ReviewPullulan: Microbial sources, production and applications
Introduction
A variety of exopolysaccharides (EPSs) are produced by a number microorganisms as an extracellular or cell surface-attached material in the form of amorphous slime (Sutherland, 1998). These EPSs can be categorized as homopolysaccharides and heteropolysaccharides (Byrom, 1991, Garcia-Ochoa et al., 1995, Jorris and Vandamme, 1993, Weiss and Ollis, 1980, Yuen, 1974). Homopolysaccharides are generally neutral glucans, while most of the heteropolysaccharides are polyanionic due to the presence of uronic acid. Microbial EPSs are propitious substitutes for the plant polysaccharides due to their unique and superior physical properties. Microbial EPSs extensively studied now a days include xantham from Xanthomonas campesteris, succinoglycan from Rhizobium, bacterial alginates from Pseudomonas spp.; Azotobacter vinelandii and pullulan from Aureobasidium pullulans with potential industrial applications. These polysaccharides (group of macromolecules) possess diverse applications in food, chemical, energy production, and pharmaceutical industries. A few fungal EPSs with interesting industrial properties are well known. Pullulan is one of such commercially emerging biopolymers and synthesized by a yeast-like fungus A. pullulans. It is a water soluble random coil glucan that serves as a paradigm for the behavior of aqueous polysaccharides (Morris, 1995, Tsujisaka and Mitsuhashi, 1993, Yalpani, 1998).
It is well established that regularly repeating structural unit of pullulan is a maltotriose trimer α-(1 → 4)Glup-α-(1 → 4)Glup-α-(1 → 6)Glup-, produced extracellularly by A. pullulans (de Bary) G. Arnaud, a mitosporic fungus formerly known as Pullularia pullulans (de Bary) Berkhout (Syn: Dematium pullulans de Bary) (Gibbs and Seviour, 1996, Leathers, 2003). However, other structures particularly the tetramer or maltotetraose α-(1 → 4)Glup-α-(1 → 4)Glup-α-(1 → 4)Glup-α-(1 → 6)Glup-, may be present in the pullulan polymeric chain (Wallenfels, Keilich, Bechtler, & Freudenberger, 1965). So far, the maximum extent to which maltotetraose subunits have been detected is 7% (Catley, Ramsay, & Servis, 1986). This is the main reason why currently and frequently in the literature the term “pullulan” is used for both the “polymaltotriose” produced by A. pullulans and the polysaccharide varieties, similar to the pullulan, produced by other microbes. A couple of theories as to how pullulan is elaborated have been proposed by various researchers. Catley (1971a) proposed that a lipid is used as a carrier in which pullulan is taken to the outside of the plasmalemma. This view was supported by other research groups i.e. Lee et al., 1999, Simon et al., 1995. Rho, Mulchandani, Luong, and LeDuy (1988) proposed two pathways, one allowing direct conversion of glucose, whereas the second pathway used an identified precursor. A thorough review of the peculiarities of the pullulan biosynthesis with various A. pullulans strains was published in 1981 (Kondratyeva, 1981). Simon, Caye-Vaugien, and Bouchonneau (1993) proposed that both pullulan and the insoluble polysaccharide are localized on an outer surface of the chlamydospores on the basis of an electron microscopic study. Simon, Bouchet, Bremond, Gallant, and Bouchonneau (1998) revealed an inverse correlation between the concentration of pullulan and the content of intracellular glycogen though the mechanism according to which glycogen is transformed into pullulan is not well understood.
The regular alternation of (1 → 4) and (1 → 6) bonds results in two distinctive properties of structural flexibility and enhanced solubility (Leathers, 1993). The unique linkage pattern also endows pullulan with distinctive physical traits along with adhesive properties and its capacity to form fibers, compression moldings, and strong, oxygen-impermeable films. Pullulan’s solubility can be controlled or provided with reactive groups by chemical derivatization. Consequently, pullulan and its derivatives have numerous potential for food, pharmaceutical and other industrial applications. Pullulan is water soluble, insoluble in organic solvents and non-hygroscopic in nature. Its aqueous solutions are stable and show a relatively low viscosity as compared to other polysaccharides. It decomposes at 250–280 °C. It is moldable and spinnable, being a good adhesive and binder. It is also non-toxic, edible, and biodegradable. Its main quality parameters are summarized in Table 1. A number of reviews on pullulan have appeared (Israilides et al., 1999, Leathers, 2002, Leathers, 2003, Shingel, 2004) and this review focuses on microbial sources, structural geometry, upstream processing, downstream processing, peculiar characteristics and applications of pullulan.
A survey of the World patent Index in 1983 revealed that over 150 inventions were related to this polysaccharide, mostly on its new applications (LeDuy, Choplin, Zajic, & Loung, 1988). Literature has been surveyed from 2000 to 2007 and it reveals ten new inventions related to production and pharmaceutical applications of the pullulan (Boyd et al., 2006, Cade et al., 2003, Gaddy and Patton, 2006, Ikewaki et al., 2005, Leung et al., 2006, Scott et al., 2005, Thorne et al., 2000, Thorne et al., 2002, Wolf, 2005). A number of methods for pullulan production have been reported (Kato and Shiosaka, 1974, Ozaki et al., 1996, Wallenfels and Bender, 1961, Zajic, 1967). Many applications of pullulan in food have been documented (Hiji, 1986, Hijiya and Shiosaka, 1975a, Hijiya and Shiosaka, 1975b, Kato and Shiosaka, 1975a). Applications of pullulan in personal care products have been summarized by Nakashio, Tsuji, Toyota, and Fujita (1976b). Numerous derivatizations of pullulan have also been patented, including esterification (Hijiya & Shiosaka, 1975c), etherification (Fujita, Fukami, & Fujimoto, 1979), hydrogenation (Kato & Shiosaka, 1976) and carboxylation (Tsuji, Fujimoto, Masuko, & Nagase, 1978).
Section snippets
Historical background
Currently only a small number of exocellular fungal α-glucans are reported in the literature. Pullulan is the best studied α-linked glucan produced by the polymorphic fungus A. pullulans. Bauer (1938) was pioneer who observed the polysaccharide production by A. pullulans. First isolated and characterized by Bernier (1958) from culture broths of A. pullulans, pullulan has become the object of an ever increasing research effort. Thorough study of this novel polysaccharide was done by Bender,
Structural geometry
The characteristic dimeric segments of pullulan are [ → x)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1→] and [ → 4)-α-d-glucopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1→], where x may be either 4 or 6 for the (1 → 4)-linked segment. The trisaccharide G3 (maltotriose), the fundamental repeating unit of pullulan contains two (1 → 4)-linkages and no (1 → 6)-linkages. For the first time pullulan was isolated from cultures of A. pullulans in 1958 and d-glucose was reported to be the main product of acid
Microbial sources
Pullulan is produced as a water-soluble, extracellular polysaccharide by certain strains of the polymorphic fungus A. pullulans (De Bary) Arnaud (formerly known as Pullularia pullulans De Bary) Berkhout or Dematium pullulans (De Bary). The microbial production of pullulan by Pullularia pullulans was discovered by R. Bauer in 1938. A. pullulans is a ubiquitous fungus isolated commonly from the environment (Cooke, 1959, Hermanides-Nijhof, 1977). It is found in soil, water and as saprophyte on
Upstream processing
Microbially produced polysaccharides have properties which are extremely useful in different industrial applications. During the fermentation process/upstream processing of EPS production, the characteristics of the liquid media change drastically. At the beginning, the liquid has a Newtonian behavior with viscosity close to that of pure water but the formation of EPSs results in a rapid increase in the apparent viscosity and a change to non-Newtonian rheology. Since the microbial
Downstream processing
Downstream processing is required to obtain pure biopolymer from the fermentation broth and it comprises of cell harvesting from culture broth after cultivation, removal of melanin pigments produced during fermentation and precipitating the polymer with a suitable solvent. Melanin is one of the major obstacle in pullulan production and it is responsible for dark green to black color of the broth. Seihr (1981) reported intracellular as well as extracellular synthesis of melanin by the
Peculiar characteristics
The regular (1 → 6) linkages in pullulan are thought to impart structural flexibility and enhanced solubility (Buliga & Brant, 1987a). Recently, Angioletti (2003) has confirmed the same. This allows pullulan to mimic synthetic polymers derived from petrochemical-derived polymers in many aspects as biocompatibility, biodegradability, and both human as well as environmental compatibility. Pullulan powders are white and non-hygroscopic that dissolves readily in hot or cold water. It is non-toxic,
Applications
Though, microbial biopolymers are known to possess useful physical properties, even then currently only a small number of biopolymers are produced commercially on large scale. A few fungal EPSs have been reported so far that possess appealing industrial applications. Pullulan, a water soluble biopolymer from A. pullulans is one of such fungal EPSs. Numerous applications of pullulan in food and pharmaceuticals manufacturing have been reviewed by Leathers (2003).
Conclusions
Research studies in the field of polysaccharides have revealed that pullulan is a unique polysaccharide with a variety of potential industrial and medical applications. Pullulan membranes/films are being used as coating and packaging materials for foods such as instant food seasonings, powdered tea and coffee. Pullulan-coated papers also decompose easily and do not contaminate the environment (Domań-Pytka & Bardowski, 2004). Pullulan production has been stable with its major applications in
Acknowledgement
Authors are thankful to Head, Department of Biotechnology, for providing necessary bioinformatics laboratory facilities.
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