Manufacture of high-molecular-weight compounds with thickener properties has been traditionally related to plants, seeds, and seaweeds. These compounds have been named gums. The rheological properties of their solutions show important alterations depending on uncontrolled variables such as weather, and their manual-collection labor cost can often influence their market price.
Production of molecules with thickener properties from microorganisms was an important advance. This production is made under control and the polymer has constant properties. Xanthan gum is one of these biopolymers first commercialized in the 1960s, and since then has played an important role in industrial gum applications (1).
Xanthan gum is a polysaccharide synthesized by Xanthomonas sp. Its structure can be seen in Fig. 1. The repetition of this structural unit forms xanthan molecules showing very high molecular weights of several millions of Daltons. The acetyl and pyruvyl contents can change depending on culture conditions and microorganism used (2). Therefore, the polymer solutions show different rheological behavior, depending on molecular weight and composition. Xanthan with a high pyruvate content (4-4.8%) shows a greater thickener behavior than that with low pyruvate content (2.5-3%) (1). Pyruvate-free xanthan is employed in enhanced oil recovery (EOR) because microgels are not formed, although in other applications this is not so important. Xanthan's solubility in water and its high stability and thickener behavior, together with the simplicity of its industrial manufacture, made this polysaccharide a gum frequently employed for water rheological behavior modification in many industries such as food, pharmaceutical, and cosmetic and also in EOR.
From: Methods in Biotechnology, Vol. 10: Carbohydrate Biotechnology Protocols Edited by: C. Bucke © Humana Press Inc., Totowa, NJ
Xanthan molecule structure.
Xanthan molecule structure.
Xanthan gum process scheme.
Xanthan gum process scheme.
A xanthan production scheme is given in Fig. 2. Two different steps can be considered: production and isolation. In this chapter both will be commented on separately.
The type of xanthan gum produced is quite different depending on the Xanthomonas species used, the production medium composition, and the
Xanthan production scheme.
Xanthan production scheme.
operational conditions employed in the fermentation (2). Most bacteria of the Xanthomonas genus produce extracellular polysaccharides as bacterial capsules (3). The different composition of these gums is related to their contents of glucose, glucuronic acid, mannose, pyruvate, and acetate, and another sugar, galactose, is introduced into the molecule by some species (2). The type of xanthan gum produced is also influenced by the operational conditions (such as temperature, pH, dissolved oxygen, and so on) employed during the process, both in the concentration or yield obtained and in its molecular structure. The media composition seems to influence the pyruvate content, and operational conditions employed in the fermentation (mainly temperature and dissolved oxygen concentration) influence the molecular weight of the product obtained (4-8).
Xanthan gum production needs several previous steps to be carried out successfully (Fig. 3). The microorganism has to be maintained in a viable form (strain maintenance) to be grown in a complex medium to build up an inoculum able to produce the gum by fermentation. All of these steps are described in depth in this chapter.
Isolation of Xanthan Gum
A typical xanthan production fermentation broth after 60-90 h, depending on the strain, medium composition, and operational conditions is composed of 1-3% (w/w) xanthan gum, 0.1-0.3% (w/w dried) Xanthomonas cells, 0.1-1 % (w/w) unused carbohydrate, salts, and other medium components. Thus, in xanthan downstream processing it is necessary to eliminate up to 95% of the fermentation broth. The objectives of downstream processing are:
• Extraction of polymer in a solid form, that is, stable and easy to handle, store, and dissolve.
• Separation of insoluble solids precipitated together with xanthan.
• Deactivation of enzymes able to degrade xanthan molecules.
According to these objectives there are different processes for the isolation of xanthan gum. These usually involve both physical and chemical separation steps (2), such as:
• Preliminary treatments for degradation and removal of the cells.
• Polysaccharide precipitation.
• Final steps, including washing, dewatering, drying, milling, and packing.
Preliminary treatments used are of thermal or chemical nature. Usually after the xanthan production process cells are lysed by a temperature increase, which also favors xanthan dissolution and decreases broth viscosity. Cells are then separated from the production broth by filtration or centrifugation. Another possibility is the use of water or chemical agents such as alcohols to dilute the xanthan fermentation broth, decreasing viscosity and allowing cell removal by filtration. When chemical reagents such as alcohols or ketones are used, cells are also lysed and xanthan separation is enhanced.
After cell removal, xanthan in solution can be isolated by precipitation, decreasing its solubility by the addition of organic chemical agents or polyvalent salts, producing xanthan polyelectrolyte neutralization and precipitation. Combinations of both agents can be used. Precipitation is a simple technique commonly used for recovering many biological products such as antibiotics, proteins and biopolymers. The tendency of these substances to precipitate is governed by many factors: solvent environment (e.g., salt concentration, dielectric constant, and pH), temperature, and the size, shape, and charge of the molecules. One of the most common strategies to induce precipitation is to alter solvent properties, for example, by adding a miscible organic solvent or an electrolyte (9). Low-molecular-weight alcohols or ketones such as methanol, ethanol, isopropanol, i-butanol, and acetone are the organic solvents more usually employed in xanthan precipitation (10). Isopropanol (IPA) is used most frequently (1,10,11), with a ratio between 1.8:1 to 2.5:1 (v/v, IPA/broth) being employed. Normally an excess of 8-25% of this quantity is used for washing. Recovery of the alcohol is essential for process economic viability (1).
Other authors (10-12) have proposed to use polyvalent cations for polysaccharide precipitation. Xanthan can be precipitated by addition of calcium salts (10) under basic pH (between 8.5 and 12), or by aluminum salts addition (12). In this way, an insoluble xanthan salt is obtained that must be converted to a soluble salt (sodium or potassium) for commercial uses (12). Other authors have proposed to employ quaternary ammonium salts (11,12),
but finally the product must also be converted to a sodium or potassium salt, and the xanthan obtained in this way is not eligible for food industry uses (2) due to the toxicity of quaternary ammonium salts and also because of their high cost.
Smith (13) and Garcia-Ochoa et al. (10) have described the joint utilization of alcohol and salt for xanthan precipitation, finding that smaller quantities of the agents are needed. A general description of the xanthan precipitation procedure is given below.
Xanthan gum that is finally obtained must fulfill commercial quality parameters, such as: acetate and pyruvate contents, ash and moisture contents, and viscosity of its aqueous solutions, the main characteristic for thickener applications.
The main material employed in xanthan gum production is the bacteria itself: Xanthomonas campestris (NRRL B-1459). This microorganism can be obtained as a lyophilized sample from the following: Microbiology Culture Collection Research, Fermentation Laboratory of U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604.
1. YM medium: 10 g/L d-glucose purissimo; 5 g/L bacteriological peptone; 3 g/L yeast extract; 3 g/L malt extract. When YM-agar medium is employed, Agaragar purissimo (20 g/L) must be added. Bacteriological peptone, yeast extract and malt extract have to be stored at 4°C.
2. YM-T medium: 12 g/L d-glucose purissimo, 2.5 g/L bacteriological peptone, 1.5 g/L yeast extract, 1.5 g/L malt extract, 1.5 g/L PO4H(NH4)2, 2.5 g/L PO4HK2, and 0.05 g/L MgSO4. The pH has to be adjusted to 7.0 by addition of HCl.
3. Production medium (8): 40 g/L sucrose, 2.1 g/L citric acid, 1.144 g/L NH4NO3, 2.866 g/L KH2PO4, 0.507 g/L MgCl2, 0.089 g/L Na2SO4, 0.006 g/L H3BO3, 0.006 g/L ZnO, 0.020 g/L FeCl3.6H2O, 0.020 g/L CaCO3, and 0.13 mL/L HCl cc. The pH has to be adjusted to 7.0 by adding NaOH. All the products must be pure.
Materials Used in Xanthan Gum Isolation
Fermentation broths obtained as previously described were precipitated using several agents (10) such as: ethanol, IPA, and acetone, all of industrial quality, that is 96% (w/w), 85% (w/w), and 98% (w/w), respectively, and salts (NaCl, CaCl2) of pure quality, around 99% (w/w) in purity.
Characterization of xanthan produced was performed by the measurement of different parameters. Acetate and pyruvate content were measured using enzymatic kits, acetate using Boehringer-Mannheim no. 148261 and pyruvate with Boehringer-Mannheim no. 124982. Rheological behavior was determined using a viscosimeter (Brookfield LVT-Synchrolectric). This viscosimeter has
a microcapsule for sample thermostation (Brookfield SC4-18/13R). A Brookfield no. 18 spindle was usually employed. Ash and moisture contents in xanthan were measured using a Dupont 951 thermogravimetric analyzer.
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