Optimization of inulinase production from low cost substrates using Plackett-Burman and Taguchi methods

Article abstract of DOI:10.1016/j.carbpol.2013.11.007

Four marine-derived fungal isolates were screened for the production of inulinase enzyme from low cost substrates under solid state fermentation (SSF), one of them identified as Aspergillus terreus showed the highest inulinase activity using artichoke leaves as a solid substrate. Sequential optimization strategy, based on statistical experimental designs was employed to optimize the composition of the medium, including Plackett-Burman and Taguchi's (L9 3 ⁴) orthogonal array designs. Under the optimized conditions, inulinase activity (21.058 U/gds) reached the predicted maximum activity derived from the taguchi methodology, which increased about 4.79-folds the initial production medium. Fructose was produced, as an end product of inulin hydrolysis proving that the enzyme produced was exoinulinase. The marine-derived A. terreus is suggested as a new potential candidate for industrial enzymatic production of fructose from low cost substrate containing inulin as an economic source.

Full text of DOI:10.1016/j.carbpol.2013.11.007

Carbohydrate Polymers 102 (2014) 261–268
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Optimization of inulinase production from low cost substrates using
Plackett–Burman and Taguchi methods
Abeer A. Abd El Aty∗, Hala R. Wehaidy, Faten A. Mostafa
Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Four marine-derived fungal isolates were screened for the production of inulinase enzyme from low cost
substrates under solid state fermentation (SSF), one of them identified as Aspergillus terreus showed the
highest inulinase activity using artichoke leaves as a solid substrate. Sequential optimization strategy,
based on statistical experimental designs was employed to optimize the composition of the medium,
Received 25 September 2013
Received in revised form 19 October 2013
Accepted 2 November 2013
Available online 1 December 2013
ˆ
including Plackett–Burman and Taguchi’s (L9 34) orthogonal array designs. Under the optimized condi-
tions, inulinase activity (21.058 U/gds) reached the predicted maximum activity derived from the taguchi
methodology, which increased about 4.79-folds the initial production medium. Fructose was produced,
as an end product of inulin hydrolysis proving that the enzyme produced was exoinulinase. The marine-
derived A. terreus is suggested as a new potential candidate for industrial enzymatic production of fructose
from low cost substrate containing inulin as an economic source.
Keywords:
Inulinase
Optimization
Aspergillus terreus
Plackett–Burman design
Taguchi Method
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
sweetening value, its physiological metabolism in human body and
its insignificant insulinogenic effects (Pandey et al., 1999).
Although microbial inulinase (E.C. 3.2.1.7) production has been
reported by many researchers, studies on inulinase production
by marine-derived fungi under SSF are relatively less. A marine
enzyme is a unique protein molecule with novel properties derived
from an organism whose natural habitat comprises saline or
brackish water. Apart from marine-derived microorganisms were
bacteria (including actinomycetes) and fungi. Properties like high
salt tolerance, hyperthermostability, barophilicity, cold adaptivity
and ease in large scale cultivation were the key interests of the sci-
entists. These properties may not be expected in terrestrial sources
as marine organisms (Ghosh, Saha, Sana, & Mukherjee, 2005).
Inulin occurs as a reserved carbohydrate polymer mainly in the
roots and tubers of jerusalem artichoke, chicory, dandelion, bur-
drock and dahlia (Chen, Chen, Chen, Xu, & Jin, 2011). It consists of
linear chains of ␤-2, 1-linked d-fructofuranose molecules termi-
nated by a glucose residue (Vandamme & Derycke, 1983). Inulin
has recently received a great interest for the production of high
fructose syrup. d-Fructose is occupying an increasingly important
position in the modern world as a sweetener because of its higher
Although inulin can be converted into fructose by chemical
approach, this is associated with some drawbacks. Microbial inuli-
nase yields 95% pure fructose after one step enzymatic hydrolysis
of inulin. Thus microbial inulinases are important class of indus-
trial enzymes that have gained much attention in recent times.
Inulinases can be produced by many of the microorganisms includ-
ing strains of Aspergillus sp., Penicillium sp. and Kluyveromyces sp.
Owing to the cost of pure inulin, alternate inulin containing raw,
inexpensive substrates are preferred for microbial inulinase pro-
duction (Trivedia, Divechab, & Shaha, 2012).
Microbial inulinases are extensively produced through sub-
merged fermentation (Kalil, Suzan, Maugeri, & Rodrigues, 2001;
Silva-Santisteban & Maugeri Filho, 2005). Few studies on the pro-
duction of inulinase by solid state fermentation have been recently
reported (Bender, Mazutti, De Oliveira, Di Luccio, & Treichel, 2006;
Selvakumar & Pandey, 1999). Inulinase production by solid state
fermentation (SSF) has attracted much attention because of high
productivity, simple operation, cost effectiveness and better prod-
uct recovery (Singhania, Patel, Soccol, & Pandey, 2009). Moreover,
the crude fermented products from SSF can be used directly as the
enzyme source for biotransformation (Chen et al., 2011).
Statistical methods are increasingly preferred for fermentation
optimization because they reduce the total number of experiments
needed and provide a better understanding of the interactions
among factors on the outcome of the fermentation (Revankar &
Lele, 2006). Some of the popular choices in applying statistical
designs are Plackett–Burman design and Taguchi’s experimental
∗
Corresponding author. Tel.: +202 33371362/433/615/933/449;
fax: +202 33370931.
E-mail addresses: aabass44@yahoo.com (A.A. Abd El Aty),
halolty2009@yahoo.com (H.R. Wehaidy), Fatenahmedalimostafa@yahoo.com
(F.A. Mostafa).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.11.007

262
A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
Table 1
design. The Plackett–Burman design provides an efficient way of a
large number of variables and identifying the most important ones
(Deshmukh & Puranik, 2010). Taguchi’s experimental design has
gained broad acceptance in fermentation optimization. It specifies
orthogonal arrays for combining the various variables and levels in
a minimum acceptable number of experimental trials. In addition,
Taguchi’s approach facilitates the identification of the influence of
individual factors and interactive effects of factors on performance
with a few well-defined experimental sets (Prasad & Mohan, 2005).
Assessment of fermentation conditions for inulinase production
is of relevance since many fermentation parameters may signifi-
cantly affect the productivity of the enzyme and thus production
cost. In this context, the present work was focused to study the
optimization of the process parameters for inulinase production on
low cost substrate under SSF using Plackett–Burman and Taguchi
methods employing a newly isolated marine-derived Aspergillus
terreus. The hydrolysis products of inulin with crude enzyme were
also determined.
The Plackett–Burman design for Physical variables and nutrient screening in inuli-
nase production by A. terreus.
Variable code
Variable
Levels
Low (−1)
High (+1)
A
B
C
D
E
F
G
H
J
Initial pH
4.5
25
1
1
1
0
0
0
0.1
0
7.5
35
3
3
3
1
1
1
0.5
5
Incubation temperature (◦C)
Wheat bran (%)
Glucose (%)
Sucrose (%)
Yeast extract (%)
NH4H2PO4 (%)
Urea (%)
KH2PO4 (%)
K
L
Mg2+ (mM)
Ca2+ (mM)
0
5
2.4. Extraction of Inulinase enzyme
When fermentation was completed, 50 ml of distilled water
were added to the fermented matter, and the mixture was mixed
thoroughly on a rotary shaker (150 rpm) at room temperature
(28 2 ◦C) for 60 min.
The mixtures were filtered through muslin cloth. After cen-
trifugation of the filtrate at 4 ◦C for 10 min, the supernatant was
collected as the crude enzyme solution (Chen et al., 2011).
2. Materials and methods
2.1. Microorganisms and medium
The marine-derived A. terreus, Aspergillus versicolor, Aspergillus
parasiticus and Penicillium brevicompactum, previously isolated
from decayed wood samples collected from Ismalia, Egypt, were
screened for inulinase production in this study. Strains were iden-
tified in the National Research Centre, Microbial Culture Collection
Unit (MCCU) based on the morphological characterization accord-
ing to its colonial and microscopic properties comparing with
fungal species described by (Kohlmeyer & Kohlmeyer, 1991; Pitt &
Hocking, 1985). The strains were maintained on malt extract agar
(MEA) medium at 4 ◦C. The medium contained the following com-
ponents (g/L): biomalt 20, agar 15, 800 ml sterile sea water and
200 ml distilled water (Höller, König, & Wright, 1999).
2.5. Inulinase activity assay
Inulinase activity was assayed by measuring the amount of
reducing sugar fructose released from inulin, using Nelson’s
method (1944). The reaction mixture containing 0.5 ml of enzyme
extract and 0.5 ml of 1% (w/v) inulin in 0.2 M sodium acetate buffer
(pH 5). The mixture was incubated at 50 ◦C for 15 min. The amount
of reducing sugars released was measured using Somogyi’s cop-
per reagent. Absorbance was read at 520 nm. One unit of inulinase
(IU) was defined as the amount of enzyme which librated 1 ␮mol of
fructose per min under the assay conditions. Results of the determi-
nation of inulinase activity were presented in units of activity/gram
solid substrate (U/gds).
The yeast Kluyveromyces marxianus NRRL 7571, known as a good
producer for inulinase enzyme used in the screening step as a pos-
itive control compare with the selected marine fungal isolates.
2.6. Screening of the supplemental nutrients using a
Plackett–Burman design.
2.2. Inoculum preparation
Inocula were prepared by incubating the cultures on MEA slants
at 30 ◦C for about 7 days, until sufficient sporulation was observed.
The spores were harvested using 15 ml sterilized sea water and 1 ml
of the suspension was used for inoculation purpose.
Plackett–Burman design, an efficient technique for medium
component optimization (Naveena, Altaf, Bhadriah, & Reddy, 2005),
was employed for screening fermentation parameters that signifi-
cantly influenced inulinase production. Each independent variable
was tested at two levels, high and low, which were denoted by
(+) and (−), respectively. In the present study, the supplemental
nutrients were screened by a Plackett–Burman design for eleven
variables at two levels (Table 1). The eleven assigned variables were
screened in twelve experimental designs. All experiments were car-
ried out in duplicate and the averages of inulinase activity were
taken as response (Table 2).
2.3. Solid-state fermentation
In the screening step, six carbon sources were used as substrates
for inulinase production. Chicory root, artichoke leaves, banana
leaves, garlic peel, orange rinds and sugarcane bagasse were air-
dried and cut in small pieces (particle size: 1.0, 1.5 and 2.0 mm).
Solid state fermentation was carried out in Erlenmeyer flasks
(250 ml) with 3 g of the solid substrates. Moisture was adjusted by
the addition of 15 ml of sea water to each flask. Each flask was cov-
ered with hydrophobic cotton and autoclaved at 121 ◦C for 20 min.
After cooling, each flask was inoculated with 1 ml spore suspension
and incubated at 30 ◦C for 7 days in a static mode.
During the preliminary screening process, the experiments were
carried out for 7 days, and it was found that at the 96 h, the max-
imum production occurs. Hence, experiments are carried out for
96 h. All the experiments were carried out in duplicate and the aver-
age values are reported as mean SD calculated using MS Excel.
2.7. Optimization of the supplemental nutrients using Taguchi
methodology
The Taguchi methodology was used to investigate the relation-
ship between variables of medium components and to optimize
their concentrations for inulinase production by the marine-
derived A. terreus.
In this study, four factors at three levels of variations (Table 3)
were used in the experiments. The factors optimized included the
initial pH level, incubation temperature and the concentrations

A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
263
Table 2
The Plackett–Burman design variables with inulinase activity as response.
Trials Physical variables
Carbon sources
Nitrogen sources
Energy source Metal ions
Mg2+ Ca2+
Inulinase activity (U/gds)
Initial pH Incubation temp. Wheat bran Glucose Sucrose Yeast extract NH4H2PO4 Urea KH2PO4
1
2
3
4
5
6
7
8
9
−1
+1
+1
−1
−1
+1
+1
−1
−1
−1
+1
+1
+1
−1
+1
+1
+1
−1
−1
+1
+1
+1
+1
−1
−1
+1
−1
+1
−1
−1
+1
−1
+1
+1
+1
−1
+1
+1
+1
+1
+1
−1
+1
−1
−1
−1
−1
−1
+1
−1
+1
+1
−1
+1
−1
−1
+1
+1
−1
+1
+1
−1
−1
−1
+1
−1
−1
−1
+1
−1
+1
+1
+1
−1
+1
−1
+1
+1
−1
+1
+1
−1
−1
+1
+1
+1
−1
−1
−1
−1
+1
0.443
1.286
4.655
4.433
0.665
9.975
13.300
0.554
0.488
1.108
6.428
10.861
−1
+1
+1
+1
−1
−1
−1
−1
−1
+1
−1
−1
+1
+1
+1
−1
−1
+1
−1
+1
−1
+1
+1
+1
−1
−1
−1
+1
−1
+1
+1
−1
−1
−1
+1
−1
10
11
12
−1
−1
−1
+1
+1
The sign +1 and −1 represent the two different levels (high and low) of the independent variable under investigation.
of KH2PO4 and Ca2+, because they have significant impact on
inulinase production as screened by Plackett–Burman design. The
various combinations of factors and levels were in accordance with
Taguchi’s L9 orthogonal array. The factor level combinations for all
the experiments are shown in (Table 3). A verification test was also
performed to check the optimum condition. An analysis of variance
(ANOVA) for the obtained results was investigated. For designing
the experiments, analysis of variance and optimization of process,
Design-Expert® 8 software from Stat-Ease, Inc., was used.
sp., Fusarium sp., Aspergillus sp.) were used for inulinase produc-
tion (Kango, 2008; Naidoo, Ayyachamy, Permaul, & Singh, 2009;
Singh & Bhermi, 2008; Souza-Motta, Cavalcanti, Porto, Moreira, &
Lima filho, 2005). In order to select the best solid substrate (inu-
line source) and the fungal strain for inulinase production, six agro
industrial wastes (low cost substrates) were checked for their effect
on inulinase production by four marine-derived fungal isolates, A.
terreus, A. versicolor, A. parasiticus and P. brevicompactum, locally
isolated from decayed wood samples collected from Ismalia, Egypt.
Results documented in (Table 4) showed that, all the tested
strains produced the enzyme on all the substrates but with dif-
ferent activities. Among the six different solid substrates, artichoke
leaves were found to be the most suitable substrate for SSF by A. ter-
reus giving maximum inulinase production of 4.433 0.121 U/gds,
greater than that obtained with K. marxianus NRRL 7571(positive
control) on the same waste. Other substrates were found to be
poor inducers for inulinase production by A. terreus. Significant
amount of inulinase was also produced on SSF of orange rinds and
sugar cane bagasse with A. versicolor and A. parasiticus, respectively.
P. brevicompactum showed the highest production with artichoke
leaves, banana leaves and sugar cane bagasse than the other sub-
strates.
2.8. Determination of hydrolysis products
The hydrolysis products of inulin with crude enzyme were
determined by paper chromatogram (Wehaidy, 2012). The paper
chromatogram spotted with reaction products after 15 min incuba-
tion was developed using (butanol:acetone:water) (4:5:1, v/v/v) as
irrigating solvent. After 48 h. the hydrolysis products (sugars) were
visualized by heating the plates at 105 ◦C for 15 min after spraying
with detection solution containing (0.91 ml aniline, 1.66 g phthalic
acid, 48 ml diethyl ether, 48 ml butanol and 4 ml water). Glucose,
fructose and sucrose were used as standards.
There for, the marine fungal isolate A. terreus was the most
promising one for inulinase enzyme production and the present
work will focused to study the optimization of the SSF using arti-
choke leaves. Also the type of the produced enzyme (endo- or exo-)
will be determined.
3. Results and discussion
3.1. Survey of low cost substrate and marine-derived strain
favouring inulinase production
Although inulinase was initially isolated from plants, it is dif-
ficult to obtain high production (Kumar, Kunamneni, Prabhakar,
& Ellaiah, 2005). In the last decades, a large number of microor-
ganisms such as bacteria, yeast and filamentous fungi (Penicillium
3.2. Time course of fermentation
The time courses of the inulinase production by the selected
fungus A. terreus during SSF of artichoke leaves (basal medium)
Table 3
ˆ
Factors and their levels which were studied by the orthogonal array of L9 (34) design for optimization of inulinase production from A. terreus by the Taguchi method.
Trial number
Factors
Response enzyme
Activity (U/gds)
Coded levels
Actual levels
A: Initial
pH
B: incubation
Temp. (◦C)
C: Initial
concent. of
KH2PO4 (%)
D: Initial
A: Initial
pH
B: incubation
Temp. (◦C)
C: Initial
concent.of
KH2PO4 (%)
D: Initial
Experimental Predicted
(actual)
concent. of
concent. of
Ca2+ (mM)
Ca2+ (mM)
1
2
3
4
5
6
7
8
9
3
3
1
2
2
1
1
2
3
3
1
1
3
2
2
3
1
2
2
3
1
1
3
2
3
2
1
1
2
1
2
1
2
3
3
3
8.5
8.5
6.5
7.5
7.5
6.5
6.5
7.5
8.5
40
30
30
40
35
35
40
30
35
0.5
0.8
0.3
0.3
0.8
0.5
0.8
0.5
0.3
5
6
5
6
5
6
7
7
7
3.547
21.058
8.246
2.66
17.955
9.532
1.995
13.300
15.295
4.029
20.374
8.729
1.975
18.438
8.847
2.197
13.502
15.497

264
A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
Table 4
Effect of different low cost substrates on inulinase production by locally isolated marine-derived fungi under solid-state fermentation.
Fungal isolates
Inulinase activity (U/gds) with different solid substrates
Chicory roots
Artichoke leaves
Banana leaves
Garlic wastes
Orange rinds
Sugar cane bagasse
Aspergillus terreus
Aspergillus versicolor
Aspergillus parasiticus
Penicillium brevicompactum
*Kluyveromyces marxianus NRRL 7571
0.133 0.063
0.177 0.125
0.199 0.141
0.886 0.626
0.222 0.157
4.433 0.121
0.178 0.125
0.177 0.125
1.241 0.877
1.773 1.254
0.366 0.423
0.205 0.031
0.321 0.173
1.219 0.157
1.352 0.188
0.022 0.031
0.399 0.067
0.587 0.329
0.665 0.156
0.886 0.376
0.776 0.470
1.917 0.016
1.341 0.862
0.853 0.047
0.443 0.313
1.108 0.521
1.319 0.141
1.773 0.627
1.217 0.052
0.088 0.012
*
Kluyveromyces marxianus NRRL 7571: used in the screening step as a positive control for inulinase production.
without any additives were monitored for 240 h. Results indicated
that, the high level of inulinase attained within 96 h. The inuli-
nase production reached about (5.680 0.183 U/gds) after 96 h of
solid-state fermentation, and after that, the activity decreased with
increasing the time of fermentation. The observed decline in inuli-
nase activity after 96 h of incubation could be a result of protease
degradation, decrease in nutrient availability in the medium and
catabolic repression of the enzyme (Kango, 2008; Wang & Zhou,
2006). This was in agreement with the previous studies on A. niger
ATCC 20611 (Dinarvand et al., 2012) indicated the maximum inuli-
nase production obtained after 96 h of incubation at 30 ◦C. Hence,
all the experiments of optimization were carried out for 96 h.
The obtained results compared well with the results for other
producer microorganisms where the inulinase activity generally
was reported to peak much earlier in the fermentation process.
For example, (Kumar et al., 2005) observed the highest inulinase
activity after 72 h of incubation.
to 13.300 U/gds. This variation reflects the importance of medium
optimization to attain higher productivity.
According to these results,
a medium of the following
composition is expected to be near optimum: initial pH 7.5, incu-
bation temperature 35 ◦C, 0.5% KH2PO4, 5 mM Mg2+, 1% glucose,
1%sucrose, 1% NH4H2PO4 by employing 3 g of artichoke leaves
(moistened with 15 ml of tris-maleate buffer, 0.1 M, pH 7.5) as
the solid substrate in 250 ml Erlenmeyer flasks for 96 h incuba-
tion period. The enzyme activity measurement on this medium
was 13.300 U/gds. This result presented about 2.34-folds increase
in the enzyme activity, when compared to (5.679 U/gds), the results
obtained in basal production medium containing only the solid
substrate and sea water after 96 h incubation time.
The analysis of variance (ANOVA) for the experiment design
showed that, A, B, D, E, F, H, J, K, L are significant model terms. But the
factor C is not significant and removed. The first order model equa-
tion developed by PB design showed the dependence of A. terreus
inulinase production on the medium constituents: Eq. (1).
R1(inulinase activity U/gds) = +4.52 + 3.23 ∗ A + 0.51 ∗ B
− 0.72 ∗ D − 0.47 ∗ E − 0.92 ∗ F
3.3. Screening of significant nutrients using a Plackett–Burman
design
+ 0.020 ∗ G − 1.22 ∗ H + 2.19 ∗ J
Plackett–Burman design is a powerful technique for screening
important variables. In the present study, it was used to analyze
the effect of eleven variables, including fermentation conditions
and medium constitution on inulinase production (Table 1). In the
experiment design, each row represents an experiment and each
column represents an independent variable (Table 2). The yield of
inulinase, determined for each experiment design, was shown in
Table 2. Where we found a wide variation in the activity from 0.443
+ 0.45 ∗ K + 0.95 ∗ L
The analysis of the regression coefficients of the 11 variables
indicated that, initial pH, KH2PO4, Ca2+, incubation temperature
and Mg2+ had positive effect on inulinase production. Urea, yeast
extract, glucose, sucrose and NH4H2PO4 were contributed nega-
tively (Fig. 1).
Design-Expert® Software
R1
Pareto Chart
A
A: Initial pH
B: Incubation temperature
1750.65
C: Wheat bran
D: Glucose
E: Sucrose
F: yeast extract
G: NH4H2PO4
H: Urea
1312.99
J
J: KH2PO4
K: Mg2+
L: Ca2+
Positive Effects
Negative Effects
875.33
H
L
F
D
437.66
0.00
B
E
K
G
1
2
3
4
5
6
7
8
9
10
11
Rank
Fig. 1. Pareto chart showed the effect of each variable on the enzyme activity (U/gds) produced by A. terreus.

A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
265
Table 5
Analysis of variance (ANOVA) for the experimental results of the Taguchi method.
Source
Sum of squares
df
Mean square
F value
p-Value Prob > F
Model
A-initial pH
B-incubation temperature
C:Initial concent.of KH2PO4 (%)
Residual
383.73
71.22
264.37
48.15
2.23
6
2
2
2
2
8
63.96
35.61
132.19
24.07
1.11
57.41
31.96
118.66
21.61
0.0172 significant
0.0303
0.0084
0.0442
Cor Total
385.96
Std. Dev. = 1.06; Mean = 10.40; C.V.% = 10.15; PRESS = 45.12; R-squared = 0.9942; Adj R-squared= 0.9769; Pred R-squared = 0.8831; adeq precision = 19.766 (the factor, D-Ca2+
removed where, p-value Prob > F = 1.0000).
The obtained results showed that, varying the initial cultiva-
tion pH of the medium between pH 4.5 and 7.5 had high effect
on inulinase production by the marine-derived A. terreus. Maxi-
mal enzyme activities were obtained only when the initial pH of
the culture medium was adjusted to 7.5. This is a logic observa-
tion because the culture used in the present study was isolated
from marine source where the pH lied in the neutral to alkaline
range. On the other hand (Dilipkumar, Rajasimman, & Rajamohan,
2011) indicated that KH2PO4 was found to be one of the most
significant nutrient components affecting inulinase production by
Streptomyces sp. in solid-state fermentation (SSF).
The obtained results also agreed with (Gouda, 2002) who indi-
cated that, the metal ions, Ca2+ was found to be more effective in
inulinase production by A. fumigatus. This effect may due to the
formation of complex with ionized inulinase resulting in chang-
ing solubility and behaviour at the conformation of protein to a
less stable form by interaction with enzyme surface charge which
could markedly affect the ionization of some amino acid residues
(Masomian, Rahman, Salleh, & Basri, 2010).
There for, the optimum combination of the variables (initial pH,
KH2PO4, Ca2+ and incubation temperature) which had the highest
significant influence on inulinase production was further analyzed
by a Taguchi method for the best solid state fermentation bringing
maximum inulinase activity. Other variables with less significant
effect were not included in the next optimization experiment, but
instead were used in all trials at their (−1) level and (+1) level, for
the negatively contributing variables and the positively contribut-
ing variables, respectively.
which significantly influenced inulinase production, were further
ˆ
investigated for their optimum combination using Taguchi’s (L9 34)
orthogonal array design. The design and results of the experiments
together with the predicted activity from the regression equation
for the combinations are shown in (Table 3). It showed that the
actual response values agree well with the predicted response val-
ues.
Final equation in terms of coded factors: Eq. (2).
Inulinase enzyme activity = +10.40 − 3.8 ∗ A[1] + 0.91 ∗ A[2]
+ 3.80 ∗ B[1] + 3.86 ∗ B[2]
− 1.66 ∗ C[1] − 1.61 ∗ C[2]
The results of experiments performed in this section showed that
the maximum average yield of inulinase was 21.058 U/gds, which
occurred when experiment’s conditions were as follows: the ini-
tial pH 8.5, incubation temperature 30 ◦C and the concentration of
KH2PO4 and Ca2+ were 0.8% and 6 mM, respectively added to 3 g of
artichoke leaves (low cost solid substrate containing inulin) moist-
ened with 15 ml buffer (0.1 M, Tris–maleate pH 8.5). Also the other
variables which not included in the Taguchi experimental design
were included at the concentrations of, 1% glucose, 1% sucrose, 1%
NH4H2PO4 and 5 mM Mg2+ which give maximum production with
Plackett–Burman design.
Analysis of variance (ANOVA) of main effects of factors indicated
that, the model F-value of 57.41 implies the model is significant.
Values of “Prob > F” less than 0.0500 indicate model terms are
significant, in this case the factors, A-initial pH, B-incubation
temperature, C:Initial concentration of KH2PO4 (%) are significant
model terms.Values greater than 0.1000 indicate the model terms
are not significant as the factor D-Ca2+. Results also showed
that, The “Pred R-Squared” of 0.8831 is in reasonable agreement
with the “Adj R-Squared” of 0.9769. An adequate precision value
greater than 4 is desirable. The adequate precision value of 19.766
3.4. Further optimization of the nutrients using Taguchi
methodology
Based on the results of Plackett–Burman design, four variables
including initial pH, incubation temperature, KH2PO4 and Ca2+
,
Design-Expert® Software
Factor Coding: Actual
Inulinase enzyme activity (U/g solid substrate)
X1 = A: initial pH
X2 = B: inc ubation temperature
Actual Factors
C: KH2PO4 = 0.8
30
20
10
0
D: Ca2+ = 6
8.5
-10
40
7.5
35
B: incubation temperature
A: initial pH
6.5
30
Fig. 2. 3D-surface plot showing the effect of initial pH and incubation temperature on inulinase activity.

266
A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
Design-Expert® Software
Factor Coding: Actual
Inulinase enzyme activity (U/g solid substrate)
X1 = A: initial pH
X2 = C: KH2PO4
Actual Factors
B: incubation temperature = 30
D: Ca2+ = 6
30
20
10
0
8.5
-10
0.8
7.5
0.5
A: initial pH
6.5
0.3
C: KH2PO4
Fig. 3. 3D-surface plot showing the effect of initial pH and KH2PO4 concentration on inulinase activity.
indicates an adequate signal and suggests that the model can be
used to navigate the design space (Table 5).
under optimized culture conditions i.e. the initial pH of the medium
8.5, incubation temperature 30 ◦C and the concentration of KH2PO4
and Ca2+ were 0.8% and 6 mM, respectively. Under these condi-
tions, 21.058 0.84 U/gds of inulinase was obtained. This value of
enzyme yield corresponds very well to the values predicted by
the model (20.37 U/gds), which proved the validity of the model.
After statistical optimization, inulinase yield was increased to
4.79-folds when compared to (4.433 U/gds), the results obtained
in the first basal production medium, without any optimiza-
tion.
The interaction effects of variables on inulinase production were
studied by plotting 3D surface plot against any two independent
variables, as in Figs. 2–4. The results illustrated graphically in these
figures indicated that the dependency of inulinase enzyme on initial
pH level and the incubation temperature followed by the con-
centration of KH2PO4 as an energy source. The inulinase activity
increased with increasing the initial pH level to 7.5 and 8.5, where
the neutral to alkaline range was observed to be most favourable
conditions for inulinase production by this strain than the acidic
conditions. This to some extend agreed with (Naidoo et al., 2009)
who found that the maximal inulinase activity of Xanthomonas
campestris pv. phaseoli KM24 mutan was obtained when the ini-
tial pH of the culture medium was adjusted to 7.0. Previous studies
indicated that Geofungi growing in marine environments appear to
be conditioned to their environment (Raghukumar & Raghukumar,
1998). The optimum temperature favouring inulinase production
is 30 ◦C, also the activity increased at 35 ◦C but decreased greatly at
the temperature 40 ◦C.
3.6. Hydrolysis products of inulin
The paper chromatogram procedure employed to characterize
the mode of action of the enzyme produced by the marine-derived
fungus A. terreus and particularly, if it is of the endo- or exo-type.
Analysis of the products of inulin hydrolysis after 15 min, showed
the presence of fructose (Fig. 5). Release of fructose as an end
product of inulin hydrolysis states that inulinase of A. terreus was
an exoacting enzyme. These results agreed with (Trivedia et al.,
2012) who reported the production of fructose as an end product
of inulin hydrolysis by a newly isolated Aspergillus tubingensis
CR16 inulinase enzyme. (Ertan & Ekinci, 2002) also reported
that the inulinases of A. alternata and A. niger hydrolyse inulin
through an exo-type reaction producing fructose. Fructose is an
3.5. Validation of the experimental model
In order to determine accuracy of the model and to verify
the optimization results, experiments were repeated in triplicates
Design-Expert® Software
Factor Coding: Actual
Inulinase enzyme activity (U/g solid substrate)
X1 = B: incubation temperature
X2 = C: KH2PO4
Actual Factors
A: initial pH = 8.5
D: Ca2+ = 6
30
20
10
0
-10
0.8
40
35
0.5
B: incubation temperature
30
C: KH2PO4
0.3
Fig. 4. 3D-surface plot showing the effect of incubation temperature and KH2PO4 concentration on inulinase activity.

A.A. Abd El Aty et al. / Carbohydrate Polymers 102 (2014) 261–268
267
which may provide other unexplored information regarding this
enzyme.
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4. Conclusions
The marine-derived, alkalophilic and salt tolerant fungus A.
terreus, isolated from marine decayed wood, showed the abil-
ity to produce maximal yield of inulinase enzyme on low cost
substrate (artichoke leaves) under SSF. After the optimization by
using Plackett–Burman and Taguchi methods, the optimal medium
composition for the inulinase production by A. terreus during the
solid-state fermentation was found to be, initial pH 8.5, incubation
temperature 30◦C, 0.8% KH2PO4 and 6 mM Ca2+ in the presence
of 1% glucose and 1%sucrose as additional carbon sources, 1%
NH4H2PO4 as nitrogen source and 5 mM Mg2+ by employing 3 g
of artichoke leaves (moistened with 15 ml of tris-maleate buffer,
0.1 M, pH 8.5). Under the optimized conditions, the maximum yield
of inulinase achieved was 21.058 U/gds, which increased about
4.79-folds the initial production medium. In the present work,
obtained results indicated the scope for economic production of
inulinase by a new marine-derived source using solid-state pro-
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optimizing inulinase enzyme production in stead of the com-
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capacity of the A. terreus inulinase enzyme, make it an attrac-
tive source for industrial application. However, further studies on
its Properties were also evaluated to predict its end applications

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