.135 0.348 0.296 0.195 0.196 0.215 0.226
0.952 0.989 0.994 0.986 0.992 0.983 0.978
Rehydrated micro¯uidized (spray-dried) n
r2
K
(0.214)a (4.094) (0.969) 0.189 5.572 0.992 0.350 1.555 0.988 0.375 1.331 0.987 0.399 1.218 0.996 0.464 0.786 0.994 0.473 0.699 0.991 0.456 0.829 0.994
Values in parentheses are for commercial ketrol F product.
from aggregation in some of the xanthan solutions examined. In addition, Muller, Anrhourrache, Lecourtier, & Chauveteau (1986) showed that dried product had a greater tendency for microgel formation than the corresponding xanthan gum obtained from the fermentation broth. This can lead to unusually high values of molecular weight (i.e. large aggregates), which in turn should aect the ¯ow properties of the solutions. Milas et al. (1996) suggested that it was the species of molecular weight 1.2106 which was synthesized by the bacterial cells that later aggregated and formed the polydisperse distributions of very high molecular weight observed in the culture media. Results (Table 2) showed that the micro¯uidization process decreased the average molecular weight of xanthan samples. The average molecular weight did not decrease linearly with the number of passes; the decrease being more important for the ®rst passes, additional passes having less and less impact on the average molecular weight (Table 2). For instance, after two passes, the average molecular weight was approximately reduced 2 times, whereas after 20 passes, the diminution was around 6 times. The average molecular weight was aected little by the micro¯uidization process Table 2 Average molecular weights of xanthan powder samples Samples
Fig. 4. Viscosity of rehydrated micro¯uidized spray-dried xanthan solutions versus shear rate.
Commercial product ketrol F SPOM spray-dried SP2M SP4M SP8M SP12M SP16M SP20M
Molecular weights (10ÿ3)
MW of P0M/MW of sample
25,871 26,288 15,149 10,889 7,971 5,396 4,858 4,485
SP spray-dried; 0±20: number of passes; M: micro¯uidized.
1.73 2.41 3.30 4.87 5.41 5.86
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beyond 12 passes. Also the average molecular weight of non-micro¯uidized spray-dried sample was higher but not very dierent than the commercial product (ketrol F). 3.3. Hydration rate measurements The Baumann apparatus did not allow a precisely measure of the equilibrium time of hydration due to diculty in: (a) the weight of xanthan powder put on the ®lter needed at least 2±12 mg, which absorbed a volume of water greater than the calibrated capillary used, and (b) increasing the volume of the capillary did not allow precise measurement of the water volume. Water uptake versus time measured by Baumann capillary apparatus for dierent weights of sample are shown in Fig. 5. Water uptake signi®cantly depended on sample weight: the absorption value appeared progressively smaller as the sample weight increased. The same phenomenon was observed for the other powder samples. The decrease in water uptake with increasing sample weight was at ®rst surprising because xanthan is known to be one of the best water binders among hydrocolloids. Xanthan gum is also among the best thickening agent. On the Baumann apparatus, the hydration of xanthan powder led to a weak gel-like formation on the membrane surface, which reduced water uptake in a more pronounced manner when increasing the sample weight. This observation underlined the importance of constant weight for this test in order to obtain comparable results. In view of the impossibility to put on the membrane a determined sample weight (the membrane must be water-saturated), we could not easily compare samples. Nevertheless, for 6±8 dierent weights of the same powder samples (over a 2±12 mg sample range), a linear relationship was found between the water uptake at a given time and the weight of the sample, regression coecients at a given time being higher than 0.95. From
Fig. 5. Water uptake versus time for dierent weights (mg) for commercial xanthan (ketrol F).
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these observations, we have drawn the curve for a weight set arbitrarily at 5 mg (Fig. 6). Hydration properties were aected by the micro¯uidization process. Powder samples showed the same hydration pro®le (hydration rate was initially rapid and slowed as equilibrium was approached), but increasing the number of passes decreased the hydration rate and the water uptake. We also noticed that, for an equivalent average molecular weight, the non-micro¯uidized spray-dried sample had both higher hydration rate and water uptake, and was more viscous than the commercial (ketrol F) solution. This observation underlines the importance of thermal history on the functional properties of xanthan samples. We argue that heating involved rearrangements of the xanthan aggregates and therefore a modi®cation of the interactions which stabilized the initial xanthan conformation. 3.4. Tentative explanations Studies have shown that, under certain conditions such as low ionic strength (Lecourtier, Chauveteau, & Muller, 1986; Stokke & Christensen, 1996), heat-treated 4 M urea solutions (Frangou, Morris, Rees, Richardson, & Ross-Murphy, 1982; Morris, Franklin, & L'Anson, 1983; Southwick, Jamieson, & Blackwell, 1982), ultracentrifugation in the presence of 4 M urea (Morris et al., 1983) and cadoxen solutions (Sato, Norisuye, & Fujita, 1984), the order±disorder transition was irreversible. The disordered form had dierent properties than the ordered form, for instance, smaller intrinsic viscosity (Milas, Rinaudo, & Tinland, 1986; Sato et al., 1984; Southwick et al., 1982) and smaller viscosity values (Frangou et al., 1982; Southwick et al., 1982; Morris et al., 1983; Milas et al., 1986; Rochefort & Middleman, 1987). Also, the degradation of low and
Fig. 6. Theoretical curves of water uptake versus time of spray-dried xanthan (sample weight set at 5 mg). SP: spray-dried; 0±20: number of passes; M: micro¯uidized.
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high average molecular weight polymers induced by high shear processing has been reported previously (Silvesti & Gabrielson, 1991; Cencia-Rohan & Silvesti, 1993). This degradation process occurs primarily in the presence of simultaneous intense shear and turbulent mixing forces and is believed to be the result of exposure of covalent bonds to mechanical stresses during viscous ¯ow under harsh conditions (Silvesti & Gabrielson, 1991). Precisely controlled shear, turbulent and cavitation forces are generated within the micro¯uid chambers. These forces have been shown to be of sucient magnitude to cause mechanical degradation of some polymers (tragacanth) (Cencia-Rohan & Silvesti, 1993; Stokke, Smidsrod, & Elgsaeter, 1989). We postulate that the turbulence, cavitation, and high shear stress of the micro¯uidization process have an eect on the conformation of the molecule and, consequently, induced an ordered±desordered conformation transition and degradation of the molecule which is irreversible. We suggest that conformational changes occurred and that the percentage of disordered form may have increased with pressure increase and the number of passes up to a limit where polymer degradation can occur; the degree of reversibility depended upon the intensity of the process. This new conformation was stabilized by intramolecular interactions. As intrinsic viscosity of the disordered form was smaller than the ordered form, its voluminosity must be less important, therefore its hydration capacity was smaller. This hypothesis may explain the water uptake results, as well as the viscosity results. Regarding the molecular weight results, it seemed that even in the disordered form or in a degraded state, xanthan molecules aggregated. It is possible that this aggregation state is associated to or at least backed up by the spray-drying process, which would explain the dierences noted on the viscosity values (the viscosity of the rehydrated spray-dried powder solutions being higher than their corresponding original solutions). The structure of the disordered conformation is still unknown. Southwick et al. (1982) suggested that the disordered conformation present in heat-treated 4 M urea solution was not a random coil but an extended chain possessing no local secondary structure. Nevertheless, recent electron microscopic studies of xanthan presumably under disordering conditions (10ÿ4 M NH4Ac) might have indicated that the disordered samples were a mixture of species ranging from purely single and perfectly matched double-stranded chains branching into their two subchains as well as dierent degrees of mismatched chains (Harrington & Zimm, 1965). Other results on the eect of mechanical treatment on biopolyiners (tragacangh) also demonstrated that micro¯uidization induced modi®cations. These modi®cations have not been associated with polymer degradation, but more to a random scission kinetics because
of the non-®rst-order reaction, or that the energies supplied by micro¯uidization are in part used to shear or tear open the long chains. After the polymer chains are open they would become more susceptible to micro¯uidization-induced degradation as a greater number of bonds are directly accessible to the mechanical stress pockets produced during micro¯uidization (Harrington & Zimm, 1965). 4. Conclusions We postulated that micro¯uidization induced an ordered±disordered conformation transition, associated with polymer degradation. This latter is irreversible or, at least, not completely reversible. Heat treatment due to spray-drying aected the properties of xanthan aggregates or xanthan molecules by changing the interactions inside and between particles. Micro¯uidized xanthan showed weaker thickening and stabilizing properties (decrease in viscosity and pseudoplastic behaviour, decrease in hydration rate and water uptake). These results are interesting for the modi®cation of the thickening properties of polysaccharides by changing the average molecular weight. It is also of interest for the formation of complexes (protein±polysacecharide) of dierent size giving the possibility of a wide range of textures. Acknowledgements The authors thank the National Science Engineering Research Council of Canada for its ®nancial support (Industrial chair program) and the industrial partners (Novalait Inc., CQVB, Ault Foods Ltd, Agropur). References Anon (1988). In Xanthan gum: natural biogum for scienti®c water control. Rahway, NJ: Kelco Division, Merk & Co. Inc. AOAC (1990). Ocial Methods of Analysis Association of the AOAC (15th ed.). Association of Ocial Analytical Chemist. Virginia, USA. Barnes, H. A., Hutton, J. F., & Walters, K. (1989). An Introduction to Rheology (p. 19). New York: Elsevier. Cencia-Rohan, L., & Silvesti, S. (1993). International Journal of Pharmacology, 95, 23±28. Chandonnet, S., Korstvedt, H., & Siciliano, A. A. (1985, February). Soap. Cosmet. Chem. Spec., 37±38. Chazeau, L., Milas, M., & Rinaudo, M. (1995). Int. J. Polym. Anal. Characterization, 2, 21±29. Cook, E. J., & Lagace, A. P. (1985). US Pat., 4, 533, 254. Frangou, S. A, Morris, E. R., Rees, D. A., Richardson, R. K., & Ross-Murphy S. B. (1982). J. Polym. Sci.: Polym. Lett. Edn, 20, 531±538. Harrington, R., & Zimm, B. (1965). Journal of Physic Chemistry, 69, 161±175.
N. Lagoueyte, P. Paquin/Food Hydrocolloids 12 (1998) 365±371 Kitamura, S., Takeo, K., Kuge, T., & Stokke, B. T. (1991). Biopolymers, 31, 1243±1255. Kneifel, W., Paquin, P., Albert, T., & Richard, J. P. (1991). J. Dairy Sci., 74, 2027±2041. Lambert, F., Milas, M., & Rinaudo, M. (1985). Int. J. Biol. Macromol., 7, 49±52. Launay, B., Doublier, J. L., & Cuvelier, G. (1986). In J. R. Mitchell & A. Ledward (Eds.), Functional Properties of Food Macromolecules pp. 1±78. London: Elsevier Applied Science. Lecourtier, J., Chauveteau, G., & Muller, G. (1986). Int. J. Biol. Macromol., 8, 306±310. Lim, T., Uhl, J. T., & Prud'homme, R. K. (1984). J. Rheol., 28, 367±379. Liu, T., Sato, T., Norisuye, T., & Fujita, H. (1987). Carbohydr. Res., 160, 267±281. Milas, M., & Rinaudo, M. (1979). Carbohydr. Res., 76, 189±196. Milas, M., & Rinaudo, M. (1981). ACS Symp. Ser., 150, 25±30. Milas, M., & Rinaudo, M. (1986). Carbohydr. Res., 158, 191±204. Milas, M., Rinaudo, M., & Tinland, B. (1986). In G. O. Phillips, D. J. Wedlock & P. A. Williams (Eds.), Gums and stabiliser for the food industry (Vol. 3, pp. 637±644). Oxford: Elsevier. Milas, M., Reed, W. F., & Printz, S. (1996). Int. J. Biol. Macromol., 18, 211±221. Mitchell, J. R. (1979). In J. M. V. Blanshard & J. R. Mitchell (Eds.), Polysaccharides in food (pp. 51±72). Butterworths, London. Morris, E. R. (1977). In R. F. Gould (Ed.), Extracellular Microbial Polysaccharides, pp. 81±89. Washington, DC. Morris, E. R., Rees, D. A., Young, G., Walkinshaw, M. D., & Darke, A. (1977). J. Mol. Biol., 110, 1±16.
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Morris, E. R., Franklin, D., & L'Anson, K. (1983). Carbohydr. Res., 121, 13±30. Muller, G., Anrhourrache, M., Lecourtier, J., & Chauveteau, G. (1986). Int. J. Biol. Macromol., 8, 167±172. Norton, I. T., Goodall, D. M., Frangou, S. A., Morris, E. R., & Rees, D. A. (1984). J. Mol. Biol., 175, 371±394. Paquin, P., & Giasson, J. (1989). Le Lait, 69, 491±498. Paquin, P., Lebeuf, Y., Richard, J. P., & Kalab, M. (1993). In Protein and fat globule modi®cations, pp. 389±396. Special issue 9303, Brussels: International Dairy Federation. Paradossi, G., & Brant, D. A. (1982). Macromolecules, 15, 874± 879. Richardson, R. K., & Ross-Murphy, S. B. (1987). Int. J. Biol. Macromol., 9, 257±264. Robin, O., Blanchot, V., Vuillemard, J. C., & Paquin, P. (1992). Le Lait, 72, 511±531. Rochefort, W. E., & Middleman, S. (1987). J. Rheol., 31, 337±369. Sato, T., Norisuye, T., & Fujita, H. (1984). Polym. J., 16, 341±350. Sato, T., Norisuye, T., & Fujita, H. (1985). Polym. J., 17, 729±735. Schorsch, C., Garnier, C., & Doublier, J. L. (1995). Carbohydr. Polym., 28, 319±323. Silvesti, S., Gabrielson, G. (1991). Int. J. Pharm., 73, 163±169. Stokke, B. T., Smidsrod, O., & Elgsaeter, A. (1989). Biopolymers, 28, 617±637. Stokke, B. T., & Christensen, B. E. (1996). Food Hydrocoll., 10, 83± 89. Southwick, J. G., Jamieson, A. M., & Blackwell, J. (1982). Carbohydr. Res., 99, 117±127.
Eects of micro¯uidization on the functional properties of xanthan gum N. Lagoueyte, P. Paquin* Centre de recherche en sciences et technologie du lait (STELA), DeÂpartement de sciences des aliments et de nutrition, Universite Laval, Pavilion Paul-Comtois, QueÂbec, QueÂbec G1K 7P4, Canada Received 5 February 1997; received in revised form 16 May 1997; accepted 19 December 1997
Abstract The eects of micro¯uidization on xanthan gum were studied by ¯ow behaviour, hydration rate, water uptake and molecular weight determinations. The eect of pressure and number of passes on the xanthan solution produced a decrease in all these functional properties. Consequently the thickening and, stabilizing properties were reduced. We argued that the high shear, turbulence forces and cavitation involved in the micro¯uidization process produced ordered±disordered conformational transition (by opening of the molecule) and polymer degradation. The opening of the molecule occurred ®rst, followed by polymer degradation due to mechanical stress. Dierences observed in viscosity between treated xanthan dispersion and their spray-dried powders is associated with reorganisation of xanthan aggregates during heating and cooling, which occurred in spray-drying, but these phenomena do not change the high pressure eect on the biopolymer. # 1998 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Xanthan gum is an hydrocolloid widely used in food industries for its thickening and stabilizing properties, and for its unusual stability towards temperature, ionic strength, pH, enzymatic and chemical reactive (Anon., 1988). It is an exocellular heteropolysaccharide consisting in a linear backbone of 1-4 linked b-d glucose residues having a trisaccharide side-chain attached to alternative d glucosyl residues, and it is produced by a distinct fermentation process of Xanthomonas campestris. Nevertheless the knowledge on its composition and secondary structure is still under discussion. Indeed, in aqueous solutions, xanthan undergoes an ordered±disordered conformational change depending on temperature, ionic strength, counterion valence and pH, as well as acetyl and pyruvyl substituent content (Lambert, Milas, & Rinaudo, 1985; Milas & Rinaudo, 1981; Morris, 1977; Paradossi & Brant, 1982). Many authors suggested that the ordered form is a double-stranded helix, and that the conformation transition induced the complete or * Corresponding author.
partial dissociation of the double helix into single chains (Kitamura, Takeo, Kuge, & Stokke, 1991; Liu, Sato, Norisuye, & Fujita, 1987; Sato, Norisuye, & Fujita, 1987). Other results suggested that the ordered conformation is a single strand (Morris, Rees, Young, Walkinshaw, & Darke, 1977; Norton, Goodall, Frangou, Morris, & Rees, 1984) and that below the melting temperature, ordered and disordered regions co-exist within the same xanthan molecule (Norton et al., 1984). Recent studies (Chazeau, Milas, & Rinaudo, 1995; Milas, Reed, & Printz, 1996) proposed that the native xanthan conformation is single-stranded whereas re-natured xanthan conformation (xanthan obtained by puri®cation including heat treatment) is a doublestranded helix. Its has been postulated (Richardson & Ross-Murphy, 1987) that rheological properties of xanthan solution are consistent with a picture of a tenuous network, which from other evidence involves non-covalent intermolecular forces. The exact nature of these forces still remains to be completely established, and possibly a balance between hydrogen bonding, hydrophilic/hydrophobic interactions and cation-speci®c interactions. However, others workers (Lim, Uhl, & Prud'homme, 1984; Milas & Rinaudo,
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1979; Schorsch, Garnier, & Doublier, 1995) have suggested that the unusual rheological properties of xanthan solutions for concentrations of 1% and above are due to the formation of liquid crystalline domains, these structures being disrupted by shear rates greater than 0.1 sÿ1. Micro¯uidization technology is a very unique type of high pressure homogenization: the reaction chamber is built in such a way that the liquid is divided into two microstreams that are projected against one another at high speed and at an angle of 180 (Cook & Lagace, 1985). This technique is currently used by the pharmaceutical and cosmetics industries to produce ®ne emulsions (Chandonnet, Korstvedt, & Siciliano, 1985). Possible applications in production of dairy-based products have also been reported (Paquin & Giasson, 1989; Paquin, Lebeuf, Richard, & Kalab, 1993; Robin, Blanchot, Vuillemard, & Paquin, 1992). Other workers have also demonstrated that micro¯uidization can aected polymeric structure for small polymers (Silvesti & Gabrielson, 1991). The aim of the present work was to study the in¯uence of the mechanical treatment (micro¯uidization) on the characteristics of xanthan. 2. Materials and methods 2.1. Micro¯uidized xanthan Xanthan gum (Ketrol F, Keleo Division of Merck and Co. Inc., San Diego, CA) was dispersed in distilled water (1.0% w/w) and stirred at room temperature for 1 h. The solution was then kept at 4 C for another 12 h to achieve complete hydration. The micro¯uidization treatment was carried out with a micro¯uidizer (model M-1102, Micro¯uidic Corporation, Newton, USA) at 75 MPa. The solution was recirculated for dierent numbers of passes (0, 2, 4, 8, 12, 16 and 20 passes). The micro¯uidizer is a recent high pressure homogenization technology developed in the early 1980s (Cook & Lagace, 1985). The principle is: the liquid is divided in two jet streams, which enter microchannels where they are subjected to shear forces. After
that, the jet streams are directed in an area where there is liquid/liquid contact, and the liquids are submitted to cavitation and turbulence due to pressure drop when the homogenized liquid passes out of the reaction chamber (Fig. 1; Paquin & Giasson, 1989). During the experience, the temperature is kept constant (23 C) by immersing in ice the reaction chambers and the coiled heat exchanger is connected at the outlet of the micro¯uidizer. The micro¯uidized solutions are spray-dried (Niro Atomizer, Copenhagen, Denmark) at an inlet temperature of 200 C and an outlet temperature of 80± 85 C. The powders are stored in sealed containers and are kept at 4 C until used. The moisture content of the dry material determined by drying at 103 C for 18 h, was 11.00.5% (AOAC, 1990). 2.2. Size exclusion chromatography (SEC) SEC characterizations were carried out on a HP 1050 apparatus (Hewlett±Packard Ltd, Mississauga, Canada) with a TSK gel 600OPWXL column (Hosohaas, Montgomeryville, USA) and a dierential refractometer as the detector (HP 10474). Powder samples were dissolved at 0.5 mg/ml in NH4NO3 (0.1 M), with 0.5 g/l Na3N as preservative, at room temperature, and stirred for 2 h. The solutions were stored at 4 C for 12 h before use. After sonication (50±60 Hz, 30 min), the solutions were ®ltered through 0.45 mm membrane ®lters (Nucleopore MF Canada Inc., Toronto). Typically, 20 ml of xanthan sample was injected for each SEC run. Molecular weights were determined by extrapolation of a calibration curve, obtained with Pullulan standards (American Polymer Standars Corporation, Mentor, USA). All measurements were duplicated and performed at 30 C with an elution rate of 0.5 ml/min. 2.3. Water binding of xanthan by the Baumann apparatus Spontaneous water uptake was determined by a modi®cation of the Baumann method according to Kneifel, Paquin, Albert, & Richard (1991). A small amount of powdered sample, in the range of 2±12 mg,
Fig. 1. Shematic representation of the reaction chamber of the micro¯uid equipment.
N. Lagoueyte, P. Paquin/Food Hydrocolloids 12 (1998) 365±371
was spread in a thin layer onto a Nucleopore MF membrane (Nucleopore Canada Inc., Toronto), which was then placed on the plastic ®lter of the apparatus. The membranes used were mixed esters of cellulose with an average pore diameter of 0.1 mm. Water uptake, as measured by the calibrated capillary (2.000.01 ml), was recorded as a function of time for 60 min. A value of 0.0130.001 ml/h water uptake was found for the blank value and attributed to evaporation. It was evaluated by measuring the water uptake by three membranes on the Baumann device. This small value was considered negligible when compared to xanthan water uptake and was not considered in further calculations.
367
likely by strengthening both polymer±polymer and polymer±water interactions during the atomization process. All the samples (ketrol F, non-micro¯uidized spraydried, and micro¯uidized) showed a typical pseudoplastic ¯ow behavior associated to the formation of a weak, tenuous, gel-like network. We observed an important dierence between the non-micro¯uidized samples (commercial product ketrol F and spray-dried product) and the micro¯uidized samples. The results presented in Fig. 3 demonstrated that the ¯ow behavior of the micro¯uidized samples is changed as the samples are submitted to more severe treatment from 0 to 20 passes through the micro¯uidizer. The dependence of the
2.4. Rheological measurements The dry powders were suspended in distilled water (1% w/w) and stirred at room temperature for 1 h. Subsequently, the solutions were stored at 4 C for 12 h before use. The ¯ow behaviour of each micro¯uidized xanthan gum solution prior to drying and rehydrated micro¯uidized powders was determined with a controlled stress viscometer (Carri-Med CS500, Westech Industrial Ltd, Mississauga, Canada), equipped with cone-and-plate geometry. The stainless steel cone employed was CP 5544 (40 mm diameter and 1.58 C). Three independent tests were carried out with a fresh sample of each solution at 25 C and the shear stress was increased linearly from 0 to 15 Pa in 10 min. 3. Results and discussion 3.1. Flow characterization In order to be able to measure functional properties (molecular weight/HPLC, hydration properties/Bauman), micro¯uidized xanthan solutions were spraydried. We also spray-dried the non-treated xanthan solution to compare it to the commercial xanthan product (ketrol F). The rehydrated non-micro¯uidized, spray-dried solution and the commercial product (ketrol F) solution show small dierences in viscosity pro®le (Fig. 2). The spray-dried xanthan higher viscosity is related to the fact that the spray-dried product gives spherical particles when it is dried, compared to commercial product that is dried by alcohol drying, which gives a ®ber-like product. Heating involved the modi®cation of the state of aggregation and, eventually, reversible conformational change. On subsequent cooling, an alternative pattern of side chain±backbone interactions, presumably dictated by kinetic rather than thermodynamic factors, was established (Milas & Rinaudo, 1986). We argue that the reorganization of the aggregates led to a stronger gel-like formation, most
Fig. 2. Viscosity of commercial and spray-dried xanthan versus shear rate.
Fig. 3. Flow behavior of rehydrated micro¯uidized spray-dried xanthan solutions.
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rheology must be related to the orientation and the deformation of the molecules and the modi®cations of the interchain interactions (Mitchell, 1979). Launay, Doublier, & Cuvelier (1986) have suggested that xanthan molecules may exist at rest in an equilibrium state of aggregation and that the extent of aggregation was reduced on shearing. The eect of shear rate on the viscosity of xanthan gum solutions may be described by the following power-law model (Barnes, Hutton, & Walters, 1989): K _ nÿ1
1
where =apparent viscosity, K=consistency index, _
=shear rate and n=¯ow behavior index. Fig. 4 shows the eect of shear rate on the viscosity of micro¯uidized xanthan gum according to Eq. (1). The viscosity decreased as the shear rate increased, the consistency index tended to decrease with the number of passes, whereas the ¯ow behavior index increased (Table 1). The pseudoplastic behaviour was less and less marked with the increase of the number of passes. With reference to K and n values, the micro¯uidization had no more signi®cant eect on viscosity beyond eight passes for the micro¯uidized xanthan dispersions and beyond 12 passes for the rehydrated micro¯uidized spray-dried solutions. These dierences were possibly associated with the fact that spray-dried samples have a larger aggregation state than the dispersion samples. 3.2. Molecular weight determination The values of average molecular weight obtained were higher than those in the literature (25106). Southwick, Jamieson, & Blackwell (1982) noted that the large discrepancies reported in the studies on molecular weight for xanthan (2106±50106) probably resulted
Table 1 Consistency index and ¯ow behavior index of xanthan gum dispersions Micro¯uidized dispersions Number of passes ketrol F 0 2 4 8 12 16 20 a
n
K
r2
0.218 0.572 0.587 0.641 0.630 0.627 0.614
4.135 0.348 0.296 0.195 0.196 0.215 0.226
0.952 0.989 0.994 0.986 0.992 0.983 0.978
Rehydrated micro¯uidized (spray-dried) n
r2
K
(0.214)a (4.094) (0.969) 0.189 5.572 0.992 0.350 1.555 0.988 0.375 1.331 0.987 0.399 1.218 0.996 0.464 0.786 0.994 0.473 0.699 0.991 0.456 0.829 0.994
Values in parentheses are for commercial ketrol F product.
from aggregation in some of the xanthan solutions examined. In addition, Muller, Anrhourrache, Lecourtier, & Chauveteau (1986) showed that dried product had a greater tendency for microgel formation than the corresponding xanthan gum obtained from the fermentation broth. This can lead to unusually high values of molecular weight (i.e. large aggregates), which in turn should aect the ¯ow properties of the solutions. Milas et al. (1996) suggested that it was the species of molecular weight 1.2106 which was synthesized by the bacterial cells that later aggregated and formed the polydisperse distributions of very high molecular weight observed in the culture media. Results (Table 2) showed that the micro¯uidization process decreased the average molecular weight of xanthan samples. The average molecular weight did not decrease linearly with the number of passes; the decrease being more important for the ®rst passes, additional passes having less and less impact on the average molecular weight (Table 2). For instance, after two passes, the average molecular weight was approximately reduced 2 times, whereas after 20 passes, the diminution was around 6 times. The average molecular weight was aected little by the micro¯uidization process Table 2 Average molecular weights of xanthan powder samples Samples
Fig. 4. Viscosity of rehydrated micro¯uidized spray-dried xanthan solutions versus shear rate.
Commercial product ketrol F SPOM spray-dried SP2M SP4M SP8M SP12M SP16M SP20M
Molecular weights (10ÿ3)
MW of P0M/MW of sample
25,871 26,288 15,149 10,889 7,971 5,396 4,858 4,485
SP spray-dried; 0±20: number of passes; M: micro¯uidized.
1.73 2.41 3.30 4.87 5.41 5.86
N. Lagoueyte, P. Paquin/Food Hydrocolloids 12 (1998) 365±371
beyond 12 passes. Also the average molecular weight of non-micro¯uidized spray-dried sample was higher but not very dierent than the commercial product (ketrol F). 3.3. Hydration rate measurements The Baumann apparatus did not allow a precisely measure of the equilibrium time of hydration due to diculty in: (a) the weight of xanthan powder put on the ®lter needed at least 2±12 mg, which absorbed a volume of water greater than the calibrated capillary used, and (b) increasing the volume of the capillary did not allow precise measurement of the water volume. Water uptake versus time measured by Baumann capillary apparatus for dierent weights of sample are shown in Fig. 5. Water uptake signi®cantly depended on sample weight: the absorption value appeared progressively smaller as the sample weight increased. The same phenomenon was observed for the other powder samples. The decrease in water uptake with increasing sample weight was at ®rst surprising because xanthan is known to be one of the best water binders among hydrocolloids. Xanthan gum is also among the best thickening agent. On the Baumann apparatus, the hydration of xanthan powder led to a weak gel-like formation on the membrane surface, which reduced water uptake in a more pronounced manner when increasing the sample weight. This observation underlined the importance of constant weight for this test in order to obtain comparable results. In view of the impossibility to put on the membrane a determined sample weight (the membrane must be water-saturated), we could not easily compare samples. Nevertheless, for 6±8 dierent weights of the same powder samples (over a 2±12 mg sample range), a linear relationship was found between the water uptake at a given time and the weight of the sample, regression coecients at a given time being higher than 0.95. From
Fig. 5. Water uptake versus time for dierent weights (mg) for commercial xanthan (ketrol F).
369
these observations, we have drawn the curve for a weight set arbitrarily at 5 mg (Fig. 6). Hydration properties were aected by the micro¯uidization process. Powder samples showed the same hydration pro®le (hydration rate was initially rapid and slowed as equilibrium was approached), but increasing the number of passes decreased the hydration rate and the water uptake. We also noticed that, for an equivalent average molecular weight, the non-micro¯uidized spray-dried sample had both higher hydration rate and water uptake, and was more viscous than the commercial (ketrol F) solution. This observation underlines the importance of thermal history on the functional properties of xanthan samples. We argue that heating involved rearrangements of the xanthan aggregates and therefore a modi®cation of the interactions which stabilized the initial xanthan conformation. 3.4. Tentative explanations Studies have shown that, under certain conditions such as low ionic strength (Lecourtier, Chauveteau, & Muller, 1986; Stokke & Christensen, 1996), heat-treated 4 M urea solutions (Frangou, Morris, Rees, Richardson, & Ross-Murphy, 1982; Morris, Franklin, & L'Anson, 1983; Southwick, Jamieson, & Blackwell, 1982), ultracentrifugation in the presence of 4 M urea (Morris et al., 1983) and cadoxen solutions (Sato, Norisuye, & Fujita, 1984), the order±disorder transition was irreversible. The disordered form had dierent properties than the ordered form, for instance, smaller intrinsic viscosity (Milas, Rinaudo, & Tinland, 1986; Sato et al., 1984; Southwick et al., 1982) and smaller viscosity values (Frangou et al., 1982; Southwick et al., 1982; Morris et al., 1983; Milas et al., 1986; Rochefort & Middleman, 1987). Also, the degradation of low and
Fig. 6. Theoretical curves of water uptake versus time of spray-dried xanthan (sample weight set at 5 mg). SP: spray-dried; 0±20: number of passes; M: micro¯uidized.
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high average molecular weight polymers induced by high shear processing has been reported previously (Silvesti & Gabrielson, 1991; Cencia-Rohan & Silvesti, 1993). This degradation process occurs primarily in the presence of simultaneous intense shear and turbulent mixing forces and is believed to be the result of exposure of covalent bonds to mechanical stresses during viscous ¯ow under harsh conditions (Silvesti & Gabrielson, 1991). Precisely controlled shear, turbulent and cavitation forces are generated within the micro¯uid chambers. These forces have been shown to be of sucient magnitude to cause mechanical degradation of some polymers (tragacanth) (Cencia-Rohan & Silvesti, 1993; Stokke, Smidsrod, & Elgsaeter, 1989). We postulate that the turbulence, cavitation, and high shear stress of the micro¯uidization process have an eect on the conformation of the molecule and, consequently, induced an ordered±desordered conformation transition and degradation of the molecule which is irreversible. We suggest that conformational changes occurred and that the percentage of disordered form may have increased with pressure increase and the number of passes up to a limit where polymer degradation can occur; the degree of reversibility depended upon the intensity of the process. This new conformation was stabilized by intramolecular interactions. As intrinsic viscosity of the disordered form was smaller than the ordered form, its voluminosity must be less important, therefore its hydration capacity was smaller. This hypothesis may explain the water uptake results, as well as the viscosity results. Regarding the molecular weight results, it seemed that even in the disordered form or in a degraded state, xanthan molecules aggregated. It is possible that this aggregation state is associated to or at least backed up by the spray-drying process, which would explain the dierences noted on the viscosity values (the viscosity of the rehydrated spray-dried powder solutions being higher than their corresponding original solutions). The structure of the disordered conformation is still unknown. Southwick et al. (1982) suggested that the disordered conformation present in heat-treated 4 M urea solution was not a random coil but an extended chain possessing no local secondary structure. Nevertheless, recent electron microscopic studies of xanthan presumably under disordering conditions (10ÿ4 M NH4Ac) might have indicated that the disordered samples were a mixture of species ranging from purely single and perfectly matched double-stranded chains branching into their two subchains as well as dierent degrees of mismatched chains (Harrington & Zimm, 1965). Other results on the eect of mechanical treatment on biopolyiners (tragacangh) also demonstrated that micro¯uidization induced modi®cations. These modi®cations have not been associated with polymer degradation, but more to a random scission kinetics because
of the non-®rst-order reaction, or that the energies supplied by micro¯uidization are in part used to shear or tear open the long chains. After the polymer chains are open they would become more susceptible to micro¯uidization-induced degradation as a greater number of bonds are directly accessible to the mechanical stress pockets produced during micro¯uidization (Harrington & Zimm, 1965). 4. Conclusions We postulated that micro¯uidization induced an ordered±disordered conformation transition, associated with polymer degradation. This latter is irreversible or, at least, not completely reversible. Heat treatment due to spray-drying aected the properties of xanthan aggregates or xanthan molecules by changing the interactions inside and between particles. Micro¯uidized xanthan showed weaker thickening and stabilizing properties (decrease in viscosity and pseudoplastic behaviour, decrease in hydration rate and water uptake). These results are interesting for the modi®cation of the thickening properties of polysaccharides by changing the average molecular weight. It is also of interest for the formation of complexes (protein±polysacecharide) of dierent size giving the possibility of a wide range of textures. Acknowledgements The authors thank the National Science Engineering Research Council of Canada for its ®nancial support (Industrial chair program) and the industrial partners (Novalait Inc., CQVB, Ault Foods Ltd, Agropur). References Anon (1988). In Xanthan gum: natural biogum for scienti®c water control. Rahway, NJ: Kelco Division, Merk & Co. Inc. AOAC (1990). Ocial Methods of Analysis Association of the AOAC (15th ed.). Association of Ocial Analytical Chemist. Virginia, USA. Barnes, H. A., Hutton, J. F., & Walters, K. (1989). An Introduction to Rheology (p. 19). New York: Elsevier. Cencia-Rohan, L., & Silvesti, S. (1993). International Journal of Pharmacology, 95, 23±28. Chandonnet, S., Korstvedt, H., & Siciliano, A. A. (1985, February). Soap. Cosmet. Chem. Spec., 37±38. Chazeau, L., Milas, M., & Rinaudo, M. (1995). Int. J. Polym. Anal. Characterization, 2, 21±29. Cook, E. J., & Lagace, A. P. (1985). US Pat., 4, 533, 254. Frangou, S. A, Morris, E. R., Rees, D. A., Richardson, R. K., & Ross-Murphy S. B. (1982). J. Polym. Sci.: Polym. Lett. Edn, 20, 531±538. Harrington, R., & Zimm, B. (1965). Journal of Physic Chemistry, 69, 161±175.
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