Pullulan: production, properties and applications

1. Chandrima Shrivastava
13FET1001

2.  Pullulan is a fungal polysaccharide, synthesized by
Aureobasidium pullulans in starch and sugar cultures.
 The name “pullulan” was proposed by Bender, who was the first
to describe the formation of this extracellular polysaccharide by
Aureobasidium pullulans (syn. Pullularia pullulans)
 Pullulan is being used extensively in the food industry as a food
ingredient in Japan since 1976, and has GRAS status in the USA.
 Pullulan has applications in several industrial sectors like the
food, pharmaceutical and cosmetic industries.

3.  Aureobasidium pullulans, called as “black yeast” (because it forms a black
pigment melanin), has been used for the production of pullulan.
 It is a ubiquitous saprophyte, found in soil, lake water, on the surface of
latex paint films, synthetic plastic materials, and foods such as cereals,
fruits, cheese and tomato.
 Other microorganisms have also been reported to produce pullulan.
Aureobasidium pullulans
Tremella mesenterica
Cytaria harioti
Cytaria darwinii
Cryphonectria parasitica
Teloschites flavicans
Rhodototula bacarum
Aureobasidium pullulans

4.  Pullulan is a linear polysaccharide (glucan) consisting predominantly of
repeating maltotriose units.
 The maltotriose units, which consist of three α-1,4-linked glucose
molecules, are linked by α-1,6-glycosidic bonds - this repeating sequence
forms a stair-step-type structure. The regular alteration of α-1,4 and α-1,6
bonds results in structural flexibility and enhanced solubility.
 Maltotetraose units may also occur, but are rarer (6%)

5.  The commercially available pullulan (Pullulan PI-20) has a
purity of >90%
 “P” stands for “pullulan”, “I” for “deionized”
 Its average MW is 200 kDa
 The INS No. of pullulan is 1204
 The chemical formula is (C6H10O5)n
 For pullulan PI-20, n corresponds to 1250 glucose units

6.  Pullulan is produced commercially by mesophilic
fermentation (22–30oC) of hydrolysed starch with a selected
non-toxic strain of A. pullulans
 The production strain
 Has high yield of pullulan
 Low production of melanin
 Does not produce aureobasidin A
 Pullulan is formed extracellularly when the cells are in the
late log phase and stationary phase of growth
 Some fermentation substrates from agro-industrial wastes,
such as sweet potato, soybean pomace, potato starch waste,
grape skin pulp extract, jaggery, carob pod, olive oil waste
effluents and molasses, have also been reported

7.  After completion of the fermentation, the fungal cells are removed
by microfiltration.
 The cell-free filtrate is heat-sterilized and treated with activated
carbon to remove pigments.
 The decolorized filtrate is cooled and deionized using cation and
anion exchange resins.
 The deionized solution is refiltered and concentrated by
evaporation to a solid content of about 30% and dried in a drum
drier.
 The dried pullulan is pulverized to a specific particle size and
packed.

9.  Carbon source
 Sucrose, maltose, fructose, glucose, corn starch
 Concentration in the range of 10-15% gives highest yield
 Nitrogen concentration
 Corn gluten, soybean proteins, peptones, nitrates and ammonium salts
 Depletion of N is necessary for initiation of pullulan synthesis
 10:1 C/N ratio most favorable
 Other ingredients
 The M.W. declines as the phosphate content of the medium increases
 Among Fe, Zn, Mn, Cu, Ca, Mo, Co and I ions, only Fe has a positive
effect on biomass yield

10.  Production environment pH
 Optimum pH of the synthetic medium: 6 - 7
 Pullulan synthesis begins at pH 5 when fungal cells enter their late
exponential phase of growth
 Oxygen concentration
 Due to high initial viscosity (>300 cP), dissolved O2 content drops
during agitation in fermentor, reducing yield of pullulan
 Temperature
 Optimum temp for sucrose as C source: 26oC
 Gradual decline in pullulan concentration at temp. lower or higher
than optimum

11. Hexokinase and
isomerase convert
different carbon
sources into the
pullulan precursor,
UDPG (uridine
diphosphoglucose)

12.  Formation of melanin pigment
 Intracellular and extracellular synthesis of melanin during the last stages of
fermentation
 Inhibitory effect caused by high sugar concentrations in the
medium
 High cost associated with pullulan precipitation and recovery
 Viscosity of the fermentation broth (initially >300 cP)

13.  Pullulan PI-20 is a white to off-white tasteless, odourless powder
 Pullulan is water soluble, insoluble in organic solvents (except
dimethylformamide and diimethylsulfoxide)
 Its solubility can be modified by esterification, etherification, or cross-linking to
make even more useful products
 It is non-hygroscopic in nature
 It begins thermal decomposition and carbonizes at 250-280oC
 Pullulan is stable in the presence of most metal ions - a significant feature is its
high stability to sodium chloride
 Films can be made by casting an aqueous pullulan solution on a smooth surface
and applying continuous drying - pullulan films have a low oxygen permeability
and dissolve readily in warm or cold water

14.  Its aqueous solutions are stable and show a relatively low viscosity (2 cP) compared
to other polysaccharides (viscosity resembles that of gum arabic solutions), and do
not form gels
 It is moldable and spinnable, being a good adhesive and binder
 It is also non-toxic, edible and biodegradeable
 Pullulan is susceptible to pullulanase and isopullulanase enzymes, that specifically
hydrolyze pullulan
Viscosity (in cP) of 1% solution at 30o C
Pullulan 2
Gum arabic 1-5
Methyl cellulose 200
Guar gum, Locust bean gum, Xanthan 2000-3000
Soduim alginate 200-700

15. Parameter Specification
Appearance White or yellowish-white
powder
Water solubility (25oC) Easily soluble
Specific optical activity [α] D2O (1% water) Min. +160o
Polypeptides (%) Max. 0.5
pH of solutions 5 – 7
Mineral residue-ash (sulphated, %) Max. 3
Moisture (loss on drying, %) Max. 6
Molecular weight (Range, kDa) 100 - 250

16.  The chemical structure, and thus the reactivity of pullulan resembles that
of maltodextrin and starch amylopectin, both of which are common
constituents of food
 Having a large molecular weight, Pullulan PI-20 is essentially non-reducing
 It is stable in aqueous solution over a wide pH range (3-8)
 Only prolonged heating at pH< 3 leads to a decrease in viscosity which is
indicative of hydrolytic depolymerization

17.  Because it does not contain any chemically reactive group, pullulan
is not expected to interact chemically with other nutrients in food.
 As it is not degraded by the digestive enzymes of the human
alimentary tract to a significant extent, pullulan remains intact in
the small intestine.
 Pullulan can be classified in the group of soluble fibres.
 Having a low viscosity and a chemical structure lacking anionic or
cationic groups, pullulan is not expected to impair the small
intestine absorption of essential nutrients such as vitamins and
minerals at recommended levels of intake.

18. Property Applications
Film formability,
spinnability and
moldability
Effective wrapping, packaging, and sealing material for
various foods
Used in coating chewing gums, candies coatings, and on
surface of eggs and fruits to extend shelf life
Oxygen barrier Pullulan films/coatings are impermeable to oxygen
Pullulan is a preferable packing material that prevents
scattering, and deterioration in taste and flavour due to
oxidation of the fat and oil content of the food

19. Property Applications
Water soluble packets Pullulan laminated paper for tea bags retains the fresh
flavour of tea
Viscosity improver and
thickener
Pullulan imparts thickness to beverages, ice creams and
sauces to improve emulsion stability of these products
Quality and texture
improver
The addition of pullulan in 0.1% is effective in retaining
shape of baked meat products, ham and sausages
Imparts texture to chewing gums

20. Property Applications
Binder and adhesive Pullulan has about double the adhesive strength of starch
To obtain meat products by binding fragments of beef, pork,
or poultry with pullulan into cubes or sheets
In products like doughnuts, it acts as a binder, texturizer and
imparts gloss
Ingredient in Foods
(Utilization of Low
Digestibility)
To prepare low-calorie foods by using mixtures of wheat
flours, amylose starch, and pullulan (like cookies, biscuits, and
wafers in which pullulan replaces amyloceous ingredients)

21. Industry Applications
Photography In photosensitive resin composition that facilitates removal of
the darkened part on exposure by dissolution
Textile As a textile sizing agent
Cosmetics As an emulsifying agent that retains moisture and improves
gelling in lipsticks, and stabilizes creams and lotions
Adhesives In stationery pastes and remoistening pastes for labels, postal
stamps, envelopes, etc.
Paints In water paints with favorable film formability
Foundry As a bounder for foundry sands in the preparation of casts and
molds
Printing As a damping water ingredient
Plywood As an adhesive

22.  Dietary exposure to pullulan as a dietary fibre could reach 1g/kg body weight per
day for children (2–5 years old) and 0.4 g/kg body weight per day for the general
population (JECFA).
 These estimates are 8 and 20 times lower, respectively, than the no-observed-
effect level (NOEL) observed in the 90-day rat study evaluated previously.
 The total mean daily intake of pullulan is calculated to be 9.4 grams/per day
 ADI: “not specified”
 In 2002, pullulan was approved by FDA and recorded in GRAS
 Bio-degradable, non-toxic, non-mutagenic
 In 2012, the FSSAI approved the use of pullulan as a food additive according to
GMP in Dairy based desserts, Fat-based desserts, pre-sooked pastas and noodles,
and in seasoning and condiments, regarding pullulan as a 'novel food'

23.  Pullulan is a unique polysaccharide with a variety of potential
industrial and medical applications.
 Pullulan has wide usage owing to its unique properties.
 However, despite having many valuable applications, the major
constraint prevailing on the use of pullulan is its cost, which is 3
times higher that the cost of other polysaccharides like dextran and
xanthan.
 Technical improvements in pullulan production, like engineering
innovations or improved strains, could reduce production cost.
 An emerging market for pullulan may be in the formulation of
capsules for dietary supplements and pharmaceuticals.

24. ThankYou!

25.  Industrial Gums – R.L. Whistler, J.N. BeMiller
 Pullulan: Chemical and Technical Assessment – Ivan Stankovic
 Pullulan: Microbial sources, production and applications – P. Singh, G.
Saini, J. F. Kennedy
 Pullulan: Production and usage in food industry – P. Oguzhan, F. Yangilar
 Studies on Downstream Processing of Pullulan – D. K. Kachhawa, P.
Bhattacharjee, R. S. Singhal
 Trends in Microbial Production of Pullulan and its Novel Applications in
the Food Industry – Anil H. Lachke, Vinay B. Rale