International Journal of Biological Macromolecules 81 (2015) 867–876

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Statistical modeling of pullulan production and its application in

pullulan acetate nanoparticles synthesis
K.R. Sugumaran ∗ , V. Ponnusami
Bioprocess Engineering Laboratory, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history: This work focused on the optimization of exo-polysaccharide, pullulan production by exploiting cassava
Received 9 April 2015 bagasse, an agricultural solid waste residue by solid state fermentation and its application in the prepa-
Received in revised form ration of pullulan acetate nanoparticles. Statistical approach was investigated to maximize the pullulan
15 September 2015
production using C/N ratio, initial pH, NaCl and ZnSO4 ·5H2 O. The optimum conditions for maximum yield
Accepted 16 September 2015
of pullulan (39.42 ± 0.62 mg/gds) were found to be: C/N = 25.94, initial pH = 5.5 and NaCl = 0.55 g/L. Using
Available online 21 September 2015
the optimized medium variables, the production of pullulan was investigated in lab scale solid state
fermentation. The pullulan produced was characterized by thermo gravimetric and XRD analysis. Also
Keywords:
Pullulan
pullulan acetate nanoparticles were synthesized from chemical modification of pullulan and the average
Cassava bagasse particle size of nanoparticles was examined by zeta particle sizer.
Pullulan acetate nanoparticles © 2015 Elsevier B.V. All rights reserved.

1. Introduction In this present work, an attempt has been made to optimize

Pullulan, exo-poysaccharide comprising matotriose units is pro-
duced by polymorphic yeast like microorganism Auerobasidium
pullulans which has high molecular weight, excellent adhesive
property and is competent of forming transparent, oxygen imper-
meable thin films [1]. Owing to excellent mechanical properties
with non-immunogenic and non-toxic behavior, pullulan is exten-
sively used in cosmetic, food, biomedical and pharmaceutical
industries [2].
Despite its excellent physical properties and wide industrial
applications, pullulan is not a favorable polysaccharide due to
its high cost of production. However, up to thirty percent of
the production cost could be reduced by employing cost effec-
tive substrates in the fermentation [3,4]. Several researchers have
employed low cost substrates for pullulan production [5–16].
Owing to the rich carbohydrate content, low cost and easy avail-
ability, cassava bagasse residue was selected as a solid substrate
[17]. Elemental analysis of cassava bagasse used in the study is as
follows: C = 39.66%; N = 1.52%; H = 5.38%; O = 53.36% and S = 0.08%
by wt [17]. In our previous study, production of pullulan was
investigated with cassava bagasse by solid state fermentation and
produced pullulan was characterized by FTIR, NMR and GPC [16].
the medium variables such as C/N ratio (without supplementary
carbon source added), initial pH and NaCl (g/L) to maximize the
yield of pullulan by statistical design of experiments.
Response surface methodology (RSM) reveals the interaction
among medium variables on response variable and enables us
to achieve global optimization with less number of experiments
compared to conventional one to one design, which in turn saves
lots of time and resources. For these reasons, this method is
more advantageous than conventional method [11,12,14,18–20].
Using the optimized medium variables, the production of pul-
lulan was investigated in lab scale solid state fermentation. The
produced pullulan was also characterized by thermogravimetric
and XRD analysis. Finally, pullulan acetate nanoparticles were
synthesized from chemically modified pullulan, which was con-
firmed by 1 H NMR spectra and then pullulan acetate nanoparticles
were prepared by emulsion-solvent evaporation method. The aver-
age particle size of nanoparticles was examined by zeta particle
sizer.
2. Materials and methods
2.1. Culture conditions

In the present work, A. pullulans (MTCC 2670) was used for

∗ Corresponding author. pullulan production. The microorganism was sub-cultured using
E-mail address: sugumaransastra@outlook.com (K.R. Sugumaran). potato dextrose agar medium. Seed culture was prepared by the

http://dx.doi.org/10.1016/j.ijbiomac.2015.09.025
0141-8130/© 2015 Elsevier B.V. All rights reserved.
868 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876

Table 1
Experimental design for pullulan production using Box–Behnken design.

Run order C/N ratio pH NaCl (g/L) Observed yield (mg/gds) Predicted yield (mg/gds)

1 5 2.6 0.45 6.51 4.64

2 25 2.6 0.45 16.78 16.80
3 5 6.6 0.45 12.56 12.53
4 25 6.6 0.45 33.12 34.98
5 5 4.6 0.1 11.23 11.26
6 25 4.6 0.1 29.45 28.57
7 5 4.6 0.8 15.46 17.31
8 25 4.6 0.8 35.64 34.62
9 15 2.6 0.1 5.12 6.46
10 15 6.6 0.1 24.78 24.27
11 15 2.6 0.8 16.78 17.28
12 15 6.6 0.8 26.89 25.54
13 15 4.6 0.45 30.98 30.69
14 15 4.6 0.45 31.24 30.69
15 15 4.6 0.45 29.87 30.69

addition of two loops of inoculumn with sterilized PDA medium for
seed culture preparation [13,14]. The seed culture was maintained
at 30 ◦ C for 48 h at 200 rpm.
2.2. Solid state fermentation and recovery of pullulan
Cassava bagasse was collected from starch processing indus-
try, Salem district, Tamilnadu, India. It was washed with distilled
water to remove the impurities and air dried for a week. After grind-
ing and sieving the cassava bagasse residue, it was stored at room
temperature in a container.
The solid state production medium was prepared according to
methods standardized earlier [16]. Then, 5% (v/v) of seed culture
was added with sterilized solid state production medium. The fer-
mentation was carried out at 30 ◦ C. After fermentation, six volume
of cold distilled water was added to the fermentation sample and
the mixture was kept in an orbital shaker for 2 h at 200 rpm. Sub-
sequently, the mixture was centrifuged at 10,000 rpm for 15 min
at 4 ◦ C. Solid material with cell debris was carefully gathered and
dried in a hot air oven at 90 ◦ C. One volume of supernatant was
blended with two volumes of ethanol at 4 ◦ C for 24 h for precipita-
tion. Then precipitated sample was re-suspended and centrifuged
at 4400 × g at 4 ◦ C for 15 min to recover the product. The pre-
cipitated sample was dried in hot air oven at 90 ◦ C. The yield
of pullulan was expressed in mg of pullulan/g of dry substrate
(mg/gds) [8].
2.3. Box–Benhken design for optimization
Statistical design of experiments was carried out to know the
interaction among the medium variables on response variable. In
this work, the medium variables such as C/N ratio (without adding
supplementary carbon source), initial pH and NaCl (g/L) were con-
sidered to maximize the production of pullulan through response
surface methodology. The ratio of C/N was changed according to the
addition of NaNO2 in the production medium with fixed amount
of cassava bagasse in the production medium. The ratio of C/N is
obtained from the following expression:
Amount of carbon in cassava bagasse
C/N =
Amount of nitrogen in cassava bagasse + Amount of nitrogen in NaNO2
(1)
Before sterilization, medium variables were added to the suit-
able buffer solution with corresponding pH (2.6, 4.6 and 6.6) to
prepare the production medium. This heterogeneous fermentation
medium was agitated manually to get uniform wetting.
Yu et al. [21] reported the influence of NaCl on pullulan produc-
tion. It was observed that production of pullulan was improved
by the addition of NaCl in the production medium. Singh et al.
[22] reported maximum concentration of pullulan using sodium
chloride as one of the medium variable in response surface
methodology. It has been observed that pullulan production was
significantly improved by sodium chloride (0.15% w/v) in the pro-
duction medium [22]. Therefore, NaCl was considered as one of the
medium variable in response surface method for pullulan produc-
tion. Cassava bagasse has higher moisture holding capacity. The
solid sate fermentation of cassava bagasse for cellulase produc-
tion by Trichoderma reesi NRRL11460 was investigated with initial
moisture content of 66% by Singania et al. [23]. Ray and Moorthy
[8] reported the production of pullulan from cassava bagasse with
a moisture ratio of 1:2 by solid state fermentation. Therefore, all
the experiments were carried out with initial moisture ratio of 1:2.
Totally, fifteen runs including three center points were carried out
(Table 1) and the yield of exo-polysaccharide (mg/gds) was esti-
mated on fourth day of fermentation. The design of experiments
and regression analysis were performed using Design-Expert (Ver-
sion 9).
2.4. Lab scale solid state fermentation
A cylindrical solid state fermenter (Murhopye Scientific Com-
pany, Mysore, India) with a total volume of 2.8 L (height: 21 cm and
diameter: 13 cm) and a working volume of 1.9 L (14 cm packed bed
height) was used (Fig. 1). The fermenter has five ports: (i) sampling
port at top (S1); (ii) gas outlet port at top (A2); (iii) Temperature
measurement port in humidification chamber (T1); (iv) Temper-
ature measurement port placed in bottom of fermenter (T2); (v)
temperature measurement port in the bed (T3). The production
media was prepared according to optimum medium variables by
response surface method using cassava bagasse (400 g dry basis)
and sterilized. After sterilization, 5% v/v of inoculum was uniformly
distributed in to the heterogeneous production medium. In order to
get uniform dispersion of cells in the solid bed, it was gently agitated
by fork like apparatus [24]. Air at a rate of 1 Lpm was introduced
through sparger immersed in humidification chamber, which was
maintained at 27 ◦ C. Then, humidified air was then passed in to
the solid bed through perforated plate. The incubation tempera-
ture was maintained at 30 ◦ C by cooling water circulation through
coil inside the reactor and passage of humidified air through the
bed. The fermentation was carried out for 8 days. The sample was
withdrawn from the solid state bioreactor through sample port at
top to analyze the yield of pullulan.
 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876
Fig. 1. Simplified diagram of Lab scale solid state fermenter. (1) Roameter; (2) air filter; (3) humidifying chamber; (4) controller; (5) chiller; (6) packed bed; (7) water inlet
for cooling; (8) air filter (outlet); T1, T2, T3 are thermocouples; S1: sampling port; A2: air outlet as described in Section 2.4.
2.5. Characterization
The characterization of produced pullulan from fermentation
and commercial pullulan (TCI Chemical, Tokyo) were done by
thermo gravimetric analysis (SDTQ600, TA instruments, USA) and
X-ray diffractometer (D8 Focus, Bruker USA) and compared.
2.6. Pullulan acetate nanoparticles formation and its
characterization
Pullulan acetate (PA) was prepared according to the method
of Motozato et al. [25]. The synthesized pullulan acetate was
characterized by 1 H-NMR spectroscopy (Bruker 300 MHz Instru-
ment, Germany) with DMSO-d6 . Teramethylsilane (TMS) was used
as an internal standard. The chemical modification of pullulan
(pullulan acetate) from produced pullulan using cassava bagasse
and commercial pullulan (TCI chemicals, Tokyo) were designated
by PA1 and PA2 respectively. The degree of substitution (DS) of
acetyl groups in pullulan was observed by the following expression
[26,27] using integration value.
˛ ˇ
= (2)
3x 7 + (3 − x)
where x = degree of substitution which corresponds to the area for
one hydrogen in a glucose unit; ˛ = integration value of acetyl pro-
tons from 1.8 to 2.2 ppm; ˇ = integration value hydroxyl ( OH) and
H-1 to H-6 protons (more than 3.5 ppm).
After preparation of pullulan acetate, pullulan acetate nanopar-
ticles (PANP’s) were synthesized by emulsion-solvent evaporation
method [28]. The procedure is as follows: Accurately weighed pul-
lulan acetate (100 mg) was dissolved in 5 mL of dichloromethane
to form an organic phase which was introduced through a syringe
positioned with needle above the surface of the medium contain-
ing 50 mL of polyvinyl alcohol (aqueous) solution (0.5% w/w). The
medium was gently agitated at 800 rpm using magnetic stirrer
869
to form a primary emulsion. Then, it was subjected to sonica-
tion (Vistronics, Chennai) for 30 min. Finally, it was centrifuged at
18,000 rpm for 30 min to obtain a pellet. The pellet so collected
was suspended in de-ionized water, centrifuged (18,000 rpm for
15 min) for three times and later the collected nanoparticles were
again re-suspended in de-ionized water, lyophilized and stored.
The zeta potential, average particle size and size distribution
of pullulan acetate nanoparticles (PANP’s1 & PANP’s2) were deter-
mined by zetasizer (Malvern Zetasizer ZEN 3600, United Kingdom).
3. Result and discussion
3.1. Statistical design
3.1.1. Box–Benhken statistical design
For this statistical design, the suitable combination of medium
variables, namely C/N ratio, initial pH and NaCl (g/L) were taken.
The design of experiment was listed out in Table 1 and obtained
data were fitted to the following equation:

n 
n 
n 
n
Y = a0 + ai xi + aii xi2 + aij xi xj (3)
i=1 i=1 i=1 j=1+1
where Y = yield of pullulan (mg/gds); a0 , aii , aij = regression
coefficients.
From Table 2a, it is found that all the terms incorporated in the
full model, except for the interaction between medium variables
C/N ratio and NaCl (C/N × NaCl), were found to be statistically sig-
nificant (p < 0.05) at 95% level of confidence [4,11,14,15]. As the
p-value was greater than 0.05, the interaction between C/N ratio
and NaCl was removed from the model [11,29–31] and regres-
sion was repeated. It could be seen that all the terms in the
reduced model were statistically significant (p < 0.05) at 95% level
870 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876

Table 2a
ANOVA for full model.

Source Sum of squares Degrees of freedom Mean square F-value p-value

Model 1434.76 9 159.42 48.51 0.0002

C/N 599.10 1 599.10 182.29 0.0000
pH 340.08 1 340.08 103.48 0.0001
NaCl 73.14 1 73.14 22.26 0.0053
C/N × pH 26.47 1 26.47 8.05 0.0363
C/N × NaCl 0.96 1 0.96 0.29 0.6120
pH × NaCl 22.80 1 22.80 6.94 0.0463
C/N × C/N 73.14 1 73.14 22.26 0.0053
pH × pH 299.30 1 299.30 91.07 0.0002
NaCl × NaCl 40.23 1 40.23 12.24 0.0173
Residual error 16.43 5 3.29
Lack of fit 15.37 3 5.12 9.68 0.0951
Pure error 1.06 2 0.53
Total 1451.19 14

R-Sq = 98.87%; R-Sq (adj) = 96.83%.

Table 2b
ANOVA for reduced model.

Source Sum of squares Degrees of freedom Mean square F-value p-value

Model 1433.80 8 179.22 61.83 0.0001

C/N 599.10 1 599.10 206.67 0.0001
pH 340.08 1 340.08 117.32 0.0001
NaCl 73.14 1 73.14 25.23 0.0024
C/N × pH 26.47 1 26.47 9.13 0.0233
pH × NaCl 22.80 1 22.80 7.87 0.0310
C/N × C/N 73.14 1 73.14 25.23 0.0024
pH × pH 299.30 1 299.30 103.25 0.0001
NaCl × NaCl 40.23 1 40.23 13.88 0.0098
Residual error 17.39 6 2.90
Lack of fit 16.33 4 4.08 7.71 0.1180
Pure error 1.06 2 0.53
Total 1451.19 14

R-Sq = 98.80%; R-Sq (adj) = 97.20%.

of significance (Table 2b). The experimental results agree well with
the model as indicated by p-value for lack of fit (0.118).
3.1.2. Response surface plot
3-D response surface plots and interaction plots were con-
structed to demonstrate the interactions C/N ratio × pH, C/N
ratio × NaCl and pH × NaCl on response variable, pullulan yield.
Generally, convex/concave surface plot denotes strong interaction
among the independent variables, whereas a flat response surface
demonstrates a weak/no interaction. Elliptical shapes on contour
also demonstrate strong interactions [32]. In Fig. 2(a), it can be
observed that the change in the response, pullulan yield with a
change pH was strongly influenced by the level of C/N ratio. Sim-
ilarly, Fig. 2(c) demonstrates that change in pullulan yield with
change in pH was affected by the level of NaCl. Thus, it is evi-
dent from Fig. 2(a) and (c) that the interactions C/N ratio × pH and
pH × NaCl strongly influence pullulan yield. This is consistent with
the ANOVA results. Similarly, the flat surface shown in 3D response
plot (Fig. 2(b)) is consistent with the ANOVA results and confirms
that the interaction C/N ratio × NaCl does not have significant effect
on pullulan yield. That is effect of C/N ratio on pullulan yield was
not affected by the level of NaCl.
3.1.3. Interaction plot
The interactions among medium variables on response variable
were further examined by interaction plot, shown in Fig. 3. The
change in response variable was significantly influenced by inter-
action terms (C/N × pH and pH × NaCl) shown in Fig. 3(a) and (c).
From Fig. 3(a) and (c), it could be seen that the solid lines shown
in red and black are not parallel to each other. The change in
C/N ratio (5–25) and pH (2.6–6.6) on variation in response vari-
able were strongly influenced by the level of pH (2.6–6.6) and
NaCl (0.1–0.8 g/L) respectively. However, in Fig. 3(c), the solid lines
shown in red and black are parallel to each other. This means
that the variation in yield of pullulan with change in C/N ratio
(between 5 and 25) is not significantly affected by the level of
NaCl (0.1–0.8 g/L). The distance between these two parallel lines
demonstrate the individual effect of NaCl, while the increasing
trends of these lines indicate the individual effect of C/N ratio.
Therefore, lower p-values (p < 0.05) corresponding to linear terms
(C/N ratio and NaCl) had shifted to greater p-value (p > 0.05) during
interaction between these medium variables (C/N ratio × NaCl) on
response variable in ANOVA. The obtained results are consistent
with previous reports [33–35].
In order to maximize the yield of pullulan, the statistical model
terms namely linear (C/N, pH, NaCl), square (C/N × C/N, pH × pH,
NaCl × NaCl) and interaction (C/N × pH, pH × NaCl) had been con-
sidered due to their p-values. The regression coefficients in coded
units were substituted in Eq. (4) and following quadratic equation
was obtained:
Yield of pullulan, mg/gds (Y )
2
= 30.69 + 8.65(C/N) + 6.52 pH + 3.02 NaCl − 4.45(C/N)
− 9.0 pH2 − 3.30 NaCl2 + 2.57 (C/N) × pH − 2.38 pH × NaCl
(4)
The above regression equation was analytically solved by
inverse matrix method so as to obtain the maximum yield of
 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876 871

40 40
Design-Expert® Software Design-Expert® Software
Factor Coding: Actual Factor Coding: Actual
Yield of pullulan (mg/gds) Yield of pullulan (mg/gds)

Y ield of pullulan (m g/gds)

Yield of pullulan (mg/gds)

35.64 30 35.64 30

5.12 5.12

20 X1 = A: C/N 20
X1 = A: C/N
X2 = C: NaCl
X2 = B: pH
Actual Factor
Actual Factor 10 10
B: pH = 5.5
C: NaCl = 0.7

0
0

0.8 25
6.6 25 0.7
0.6 20
5.6 20 0.5
15
0.4
4.6 15 0.3 10
C: NaCl (g/l) 0.2 A: C/N
3.6 10
B: pH A: C/N 0.1 5
2.6 5
(b)
(a)

Design-Expert® Software 40
Factor Coding: Actual
Yield of pullulan (mg/gds)
Yield of pullulan (mg/gds)

35.64 30
5.12

X1 = B: pH 20
X2 = C: NaCl

Actual Factor
A: C/N = 20 10

0.8 6.6
0.7
0.6 5.6
0.5
0.4 4.6
0.3 3.6
C: NaCl (g/l) 0.2 B: pH
0.1 2.6

(c)

Fig. 2. 3D response surface plot representing interactions (a) C/N × pH (b)C/N × NaCl (c) pH × NaCl.

pullulan. The optimum values of C/N ratio, NaCl and pH were pre- 3.2. Production of pullulan in Lab scale fermenter
dicted to be 25.94, 0.55 g/L and 5.5 respectively. Corresponding
maximum pullulan yield predicted was 40.76 mg/gds. The exper- Validation experiments were carried out in lab scale solid
imentation was carried out under this condition to validate the state fermenter (20–400 g dry substrate). Fig. 4 explains influence
model. The maximum yield of pullulan 39.42 ± 0.62 mg/gds was of fermentation time on pullulan yield with optimized medium
obtained was obtained on the fourth day of fermentation which variables. It was found that yield of pullulan increased till 4th
was very close to the predicted yield. The obtained yield of pullu- day and then decreased. The reduction in pullulan yield in later
lan by statistical design of experiments was 12.31% more than our stage of fermentation might be due to glucoamylase activity on
previous report (33.4 mg/gds) using cassava bagasse alone (with- synthesized pullulan in the production medium [36]. The maxi-
out 5% w/v mannose). The obtained result was also compared with mum pullulan yield of 34.65 ± 0.925 mg/gds (15.9 ± 0.39 g/L) was
existing reports (Table 3). observed on fourth day of fermentation. This was lower than Erlen-
The comparison of pullulan yield was presented in Table 3. Our meyer flask experiments under optimum condition due to the
findings are consistent with the previous reports. limitation of surface area availability for microorganism growth,

Table 3
Comparison of pullulan production from various carbon sources.

S. No. Substrate Microorganism Type of C/N ratio Moisture Maximum yield of Ref.
fermentation content (%) pullulan (mg/gds)

1 Jack fruit seed A. pullulans NCIM 1049 Solid state Nil 47.9 34.22 [14]
2 Cassava bagasse A. pullulans MTCC 1991 Solid state 25.72 66.67 27.5 [8]
3 Cassava bagasse without A. pullulans MTCC 2670 Solid state 26.49 66.67 33.4 [16]
supplementary carbon source
4 Asian palmyra palm kernel A. pullulans MTCC 2670 Solid state 28.30 50 28.7 ± 0.3 [4]
5 Cassava bagasse without A. pullulans MTCC 2670 Solid state 25.94 66.67 39.42 ± 0.62 In this work
supplementary carbon source
872 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876

Fig. 3. Interaction plot representing interactions (a) C/N × pH; (b)C/N × NaCl; (c) pH × NaCl.

40

35

30
Yield of pullulan (mg/gds)

25

20

15

10

0
0 1 2 3 4 5 6 7 8
Fermentation time, d
Fig. 4. Effect of fermentation time on pullulan yield in lab scale solid state fermentation (amount of substrate = 400 g; C/N ratio: 25.94; NaCl: 0.55 g/L; pH: 5.5; moisture
content: 1:2).
 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876 873

Fig. 5. (a) Thermogravimetric analysis of commercial pullulan (sample heating rate of 10 ◦ C per minute till 1000 ◦ C under nitrogen flow of 100 mL/min). (b) Thermogravimetric
analysis of pullulan from cassava baggase (sample heating rate of 10 ◦ C per minute till 800 ◦ C under nitrogen flow of 100 mL/min).

bed height, heterogeneity, heat and mass transfer limitations
[37,38].
3.3. Thermo gravimetric analysis and XRD analysis
For industrial applications, thermostability of pullulan is an
important property and hence it is essential to estimate the
thermostability of pullulan obtained [4]. The thermo gravimetric
analysis of commercial pullulan and pullulan produced from cas-
sava bagasse are shown in Fig. 5(a) and (b).
The commercial pullulan sample and pullulan produced from
fermentation exhibited the decomposition temperatures (Td ) of
303.78 ◦ C and 296.72 ◦ C respectively. Thermal stability of the pul-
lulan obtained in this study was thus comparable with previous
reports [4,38–42]. On the other hand, thermal decomposition point
can be further improved by appropriate chemical modification of
pullulan [4,26].
The XRD patterns of commercial pullulan and produced pul-
lulan from solid state fermentation had showed a broad peak at
2 = 14 ◦ and 2 = 15 ◦ respectively (Fig. 6(a) and (b)) indicating the
amorphous nature [4,43,44].
874 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876

Fig. 6. (a) XRD analysis for commercial pullulan (scanning range (2) = 10 and 60◦ ; scanning speed = 0.01◦ /s). (b) XRD analysis for pullulan from cassava bagasse (scanning
range (2) = 10 and 60◦ ; scanning speed = 0.01◦ /s).

3.4. Preparation of pullulan acetate nanoparticles
Fig. 7(a) and (b) shows 1 H NMR spectra of PA1 and PA2 using
DMSO solvent. The peak appeared at 1.8–2.2 ppm was due to acetyl
group present in the sample [26]. Hydroxyl ( OH) proton signals
of pullulan acetate at 4–5.5 ppm decreased [27]. Using the proton
NMR spectra, the degree of substitution (DS) of PA1 and PA2 were
obtained to be 2.71 and 2.51 respectively. High DS value indicates
higher hydrophobic nature which would increase an encapsulation
efficacy of drug on polymer matrix [28].
Because of solubility in various solvents, control drug- delivery
and bio-compatibility, pullulan acetate nanoparticles (PANs) serve
as a drug carrier in biomedical industry due to their control
and long term drug delivery [45]. The efficient immobilizations
of hydrophopic compounds with synthesized nanoparticles are
extensively used in tissue engineering and potential drug delivery
applications [27,28]. In this work, the average particle sizes of
PANP’s1 and PANP’s2 were found to be 219.5 nm (polydispersity
index = 0.495) and 203.8 nm (polydispercity index = 0.353) respec-
tively (Fig. 8(a) and (b)). The mean zeta potential value of PAN’s 1
and PAN’s 2 were found to be −16.9 mV and −16.0 mV respectively
[Fig. not shown]. The negative sign of zeta potential was due to the
presence of acetyl groups on polymer surface. Similarly, Santhosh
Kumar et al. [45] reported the delivery of hudrophobic drug (Sily-
marin) using pullulan acetate nanoparticles with average size of
581 nm. Zhang et al. [27] prepared pullulan acetate nanoparticles
ranging from 185.7 nm to 423 nm for epirubicin drug delivery.
The pullulan acetate particle size of 197 nm has been used as a
carrier for lopinavir delivery [28]. Pullulan acetate nanoparticles
(250–333 nm) have been used for Epirubicin drug delivery to
human throat epidermal carcinoma cell line [46]. Our findings are
consistent with existing reports.
 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876 875

Fig. 7. (a) 1 H NMR spectra for pullulan acetate from obtained pullulan after fermentation (solvent = DMSO-d6 ). (b) 1 H NMR spectra for pullulan acetate from commercial
pullulan (solvent = DMSO-d6 ).

Fig. 8. Particle size distribution of pullulan acetate nanoparticles: (a) PAN’s1 from produced pullulan (average particle size = 219.5 nm); (b) PAN’s2 from commercial pullulan
(average particle size = 203.8 nm).

4. Conclusion C/N ratio, initial pH and concentration of NaCl were optimized by

Box–Benhken design of experiments. The maximum pullulan yield
The production of exo-polysaccharide, pullulan was successfully of 39.42 ± 0.62 mg/gds was predicted using C/N ratio (25.94), NaCl
investigated from cassava residue by solid state fermentation. In (0.55 g/L) and pH (5.5). Under this optimized medium variables, the
order to maximize the yield of pullulan, medium variables namely maximum yield of pullulan (34.65 ± 0.925 mg/gds) was obtained in
876 K.R. Sugumaran, V. Ponnusami / International Journal of Biological Macromolecules 81 (2015) 867–876

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