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Appl Microbiol Biotechnol (2003) 62:468-473

DOl 10.1007/s00253-003-l386-4
MINI-REVIEW
T. D. Leathers
Biotechnological production and applications of pullulan
Received: 11 March 2003 / Revised: 19 May 2003 / Accepted: 21 May 2003 / Published online: 27 June 2003
Springer-Verlag 2003
921
Abstract Pullulan is a unique biopolymer with many
useful traits and hundreds of patented applications.
However, despite the fact that pullulan has been in
commercial production for more than 25 years, few of
these potential uses have been widely adopted. In large
part this may be due to the relatively high price of
pullulan. Nevertheless, the last few years have seen a
resurgence in interest in pullulan, particularly for higher-
value health and pharmaceutical applications.
Introduction
Pullulan is a linear homopolysaccharide of glucose that is
often described as a-(1 ~ 6 linked maltotriose, secreted
primarily by strains of the fungus Aureobasidium pullu-
lans. The unique linkage pattern of pullulan endows the
polymer with distinctive physical traits, including adhe-
sive properties and the capacity to form fibers, compres-
sion moldings, and strong, oxygen-impermeable films.
Pullulan can be chemically derivatized to alter its water
solubility or provide reactive groups. Pullulan and its
derivatives have numerous demonstrated uses in foods,
pharmaceuticals, manufacturing, and electronics. This
review focuses on commercial production methods and
potential applications for pullulan. More comprehensive
reviews include those by Deshpande et al. (1992), Pollock
Names are necessary to report factually on available data; however,
the USDA neither guarantees nor warrants the standard of the
product, and the use of the name by USDA implies no approval of
the product to the exclusion of others that may also be suitable
T. D. Leathers ~
Fermentation Biotechnology Research Unit,
National Center for Agricultural Utilization Research,
Agricultural Research Service,
United States Department of Agriculture,
1815 N. University St., Peoria, 1L 61604, USA
e-mail: leathetd@ncaur.usda.gov
Tel.: +1-309-6816377
Fax: +1-309-6816427
(1992), Seviour et al. (1992), Tsujisaka and Mitsuhashi
(1993), and Leathers (2002).
Historical outline
Bauer (1938) made early observations on extracellular
polymer formation by A. pullulans, and Bernier (1958)
isolated and began to characterize the polysaccharide.
Bender et al. (1959) studied the novel glucan and named it
"pullulan." During the 1960s, the basic structure of
pullulan was resolved (Wallenfels et al. 1961, 1965;
Bouveng et al. 1962, 1963; Sowa et al. 1963; Ueda et al.
1963). Bender and Wallenfels (1961) discovered the
enzyme pullu1anase, which specifically hydrolyzes the a-
(1~ 6 linkages in pullulan and converts the polysaccha-
ride almost quantitatively to maltotriose. On this basis
pullulan is commonly viewed as an a-(1 ~ 6 linked
polymer of maltotriose subunits (Fig. 1). However,
pullulan can be considered to be a polymer of panose or
isopanose subunits, which may reflect more accurately
the biosynthetic origins of the molecule. Catley and
coworkers subsequently established the occurrence of a
minor percentage of randomly distributed maltotetraose
subunits in pullulan (Catley et al. 1966; Catley 1970;
Catley and Whelan 1971; Carolan et al. 1983).
Relatively little is understood about the mechanism of
pullulan biosynthesis. Unlike bacterial dextrans, which
are synthesized extracellularly by secreted glucansucras-
es, pullulan is synthesized intracellularly and secreted by
A. pullulans. Studies have suggested that synthesis
involves UDP-glucose, requires ATP, and proceeds
through lipid intermediates (Ueda and Kono 1965;
Taguchi et al. 1973; Ono et al. 1977). Catley and
McDowell (1982) found that A. pullulans incorporates
14C-labeled glucose into lipid-linked glucose, isomaltose,
panose and isopanose, and proposed a reaction mecha-
nism in which pullulan is formed by the polymerization of
either panosyl or isopanosyl moieties. An occasional
direct linkage of panosyl and isopanosyl moieties would
form the minor maltotetraosyl elements. However, none
Fig. 1 Chemical structure of a
representative portion of puUu-
lan, illustrating the primary
structure of repeating linkages.
Figure courtesy of Dr. Gregory
L. Cote, USDA
469

HO \ ' 0


HO
0,-

HO \' 0


HO

HO \ 0

HO '.


\ \
HO
0,-
SH,


HO i
SH2
0H


HO
of the enzymes involved in pullulan biosynthesis have
been identified or characterized.
Commercial production of pullulan began in 1976 by
the Hayashibara Company, in Okayama, Japan (Tsujisaka
and Mitsuhashi 1993). Pullulan production was a natural
outgrowth of Hayashibara's original business of starch
syrup production, founded in 1883. Pullulan films were
commercialized by Hayashibara in 1982. The company
remains the principle commercial source of pullulan
today. Space has been provided in their facility for the
future expansion of pullulan production, and growth is
anticipated during the next 2 years (personal communi-
cation, S. Endo, Hayashibara).
Production
Methods and conditions for pullulan production have
been detailed (Wallenfels and Bender 1961; Zajic 1967;
Yuen 1974; Kato and Shiosaka 1974, 1975b; Sugimoto
1978; Catley 1979; Tsujisaka and Mitsuhashi 1993; Ozaki
et al. 1996). In commercial production, A. pullulans is
cultivated batch-wise on medium containing starch
hydrolysates of dextrose equivalent 40-50, at 10-15%
concentration (Tsujisaka and Mitsuhashi 1993). The
medium includes peptone, phosphate and basal salts.
Culture pH is initially adjusted to pH 6.5, and falls,
especially during the first 24 h, to a final pH of
approximately pH 3.5. Maximal culture growth occurs
within 75 h, and optimal pullulan yields are obtained
within about 100 h (Tsujisaka and Mitsuhashi 1993).
Cultures are stirred and aerated, and the temperature is
held at 30C. Yields of greater than 70% of initial
substrate are claimed. Culture conditions and strain
selection are important in obtaining high molecular
weight pullulan that is relatively free of melanin. A.
pullulans cells are removed by filtration of diluted culture
broth. Melanin is removed by treatment with activated
charcoal, and pullulan is recovered and purified by
precipitation with organic solvents, particularly alcohols.
Pullulan may be further purified through the use of
ultrafiltration and ion exchange resins.
Aureobasidium is considered to be one of the "black
yeasts" and melanin removal is a unique problem that
may add to the cost of pullulan production. Another
peculiar quality control issue is the tendency for enzymes
from Aureobasidium to reduce the molecular weight of
pullulan in late cultures (reviewed in Leathers 2002).
Much of the published research on pullulan in recent
years has been concerned with improving the economics
of production, mainly by identifying even less expensive
feedstocks, isolating improved production strains, or
developing alternative fermentation schemes (reviewed
in Leathers 2002). Nevertheless, it seems likely that the
470
limited scale of pullulan production is a major factor in
determining production costs.
Pullulan production at Hayashibara is currently at
approximately 300 metric tons per year (personal com-
munication, S. Endo, Hayashibara). Food grade pullulan
(PF-20) wholesales in Japan for approximately US $20/
kg, and pharmaceutical grade (deionized) pullulan (PI-20)
sells for approximately US $25/kg (personal communi-
cation, S. Endo, Hayashibara).
Properties and applications
The regular occurrence of a-(l-e,6) linkages in pullulan
interrupts what would otherwise be a linear amylose
chain. This unique linkage pattern is believed to be
responsible for the structural flexibility and solubility of
pullulan, resulting in distinct film- and fiber-forming
characteristics not exhibited by other polysaccharides. In
fact, pullulan films and fibers resemble certain synthetic
polymers derived from petroleum. Although more expen-
sive than these plastics, pullulan products are edible and
biodegradable, and underivatized pullulan is highly
water-soluble.
Dry pullulan powders are white and non-hygroscopic
and dissolve readily in hot or cold water. Pullulan is non-
toxic, non-mutagenic, odorless, tasteless, and edible (Fujii
and Shinohara 1986; Kimoto et al. 1997). Presumably
because of its resistance to mammalian amylases, it
provides few calories and appears to be treated as dietary
fiber in rats and humans (Oku et al. 1979; Yoneyama et
al. 1990). Studies suggest that dietary pullulan functions
as a prebiotic, promoting the growth of beneficial
bifidobacteria (Mitsuhashi et al. 1990; Yoneyama et al.
1990; Sugawa-Katayama et al. 1994). Pullulan can be
used as a partial replacement for starch in pastas or baked
goods (Yuen 1974; Hijiya and Shiosaka 1975b; Kato and
Shiosaka 1975a; Hiji 1986).
Pullulan solutions are of relatively low viscosity,
resembling gum arabic (Tsujisaka and Mitsuhashi 1993).
Pullulan can be used as a low-viscosity filler in beverages
and sauces. The viscosity of pullulan solutions is stable to
heating, changes in pH, and most metal ions, including
sodium chloride. Pullulan has demonstrated uses in
cosmetics, lotions, and shampoos (Nakashio et al.
1976b). Pullulan and its derivatives exhibit adhesive
properties (Hijiya and Shiosaka 1975a) and can be used in
wound-healing compositions (Leung et al. 2001). Pullulan
can be used as a denture adhesive, a binder and stabilizer
in food pastes, and to adhere nuts to cookies.
Pullulan films are formed by drying a pullulan solution
(usually 5-10%) onto an appropriate smooth surface.
Films can be as thin as 5-60 /lm. Pullulan films are clear,
highly oxygen-impermeable and have excellent mechan-
ical properties (Yuen 1974). Underivatized films readily
dissolve in water, and thus melt in the mouth as edible
food coatings (Conca and Yang 1993). Specialty films
may include colors or flavors, and decorative pullulan
chips are produced for food uses. Alternatively, pullulan
can be applied directly to foods as a protective glaze.
Pullulan and its derivatives have many potential
pharmaceutical, clinical, and health care uses. Pullulan
can be used in pharmaceutical coatings, including
sustained-release fonnulations (Miyamoto et al. 1986;
Izutsu et al. 1987; Childers et al. 1991). Oral care
products based on pullulan films have recently been
commercialized (Leung et al. 2000; Anonymous 2001). In
addition, pullulan and its derivatives have photographic,
lithographic, and electronic applications (Sano et al.
1976; Tsukada et al. 1978; Shimizu et al. 1983; Sasago et
al. 1988; Vermeersch et al. 1995).
Most pullulan derivatizations are intended to reduce its
water solubility or to introduce charged or reactive groups
for functionality. The water solubility of pullulan can be
progressively reduced by esterification (Hijiya and Sh-
iosaka 1975a; 1975c) or etherification (Fujita et al.
1979a). Hydrogenation reportedly increases the heat
stability of pullulan (Kato and Shiosaka 1976), and
carboxylation enhances its solubility in cold water (Tsuji
et al. 1978). Cross-linked pullulan beads are useful in gel
permeation chromatography (Nagase et al. 1979; Mo-
tozato et al. 1986), and polyanionic and cationic deriva-
tives have been prepared (Fujita et al. 1979b; Oonishi
1985). Cyanoethylated pullulan has potential uses in
electronic devices (Onda et al. 1982). Pullulan has been
sulfated (Mocanu et al. 1985), chlorinated (Mayer et al.
1990), sulfinethylated (Imai et al. 1991), and chloroalky-
lated (Mocanu et al. 1999). Azidopullulan (Mayer et al.
1990) and siloxane derivatives have been prepared
(Uchida et al. 1996). Pullulan substituted with cholesterol
or fatty acids can be used to stabilize fatty emulsions
(Yamaguchi and Sunamoto 1991). Pullulan derivatives
are promising as non-toxic conjugates for vaccines
(Yamaguchi et al. 1985; Mitsuhashi and Koyama 1987)
and interferon (Suginoshita et al. 2002) and can facilitate
liposome delivery (Takada et al. 1984; Sunamoto et al.
1987). Pull ulan-based hydrogels and nanoparticles may
have a variety of uses (Masci et al. 2002; Na et al. 2003).
Outlooks and perspectives
Pullulan is a unique polysaccharide with a multitude of
demonstrated practical applications. The major constraint
on the use of pullulan appears to be its price. As
illustrated in Fig. 2, many of the earliest pullulan patents
concern bulk industrial applications, for example as a
flocculating agent (Zajic 1967; Zajic and LeDuy 1973) or
in the production of paper or paint (Nakashio et al. 1975a,
1976a; Nomura 1976). Similarly, pullulan has been touted
as a biodegradable substitute for nylon or rayon fibers and
molded polystyrene or polyvinyl alcohol (Hijiya and
Shiosaka 1974, 1975c; Nakashio et al. 1975b; Matsunaga
et al. 1976, 1977; Tsuji et al. 1976). However, pullulan is
too expensive for many of these potential uses, including
those in which it serves as biodegradable replacement for
petroleum-derived polymers. Although pullulan is effi-
I
I
/
1995-2003
19911995
19861990
Fig. 2 Survey of United States Patents 1976-2002 contammg
"pullulan" in the title or abstract, categorized into application fields
ciently produced by fermentation of relatively inexpen-
sive substrates, it currently sells for approximately three
times the price of dextran or xanthan, other microbial
gums produced by fermentation. Perhaps accordingly,
more recent patents have increasingly concerned higher-
value applications for pullulan, especially those related to
human health (Fig. 2). Technical improvements in
pullulan production, such as engineering innovations or
improved production strains, could reduce the cost of
production. Molecular genetic approaches to improved
production are currently limited by a lack of fundamental
knowledge about pullulan biosynthesis. Costs may be
determined to a large extent by the modest production
scale of pullulan. Pullulan production levels have been
fairly stable for a number of years, with specialty food
applications as a major market. The recent commercial-
ization of pullulan-based oral care products is encourag-
ing for the future of pullulan, although competing
formulations have appeared (e.g., Barkalow et al. 2002).
Another emerging market for pullulan may be in the
formulation of capsules for dietary supplements and
pharmaceuticals (Anonymous 2002). Increased demand
for pullulan could justify expanded production, resulting
in new viable market niches for this unique biopolymer.
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