Microsoft Word - 8-revisado-558.doc Original Article Biosci. J., Uberlândia, v. 23, n. 4, p. 67-73, Oct./Dec. 2007 67 XANTHAN SYNTHESIZED BY STRAINS OF Xanthomonas campestris pv pruni: PRODUCTION, VISCOSITY AND CHEMICAL COMPOSITION XANTANA SINTETIZADA POR CEPAS DE Xanthomonas campestris pv pruni: PRODUÇÃO, VISCOSIDADE E COMPOSIÇÃO QUÍMICA Caroline Dellinghausen BORGES1, Claire Tondo VENDRUSCOLO1, 2 1. Departamento de Ciência e Tecnologia Agroindustrial, Universidade Federal de Pelotas – UFPel , Pelotas – RS. caroldellin@bol.com.br . 2. Centro de Biotecnologia - UFPel ABSTRACT: The aim of this work was to characterize the xanthan produced by 30 strains of the Xanthomonas campestris pv pruni, evaluating the production, viscosity and chemical composition. Xanthan production by each strain was evaluated through weight of the dry product per volume of broth fermented (g.l-1); the viscosity of 3 % (w/v) aqueous solution of the biopolymers was measured in rheometer rotating mode at 25ºC and the chemical composition was determined by comparative thin-layer chromatography (TLC). Xanthan production was strain dependent and the highest production reached 9.2 g.l-1 for strain 83 and the lowest 3.8 g.l-1 for strain 51. Strain 107 did not produce xanthan. The aqueous solutions of the xanthans produced showed pseudoplastic behavior. The xanthan synthesized by strain 101 showed the highest viscosity and strain 83 produced the xanthan with the lowest viscosity. All the xanthans synthesized contained glucose, rhamnose, mannose and glucuronic acid in their chemical composition, differently from commercial xanthan, that do not contain rhamnose. There was not relation between yield, viscosity and chemical composition. The yield obtained was not appropriate for industrial production according to the literature, but the viscosity of the aqueous solutions of the xanthans synthesized by sixteen strains was higher than the commercial xanthan, compensating for the low production. KEYWORDS: Chemical composition. Production. Viscosity. Xanthan. Xanthomonas campestris pv pruni. INTRODUCTION Xanthan is an extracellular polysaccharide produced by several pathovars of Xanthomonas campestris and by other species of Xanthomonas (SUTHERLAND, 1993). The isolation and screening of Xanthomonas strains from natural habitats is still the most efficient method of identifying strains with high capacity of xanthan production and/or high rheological quality (TORRESTIANA; FUCIKOVSKY; GALINDO, 1990; NITSCHKE; THOMAS, 1995; GUPTE; KAMAT ,1997; SÁNCHEZ et al., 1997; MOREIRA et al., 2001; ANTUNES et al., 2003). Other criteria have been questioned for the selection of strains such as colony morphologic characteristics and virulence (GALINDO, 1994a; NITSCHKE; RODRIGUES, 2000). Modern methods of molecular biology as the techniques of Amplified Fragment Length Polymorphism (AFLP) and Random Amplified Polymorphic (DNA-RAPD) were used without success to correlate the genetic profile with xanthan production, chemical composition and rheological behavior (TESSMANN, 2002; MAYER, 2006). According to Sánchez et al. (1997), most of the literature regarding xanthan gum production is based on strains obtained from culture collections, mainly Xanthomonas campestris NRRL B – 1459 and its derivatives. There are very few reports dealing with the characterization of novel Xanthomonas strains. The results obtained previously have shown that several strains of X. campestris pathovar pruni have potential for commercial production of xanthan polymer (SOUZA; VENDRUSCOLO, 1999; MOREIRA et al., 2001; ANTUNES et al., 2003), justifying the importance of research with this pathovar. Xanthan, synthesized by the bacterium X. campestris pv campestris NRRL B-1459, is constituted by D-glucose, D-mannose, D-glucuronic acid and residues of acetic and pyruvic acid (SLONECKER; JEANES, 1962). However, some species and pathovars produce gums with other sugars in their constitution (ORENTAS; SLONEKER; JEANES, 1963; KONICEK; LASÍK; WURST, 1977; LOWSON; SYMES, 1977; SOUZA; VENDRUSCOLO, 1999). The commercial interest in the xanthan gum is due to its rheological properties (ROCKS, 1971; GALINDO, 1994a; GARCÍA-OCHOA et al., 2000). These properties are sometimes measured as an indicator of product quality (MARCOTTE; HOSHAHILI; RAMASWAMY, 2001). The species, pathovar and strain influence of Xanthomonas in the rheological behavior of polymer produced has been investigated (TORRESTIANA; FUCIKOVSKY; Received: 04/12/06 Accepted: 11/09/07 Xanthan synthesized... BORGES, C. D. ; VENDRUSCOLO, C. T. Biosci. J., Uberlândia, v. 23, n. 4, p. 67-73, Oct./Dec. 2007 68 GALINDO, 1990; SÁNCHEZ et al., 1997; NITSCHKE; RODRIGUES, 2000; ANTUNES et al., 2003). The objective of this work was to evaluate the xanthan production, in orbital shaker, for thirty strains of the bacterium Xanthomonas campestris pv pruni, determining the production, viscosity and chemical composition of the biopolymers obtained. MATERIAL AND METHODS Microorganism Strains 12, 19, 25, 26, 27, 30, 38, 42, 51, 53, 55, 61, 77, 78, 81, 83, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 112, 113, 114 and 115 of Xanthomonas campestris pv pruni had not been tested for the biopolymer production. These strains were isolated from plum and peach leaves and fruits at the Centro de Pesquisa Agropecuaria de Clima Temperado (EMBRAPA, Pelotas, Brazil). The standard strain NRRL B-1459 of Xanthomonas campestris obtained from of Foundation for Research and Technology André Tosello, Campinas, Brazil, was used for comparison of the production results. Culture media A) Maintenance of cells in agar SPA, containing (g.l-1): sucrose 20.0; peptone 5.0; K2HPO4 0.5; MgSO4 7H2O 0.25; agar 16.0 (HAYWARD, 1964). B) Production of cells in liquid media YM, containing (g.l-1): malt extract 3.0; yeast extract 3.0; peptone 5.0; glucose 10.0 (HAYNES; WICKERHAM; HESSELTINE, 1955). C) Medium of biopolymers production MPII, containing (g.l-1): (1) NH4H2PO4 1.5; K2HPO4 2.5; MgSO4 7H2O 0.2; (2) sucrose 50.0 (CADMUS et al, 1978 modified by VENDRUSCOLO et al., 2000). The media (1) was adjusted to pH 7.0 and the two solutions were sterilized separately at 121ºC for 15 min. Commercial xanthan Commercial xanthan was used for comparing of the viscosity results and chemical composition. Xanthan production The biopolymer production was made by aerobics fermentation in batch, in orbital shaker (New Brunswick Scientific model Innova 4230), at 28°C and 200 rev. min–1 for 72 h (SOUZA; VENDRUSCOLO, 1999). The experiment was carried out in triplicate. The fermented broth was centrifuged for cell removal (Sorvall Instruments model RC-5C) at 4oC, 16 000 g during 30 min. Ethanol 96 GL was added to the supernatant in the proportion of 1:4 (v/v) for recovery of the biopolymer. These were dried at 56°C until reaching constant weight and subsequently ground in a disk mill (Fritsch model Pulverisette), until granulometry of 0.5 µm. The production of the biopolymers of each strain was evaluated by the weight of the dry product per liter of fermented broth and the averages expressed in g.l-1. Viscosity The viscosity of 3 % (w/v) aqueous solutions of the xanthans synthesized by 29 strains of X. campestris pv pruni was compared with the viscosity of xanthan synthesized by standard strain X. campestris NRRL B-1459 and with dialyzed and not dialyzed samples of a commercial xanthan. The sample was dialyzed to remove its salt contents, against ultra pure water for 48 h, at 4°C. The solutions of the xanthans were agitated for 2 h, heated up to 60ºC during 20 min and kept in rest at room temperature for 24 h (XUEWU et al., 1996). The viscosity was determined in rheometer rotating mode (HAAKE model RS150), at 25ºC. A plate- plate system was used, with a sensor PP35Ti and shear rate of 0.01-100 s-1; the time of each assay was 300 s. Chemical composition The polymers synthesized by the 29 strains of X. campestris pv pruni and the dialyzed and not dialyzed samples of commercial xanthan were hydrolyzed using 2 N HCl [3:100 (w/v)] at 80ºC for 16 h. The chemical composition of the biopolymer was determined through comparative thin-layer chromatography (TLC) to the comparison with authentic samples of glucose, rhamnose, mannose and glucuronic acid. Three µL were applied from the hydrolyzed samples and from the standards in silica gel 60 F254 plates (Merck) and eluted with chloromethane: methanol: acetic acid: water, in the proportion of 40:40:10:10 (v/v/v/v). For revelation the reagent used was sulfuric-anisaldehyde (MOREIRA; SOUZA; VENDRUSCOLO, 1998). RESULTS AND DISCUSSION Production The analysis of the results showed that the xanthan production by X. campestris pv pruni was influenced by the strain. The values in g.l-1 varied between 3.8 (strain 51) and 9.2 (strain 83), the strain 107 did not produce xanthan, as shown in Figure 1. Xanthan synthesized... BORGES, C. D. ; VENDRUSCOLO, C. T. Biosci. J., Uberlândia, v. 23, n. 4, p. 67-73, Oct./Dec. 2007 69 6,4 5,5 5,0 4,84,74,8 5,8 7,4 6,4 6,1 7,4 4,6 6,5 4,1 4,6 5,2 0,0 6,8 5,8 7,8 4,4 6,66,6 7,0 6,9 9,2 3,6 6,3 5,0 4,0 5,0 0 1 2 3 4 5 6 7 8 9 10 1 2 1 9 2 5 2 6 2 7 3 0 3 8 4 2 5 1 5 3 5 5 6 1 7 7 7 8 8 1 8 3 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 1 0 7 1 0 8 1 0 9 1 1 2 1 1 3 1 1 4 1 1 5 1 4 5 9 Strain Figure 1. Xanthan production (g.l-1) by strains of X. campestris pv pruni and standard strain X. campestris NRRL B-1459. These results agree with the previous findings of others. Torrestiana et al. (1990) studied xanthan production by six isolated of X. campestris pv campestris and obtained yields varying from 0.0 to 8.0 g.l-1. Sánchez et al. (1997) evaluated xanthan production by several Xanthomonas campetris pathovars. For campestris pathovar the production oscillated between 3.4 and 15.3 g.l-1, production by juglandis pathovar varied from 0.0 to 7.3 g.l-1 and by pruni and manihotis pathovars xanthan production reached 7.4 and 9.0 g.l-1, respectively. Moreira et al. (2001) in a study with 18 strain of X. campestris pv pruni and under the same fermentation conditions used in this work, found similar values. The lowest value 2.3 g.l-1 for strain 44 and the highest 8.4 g.l-1 for strain 73. The production of a certain strain can be strongly influenced by the production medium. Souza and Vendruscolo (1999) analyzed the xanthan production by strain 58 of X. campestris pv pruni in four different production media, and in one of them, also there was not xanthan production. The influence of the production medium on the xanthan yield by the standard strain NRRL B - 1459 was observed when comparing the value obtained in this experiment, 6.4 g.l-1, with the values obtained by other authors. Galindo et al. (1994b) in a the study of xanthan production by X. campestris strain B-1459 in a different medium, obtained 9.3 g.l-1, while Nitschke and Thomas (1995) in another medium obtained 11.3 g.l-1. The results of previous studies cited show that the production is dependent on the strain and that was also confirmed in this work. It can be stated, therefore, that the selection of strain should be the first stage in the xanthan search with the highest yield and quality. Viscosity The aqueous solutions of all xanthans showed pseudoplastic behavior (Table 1). The aqueous solutions of the xanthan synthesized by strain 101 presented the highest viscosity (12.700 mPa.s in 10 s-1), while the xanthan produced by strain 83 presented the lowest value of viscosity (1.370 mPa.s) in the same shear rate. Souza and Vendruscolo (1999) determined the viscosity of the 3 % (w/v) aqueous solutions of xanthan produced by X. campestris pv pruni strain 24 and 58, obtaining approximately 5.000 mPa.s and 300 mPa.s, respectively in the shear rate 10 s-1. Among the analyzed strains, sixteen produced xanthan with higher viscosity than the commercial xanthan (Table 1). Antunes et al. (2000) evaluated xanthan viscosity produced by Xanthomonas campestris pv pruni strains in conventional and alternative medium. The xanthan produced in conventional media (MPI and MPII) showed comparable viscosity with commercial xanthan. The viscosity of the commercial xanthan after dialyzis decreased of from 3.400 mPa.s to 2.770 mPa.s in shear rate 10 s-1, probably due to the elimination of salts that are added to facilitate the solubilization of the polymer and to increase its viscosity (JEANES; PITTSLEY; SENTI, 1961; MORRIS, 1996). When comparing the viscosity of the xanthan synthesized for X. campestris NRRL B- 1459 in the shear rate 10 s-1, it reached 2.700 mPa.s, equivalent to the value found for the viscosity of the dialyzed commercial xanthan, 2.770 mPa.s in the same shear rate. The results of the viscosity of aqueous solutions of the polymers showed that the behavior X an th an p ro d u ct io n (g .l- 1 ) Xanthan synthesized... BORGES, C. D. ; VENDRUSCOLO, C. T. Biosci. J., Uberlândia, v. 23, n. 4, p. 67-73, Oct./Dec. 2007 70 was dependent on the used strain. However, no relation was found between viscosity and yield. Table 1. Viscosity (mPa.s) at 25°C of 3 % (w/v) aqueous solutions of xanthan synthesized by 29 strains of Xanthomonas campestris pv pruni, standard strain NRRL B-1459 of X. campestris and dialyzed and not dialyzed commercial xanthan Strain Viscosity (mPa.s) Shear rate 10 s-1 30 s-1 60 s-1 100 s-1 12 2.150 0.840 0.500 0.350 19 2.350 0.880 0.530 0.360 25 2.390 0.990 0.580 0.380 26 8.930 2.980 1.620 1.010 27 7.600 2.550 1.340 0.840 30 6.120 2.210 1.210 0.770 38 7.660 2.560 1.410 0.890 42 2.530 0.950 0.550 0.360 51 2.050 0.840 0.490 0.320 53 2.270 0.850 0.500 0.340 55 2.220 0.920 0.540 0.360 61 2.110 0.790 0.460 0.320 77 1.810 0.730 0.440 0.300 78 2.390 0.860 0.490 0.330 81 2.040 0.820 0.490 0.340 83 1.370 0.570 0.340 0.220 100 7.010 2.530 1.400 0.870 101 12.700 4.080 2.080 1.290 102 7.210 2.570 1.440 0.910 103 2.590 1.090 0.650 0.430 104 12.100 3.900 2.050 1.240 105 7.920 2.700 1.480 0.930 106 9.930 3.270 1.750 1.090 108 11.200 3.750 1.970 1.200 109 9.600 3.230 1.770 1.110 112 7.830 2.770 1.490 0.940 113 8.100 2.860 1.570 0.990 114 7.270 2.720 1.520 0.950 115 10.300 3.500 1.870 1.170 X* 3.400 1.190 0.650 0.430 XD** 2.770 0.990 0.560 0.380 1459*** 2.700 1.090 0.650 0.440 *Not dialyzed commercial xanthan; ** Dialyzed commercial xanthan; *** Standard strain NRRL B-1459 of X. campestris Chemical composition In the chromatograms could be observed that all the samples contained rhamnose, mannose, glucose and glucuronic acid, as expected for the pruni pathovar (SOUZA; VENDRUSCOLO, 1999; VENDRUSCOLO et al., 2000; MOREIRA et al., 2001; ANTUNES et al., 2003). The polymer of all the strains differed from the commercial xanthan (dialyzed and not dialyzed) by the presence of rhamnose. There were no differences in the chromatograms from the samples dialyzed and not dialyzed of commercial xanthan. Differences in xanthan structure have been evaluated by other authors. The polymer produced by Xanthomonas campestris pv vesicatoria contains galactose instead of mannose (ORENTAS; SLONEKER; JEANES, 1963). Lowson and Symes (1977) reported the rhamnose presence in the xanthan produced by X. campestris pv juglandis and xylose in the polysaccharide synthesized by the phaseoli pathovar. Konicek et al. (1977) found the galactose and ribose presence in the polysaccharide synthesized by mutant strains of X. fuscans. The difference was visible in the intensity and size of the spots of the components in the chromatograms; however there was no relation between chemical composition with yield and viscosity. According to Antunes et al. (2000) the differences in the intensity and in the size of the spots could explain why some strains produced Xanthan synthesized... BORGES, C. D. ; VENDRUSCOLO, C. T. Biosci. J., Uberlândia, v. 23, n. 4, p. 67-73, Oct./Dec. 2007 71 biopolymers with higher viscosity than others. According to these authors the polymers with more mannose and glucuronic acid showed greater thickenig properties. Moreira et al. (2001) found a relation between the chemical composition and the rheological behavior. Biopolymers that contained greater mannose concentration maintained or incresead their viscosity with increasing temperature. CONCLUSIONS The yield obtained was not appropriate for industrial production (10 – 20g.l-1), according to the literature, but the viscosity of the aqueous solutions of the xanthan synthesized by sixteen strains was higher than the commercial xanthan, compensating for the low production. These strains should be revaluated in other media and production conditions in an attempt to increase yield. ACKNOWLEDGEMENTS The authors acknowledge CAPES for the financial support and CPACT-EMBRAPA for supplying the strains. RESUMO: O objetivo deste trabalho foi caracterizar a xantana produzida por 30 cepas de Xanthomonas campestris pv pruni, avaliando a produção, viscosidade e composição química. A produção de xantana por cada cepa foi avaliada pelo peso do produto seco por volume de caldo fermentado (g.l-1); a viscosidade das soluções aquosas dos biopolímeros a 3% (m/v) foi mensurada em reômetro no modo rotativo a 25ºC e a composição química foi determinada por cromatografia comparativa de camada delgada (CCDC). A produção de xantana foi cepa dependente e a maior produção alcançada 9,2 g.l-1 para cepa 83 e a menor 3,8 g.l-1 para a cepa 51. A cepa 107 não produziu xantana. As soluções aquosas das xantanas produzidas apresentaram comportamento pseudoplástico. 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