Xanthan Gum Production from Hydrolyzed Rice Bran

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hydrolysis, the total sugar content of rice bran solution was determined by ... 0.507, Na2SO4 0.089, H3BO3 0.006, ZnO 0.006, FeCl3·6H2O. 0.020 and CaCO3 ...
Korean J. Microbiol. Biotechnol. Vol. 40, No. 4, 356–363 (2012) http://dx.doi.org/10.4014/kjmb.1205.05023 pISSN 1598-642X eISSN 2234-7305

Xanthan Gum Production from Hydrolyzed Rice Bran as a Carbon Source by Xanthomonas spp. Demirci, Ahmet Sukru*, Muhammet Arici, and Tuncay Gumus Department of Food Engineering, Faculty of Agriculture, Namik Kemal University, 59030 Tekirdag, Turkey Received : May 17, 2012 / Revised : August 1, 2012 / Accepted : September 3, 2012

The aim of this study was to utilize rice bran, the main waste product of paddy processing, in xanthan gum production by Xanthomonas campestris fermentation. Deffated rice bran was enzymatically hydrolyzed using cellulase, gluco-amylase, alpha-amylase and xylanase at various pHs and temperatures within 0-12 h. The highest sugar content reached at 35oC, pH 5.5 in 6 h with 41.66%. The enzymatic hydrolysate was used as the carbon source for xanthan gum production by X. campestris NRRL B-1459 and X. campestris pv. campestris. The highest productivities obtained were 21.87 and 17.10 g/L, respectively. Viscosity measurement for the obtained xanthan gums and commercial gum was carried out in gum solutions at various pHs and temperatures. The highest viscosity was reached with 1% gum solutions at 20oC and pH 5.5 for all gums with viscosity values of 470, 131 and 138 mPa sec, respectively. This work has provided relevant scientific information about the use of rice bran, an abundant agroindustrial residue, to produce xanthan gum. Key words: Rice bran, xanthan gum, Xanthomonas spp.

Introduction Xanthan gum is an extracellular polysaccharide produced by pathovars of Xanthomonas campestris and by other Xanthomonas species [32]. Xanthan gum, a cream-colored, odorless, free-flowing powder, hydrates rapidly in cold and hot water to give a reliable viscosity even at low concentrations. It is highly resistant to enzymatic degradation, extremely stable over a wide pH range, and forms highly pseudoplastic aqueous solutions [18]. It is widely used in a broad range of industries, such as in foods, toiletries, oil recovery, cosmetics, in water-based paints, etc., due to its superior rheological properties and is used as a rheological control agent in aqueous systems and as stabilizer for emulsions and suspensions. The important properties of xanthan gum is the ability to form high viscosity solutions at low shear forces, which are highly pseudoplastic and may also display a viscosity yield value [27]. Other than its wide applications in the chemical industry, the major application of this product in the food industry is as a

*Corresponding author Tel: +90 (282) 293 1442, Fax: +90 (282) 293 1480 E-mail: [email protected]

suspending and thickening agent [18]. Currently, the worldwide consumption of xanthan gum is approximately 23 million kg per annum, of which approximately 5 million kg is used as a drilling fluid viscosifier in the oil industry [14, 35]. Xanthan gum is produced industrially from sucrose or glucose by fermentation using the gram-negative bacterium X. campestris. Commercially available xanthan gum is relatively expensive due to glucose or sucrose being used as the sole carbon source and the very stringent purity standards of the Food and Drug Administration for foods [23]. Cost reduction could be achieved by using waste agricultural products, such as rice bran. Rice bran is a solid residue from rice polishing that is used in animal nutrition and rice oil production. Rice bran, an agricultural waste, can be recycled as raw materials for developing novel biotechnological process. It is abundant in the world [10]. The dry mass ratio of the bran to whole rice particle is about 10%. After squeezing rice oil, the residual defatted rice bran powder contains a significant proportion of starchy and cellulosic polysaccharides. The components of the rice bran are starch and dextrin (46.7%), cellulose and hemicellulose (11.3%), protein (18.4%), lipid (1.4%), ash (10.4%), and others (11.8%) [33]. The fermentation medium for xanthan production contains

XANTHAN GUM PRODUCTION FROM RICE BRAN

a carbohydrate and a nitrogen source such as ammonium ion, trace elements and other growth factors [16, 28]. Defatted rice bran contains sugars in the forms of polysaccharides such as starch and holocellulose [33]. Since rice bran not only contains a large amount of polysaccharides but is also 18% protein, defatted rice bran may serve as sources of both carbon and nitrogen necessary for the production xanthan gum. The cost of the fermentation medium represents a critical aspect of the commercial production of xanthan. The use of cheaper substrates such as waste agricultural products, instead of the commonly used glucose or sucrose, might result in a lower cost of the final product [37]. Most commercial production methods for xanthan gum use glucose or invert sugars, and most industries prefer batch processes to continuous processes [19]. Other substrates have also been tested, such as sucrose, barley and corn flour, acid whey, sugar cane molasses, coconut juice, sugar cane, etc., but glucose is still the best in terms of product yield, supply, and product quality [1, 11, 17, 27]. In the present study, hydrolyzed rice bran was tested for its suitability as a substrate for xanthan gum production, since this by-product is obtained in a large amount in paddy processing. As an example, rice bran production in Turkey is about 30,000-50,000 tons per year [6]. The disposal of this residue on the ground may cause serious environmental impacts, since this by-product has a high biochemical oxygen demand (BOD) [30]. In this context, the main objective of this work was to evaluate the production of xanthan gum using hydrolyzed rice bran as a low cost alternative substrate by two strains, X. campestris NRRL B-1459 and X. campestris pv. campestris. The rheological characteristics of the polysaccharide produced and the influence of the addition of water and the apparent viscosity of xanthan were also presented.

Materials and Methods Rice bran and enzymes Rice bran, used as the source of carbon, was provided by a rice mill in Edirne, Turkey. Enzymes (Cellulase (CELLUCLAST BG), Gluco-amylase (AMG 800 BG), Alpha-amylase (FUNGAMYL 2500 SG), Xylanase (PENTOPAN 500 BG); NOVOZYMES, Denmark) used for enzymatic hydrolysis were obtained from POLEN Food Company Istanbul, Turkey.

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Extraction of rice bran Rice bran oil was extracted from bran by soxhlet extraction for 4 h at 60oC using hexane as the solvent. The defatted rice bran was then dried overnight to remove the residual solvents. Saccharification of polysaccharides in the defatted rice bran by enzymes Saccharification of defatted rice bran was investigated by enzymatic hydrolyses at (25, 30, 35 and 40oC), pH (4.5, 5.0 and 5.5) with 5% w/v solid substrates, using (125-1250 FAU/L alpha-amylase, 40-400 AGU/L glucoamylase, 1751750 EGU/L cellulose, 135-1350 FXU/L xylanase) within 0-12 h. Hydrolytic profiles were studied by using 50 g kg-1 medium of hydrolized rice bran (HRB) in 1.5 L fermentor (results are the average of duplicate cultures) contaning 1 L working volume at 175 rpm rotary. Rice bran hydrolysates were filtered through a filter paper and the pH of the filtrates was adjusted at pH 6.8-7 with NaOH (1.0 N) and filtered again, to remove the precipitate. After filtration, the enzymatic hydrolysates were used for reducing sugar measurement and xanthan production. To adjust the final carbohydrate concentration in the media, the total carbohydrate content was determined in the hydrolysate. After hydrolysis, the total sugar content of rice bran solution was determined by Lane-Eynon method. Microorganisms X. campestris NRRL B-1459 was obtained from United States Department of Agriculture, Research- Education and Economics Agricultural Research Service, Microbial Genomics and Bioprocessing Research Unit (USA). X. campestris pv. campestris obtained from Dr. M. Mirik (Namýk Kemal University, Tekirdag, Turkey), was isolated from infected cabbage leaves (plant pathogen). The culture was maintained on GYE agar slants containing (g/L) glucose 20, yeast extract 10, calcium carbonate 20, and agar agar 15. The pH was maintained at 7.0 and the cultures were grown at 28oC for 48 h, stored at 4oC and transferred every 14 days. Inoculum preparation Actively growing cells from a newly prepared slant were inoculated into 100 mL YM (Yeast Extract-Malt) liquid medium containing (g/L), peptone 5.0, malt extract 3.0, glucose 20.0 at pH 7.0 in a 250 mL Erlenmeyer flask. The

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culture was incubated at 28-30oC for 36-48 h in an incubator shaker. The liquid culture was used to inoculate the final fermentation medium. For the gum production assay, 10% (v/v) of the inoculum was added to the production medium (containing approximately 108 c.f.u./ml) [37]. Fermentation For fermentation, the standard sucrose medium consisted of (g/L) sucrose 40, citric acid 2.1, KH2PO4 2.866, MgCl2 0.507, Na2SO4 0.089, H3BO3 0.006, ZnO 0.006, FeCl3 · 6H2O 0.020 and CaCO3 0.020. Hydrolyzed rice bran filtrate was used as a carbon source substitute of sucrose to provide carbohydrate concentrations of 4% for the fermentation studies. Fermentation experiments was carried out in a 1.5 L work volume fermentor (Sigma Braun Model Biostat B, Germany) with 1.0 L of medium containing hydrolyzed rice bran solution or sucrose under these operational conditions: temperature 30ºC, pH 7.0 (controlled with 0.1 N NH4OH and 0.1 N HCl), stirrer speed 200 rpm and airflow rate 1 L/min for 72 h [20]. The fermentation parametres were selected based on preliminary experiments and the work of Rosalan and England [27]. Analysis of xanthan The final fermentation broth was centrifuged at 10.000 rpm for 30 min at 4oC to remove the cells. The supernatant was collected and the gum was precipitated by using isopropanol (2 vol of isopropanol/vol of supernatant) then filtered through a filter paper. The gum was dried at 5060oC overnight. The dry mass of the gum was determined [15, 31]. Preparation of gum solutions and viscosity measurement Each sample was prepared in 100 mL water and was vigorously stirred using a hotplate for 30 min at 20oC before rheological measurements. The concentration of sample solutions was prepared as follows: xanthan gum 0.25, 0.50 and 1% (w/w) [9]. Viscosity measurements for the obtained xanthan gums and commercial gum were carried out using different (0.1, 0.25, 0.50, 1%) gum solutions at various pH (pH 3.5, 5.5, 7.0) and temperatures (20-100oC). The viscosity of the samples were measured using vibro-viscometer (AND, SV10 model, Japan).

Statistical Analysis The yield data of gum production was obtained from five replicates and viscosity data of the gums was obtained from three replicates. The data were analyzed by ANOVA using the SPSS statistical package program, and differences among the viscosity means were compared using the Duncan’s Multiple Range test [2].

Results and Discussion Saccharification of defatted rice bran Defatted rice bran contains starch and dextrin (46.7%), cellulose and hemicellulose (11.3%) [33] and can serve as a low-cost feedstock for xanthan production. They should be converted to glucose for bacteria to utilize them efficiently. The mixture of alpha amylase, gluco amylase, xylanase and cellulase hydrolyzed the starchy and cellulosic polysaccharides of the rice bran effectively. Results (average of three replicates) of saccharification of the polysaccharides in the defatted rice bran by enzymes are given in Fig. 1 and Fig. 2. Maximum reducing sugar content was obtained with the addition of 1250 FAU α-amylase + 400 AGU glucoamylase + 1750 EGU cellulase + 1350 FXU xylanase/L at 35oC, pH 5.5 in 6 h. Reducing sugar concentration at the beginning of the hydrolysis was 8% and evolved slowly to reach 41.66% after 6 h (Fig 1). The enzymatic hydrolysis of rice bran (HRB) substrate showed similar with first experiment as 39.75% saccharification after 6 h incubation at 35oC, pH 5.5 second experiment concentration of 625 FAU α-amylase + 200

Fig. 1. Saccharification of rice bran with 1250 FAU α-amylase + 400 AGU glucoamylase + 1750 EGU cellulase + 1350 FXU xylanase/L at different pHs and temperatures.

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nutrient components. Enzyme mixture of 6.7 g m-3 amylase and 3.3 g m-3 cellulase were used at 4.5 pH to saccharify the polysaccharides in the rice bran and concentrations of total soluble sugar increase to approximately 20 kg L after 8 h and then decreased. It is indicated that enzymatic hydrolysis of rice bran made it possible to obtain a saccharifiation yield of about 40% after an experimental time of about 6 h. Based on previous works, to make fermentation medium, the enzymatic hydrolysate adjusted to 4% (w/v) of reducing sugar, was supplemented with other nutrients. Fig. 2. Saccharification of rice bran with 625 FAU α-amylase + 200 AGU gluco amylase + 875 EGU cellulase + 675 FXU xylanase/L at different pHs and temperatures.

AGU gluco amylase + 875 EGU cellulase + 675 FXU xylanase/L (Fig 2). The halving of the all enzyme concentrations resulted in only 1.91% decrease in the total soluble sugar concentration. Thus, we have used the enzyme mixture of 625 FAU alpha-amylase + 200 AGU gluco amylase + 875 EGU cellulase + 675 FXU xylanase/L to saccharify the rice bran in this study. Further two saccharification experiments were carried out using lower concentrations of enzymes and resulted in low sugar concentraions and these sugar contents are not adequate for xanthan production. The maximum total sugar concentrations decreased to 32.50% and 23.90% by the additions of the 250 FAU alpha-amylase + 80 AGU gluco amylase + 350 EGU cellulase + 270 FXU xylanase/L and 125 FAU alpha-amylase + 40 AGU gluco amylase + 175 EGU cellulase + 135 FXU xylanase/L respectively. Generally, at the all of the enzyme concentrations, the concentration of total sugar initially increased and then decreased after 6 h. The optimum temperature and pH parametres for hydrolysis were found to be 35oC and 5.5 respectively. At lower (25oC) and higher (40oC) temperatures, using lower concentrations of enzymes the sugar yield was determined low. Formation rates were higher at pH 5.5 than at pH 4.5 and 5.0. Tanaka et al. [33] were coupled defatted rice bran saccharification with amylase and cellulase to lactic acid fermentation. The yield based on the amount of sugars soluble at 5 pH after 36 h hydrolysis of the bran by amylase and cellulase (36 kg m-3 from 100 kg m-3 of the bran) was 78%. Taniguchi et al. [34] studied to production of L-lactic acid by simultaneous saccharifcation and fermentation using unsterilized defatted rice bran as a carbon source and

Xanthan Gum Production Preliminary experiments were performed with two bacterial strains (X. campestris NRRL B-1459 and Xanthomonas campestris pv. campestris). A comparison was carried out of xanthan gum production using sucrose production medium and hydrolyzed rice bran (HRB) production medium by X. campestris NRRL B-1459 and X. campestris pv. campestris by fermentation using predetermined parameters. The amount of xanthan gum produced in excess of sucrose fermentation was determined by comparing the amount of xanthan gum produced by HRB fermentation. Results (average of five replicates) of xanthan fermentation experiments in which HRB was compared to standard sucrose media were given in Table 1. The HRB fermentation resulted in xanthan production that was higher than sucrose fermentation for all isolates. Table 1 shows that the highest yield of xanthan gum among the two strains was obtained using the HRB media by X. campestris NRRL B-1459 (21.87±1.144 g/L), followed by Xanthomonas campestris pv. campestris (17.1±0.565 g/L gum). The high yield of HRB may be due to its sugar composition which includes a reducing sugar (glucose). Research related to xanthan gum production using agroindustrial residues have been reported in the literature. Lopez et al. [21] tested four strains of X. campestris to Table 1. Xanthan production using sucrose and HRB as the carbon source for two strains. Isolates X. campestris NRRL B-1459 X. campestris pv. campestris

Xanthan Gum Production from (g/L) Sucrose

HRB

12.35±0.212c 11.00±0.849d

21.87±1.144a 17.10±0.565b

Standard errors of the mean (n=5) a,b Means with different superscript letters are different (p