Patel et al.

Many of them contain α-(Pullulan) or β-linked

Basal Medium: (g/l): Glucose 0.5 Yeast Extract 0.5

(e.g.

Scleroglucan,

Schizophyllan)

glucose

units.

NaNO ₃ 2.0, KH ₂ PO 4 1.0, MgSO 4 . 7H 2 0 0.05, KCl 0.5 and

The branched β-glucans are biologically active and

pH 5.3.

consequently are used in medicine and biotechnology, as

ABTS (2,2’-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic

well as additives in food and cosmetics. Cultivation of

acid was procured from Sigma Pvt. Ltd. All the other

mushrooms requires several weeks to complete fruiting

chemicals used were of analytical grade with highest

body formation and thus delaying the production of the

purity.

required compounds. However, submerged cultivation

of fungi offers an advantage for the production of

Micro-organisms and culture conditions

mycelial biomass and bioactive polysaccharides (Zhong

and Tang, 2004). The compounds of fungal origin have

The white rot fungal samples were collected from garden

attracted special attentions from researchers due to their

and surrounding area of ADIT Campus, New Vallabh

various pharmacological and biological activities, such

Vidyanagar, Gujarat. The collected fungi were transferred

as antitumor, antidiabetic, antimicrobial and immune-

on malt extract agar plate amended with streptomycin

stimulatory.

Among

these

compounds,

bioactive

(25 µg/ml) and incubated at 28 º C for 6-7 days. Potential

polysaccharides isolated from higher basidiomycota are

isolates were sub-cultured and maintained at 4°C on malt

the best known and appear to have strongest anticancer

extract agar slants.

activity among mushroom-derived substances.

Culture process and EPS production

The white rot fungi belong to the Basidiomycetes family.

They are mainly grown on decaying wood material.

Erlenmeyer flasks (250 ml) containing 100 ml of basal

Wood presents a nutritionally challenging substrate for

culture medium were inoculated with 8 agar plugs of

fungal growth due to its low nitrogen content and the

size 7 mm diameter punched from the edge of pre-grown

complexity of the bulk carbon present (Bridžiuvienė and

fungal culture on ME plates and incubated on a rotatory

Raudonienė, 2013).

incubator shaker (140 rpm) at 28°C for 7 days.

EPS production rate and productivity were found varying

Determination of exopolysaccharide and mycelial

with environmental conditions and nutrient medium.

biomass

Thus, we undertook the present study with following

The samples were harvested at different time interval

objectives:

from the shake flasks. The cell biomass was separated

ˆ Screening and isolation of exopolysaccharide

by centrifugation at 6000 rpm for 10 min and resulting

producing fungi from natural habitats.

supernatant was used to determine EPS. For the

determination of the EPS (exopolysaccharide), equal

ˆ To optimize various parameters for high yield of

volume of isopropyl alcohol was mixed with the cell

exopolysaccharide.

free supernatant, stirred vigorously and left overnight

ˆ To characterize physico-chemical properties of

at 4ºC for precipitation. The resultant precipitates were

EPS.

separated by centrifugation at 6000 rpm for 10 min. The

precipitants were dried to a constant weight at 70ºC. The

ˆ To

determine

various

applications

of

yield of cell biomass and EPS were expressed as the g/l

exopolysaccharide produced by newly isolated

of the culture medium. The precipitates collected (crude

fungi.

EPS fraction) was washed twice with isopropanol and

2. Materials and Methods

dried to a constant weight at 70°C. The Separated cell

biomass was dried at 70ºC in hot air oven till constant

Media and Chemicals

weight. The yield of mycelial biomass and EPS were

expressed as gram per liter.

Malt extract broth and agar were procured from Hi-media

Labs. (Mumbai, India).

512