Pullulan: Microbial sources, production and applications

Abstract

Pullulan is a water-soluble glucan gum produced aerobically by growing a yeast like fungus Aureobasidium pullulans. It is a regularly repeating copolymer with the chemical structure {→ 6)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 →}_n. Thus the polysaccharide is viewed as a succession of α-(1 → 6)-linked (1 → 4)-α-d-triglucosides i.e. maltotriose (G₃). Pullulan have a wide range of commercial and industrial applications in many fields like food science, health care, pharmacy and even in lithography. Due to its strictly linear structure, pullulan is also very valuable in basic research as well as a well-defined model substance. This review attempts to critically appraise the current literature on fungal exopolysaccharide (EPS) ‘pullulan’ considering its microbial sources, structural geometry, upstream processing, downstream processing, peculiar characteristics 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).