Advances in Microbiology, 2012, 2, 277-283 Published Online September 2012 (
Development of Efficient Fermentation Process at
Bioreactor Level by Taguchi’s Orthogonal Array
Methodology for Enhanced Dextransucrase Production
from Weissella confusa Cab3
Shraddha Shukla, Arun Goyal*
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, India
Email: *
Received April 20, 2012; revised May 19, 2012; accepted May 28, 2012
The influence of medium ingredients on extracellular dextransucrase production by a new bacterial strain Weissella
confusa Cab3 (Genbank Accession Number JX649223) was evaluated using fractional factorial design of Taguchi’s
orthogonal array. Four metabolism influencing factors viz. sucrose, yeast extract, K2HPO4 and Tween80 were selected
to optimize dextransucrase production by W. confusa Cab3 using fractional factorial design of Taguchi methodology.
Based on the influence of interaction components of fermentation, least significant factors of individual level have
higher interaction severity index and vice versa for enzyme production from Weissella confusa Cab3. Sucrose and yeast
extract were found to be the most significant factors which positively influenced the dextransucrase production. The
optimized medium composition consisted of sucrose—5%; yeast extract—2%; K2HPO4—1.0%; Tween80—0.5, based
on Taguchi orthogonal array method. The optimized composition gave an experimental value of dextransucrase activity
of 17.9 U/ml at shake flask level which corresponded well with the predicted value of 17.54 U/ml by the model. The
optimized medium by Taguchi method gave significant (3 fold) enhancement of dextransucrase activity as compared to
unoptimised enzyme activity of 6.0 U/ml. The dextransucrase production was scaled up in lab scale bioreactor resulting
in further enhancement of enzyme activity (22.0 U/ml).
Keywords: Lactic Acid Bacteria; Weissella confusa; Dextransucrase; Taguchi’s Orthogonal Array Method; Bioreactor
1. Introduction
The enzymes synthesizing dextran from sucrose are
known as dextransucrase (1,6-α-d-glucan-6-α-glucosyl-
transferase, EC They catalyze the transfer of
glucosyl residues from sucrose to dextran polymer and
liberate fructose [1]. Dextransucrase is produced by
various Leuconostoc and Streptococcus species [2,3] and
by the mold Rh izopus spp. [4]. Dextransucrase is an in-
ducible enzyme requiring sucrose in the medium for the
induction with the exception of recently isolated consti-
tutive mutant strains viz. B-512 FMC [5], B-742 [6],
B-1299 [7] and B-1355 [8]. Streptococcus species are
generally constitutive and do not require sucrose in the
growth media for enzyme expression [9]. The dextransu-
crase is a large protein consisting of approximately 1600
amino acid sequence [10]. Schematic structure of dex-
transucrases for which encoding genes have been cloned
suggests that dextransucrase consists of A, signal peptide;
B, variable region; C, N-terminal catalytic domain; D,
C-terminal glucan binding domain. N terminal part con-
tains typical signal peptides of Gram-positive bacteria
[11]. The main characteristic of the structure of glucan-
sucrase signal peptides is that it is well conserved [11]. N
terminal signal peptide aids in the translocation of dex-
transucrase across bacterial membrane. The non-con-
served region located just downstream of the signal pep-
tide tends to have no important role in the enzyme
mechanism. Its deletion does not affect the enzyme ac-
tivity [12]. As shown by protein sequence alignments of
different dextransucrases, the N-terminal domain is
highly conserved and was catalytic domain [13]. The
C-terminal domain is a functional glucan binding domain
and consists of a series of direct repeating units [10].
Dextransucrase catalyses dextran biosynthesis by trans-
ferring glucosyl residues coming from sucrose cleavage
enzymes and are of high-molecular-mass (107 to 108 Da).
The dextran synthesis reaction occurs by successive
transfer of glucosyl units to the polymer. Dextran is
*Corresponding author.
opyright © 2012 SciRes. AiM
composed of a linear chain of glucosyl residues linked
through α(16) glucosidic bonds and several α(12),
α(13), or α(14) branched linkages. Dextrans are
useful in various industries because of their inertness,
porous structure and gelling properties [14]. These are
used as food syrup stabilizers, matrix of chromatography
columns, blood plasma substitutes, antithrombogenic agents,
treatment for iron deficiency anaemia, drug carriers
Microorganisms utilize various substrates as nutrient
source to avail their growth and metabolic activities. By
utilizing the nutrients microorganisms subsequently pro-
duce various metabolism-related products. However,
fine-tuning of nutrient concentrations is an essential aim
to regulate the microbial metabolism and associated
metabolic product production. For the fine tuning of the
optimization of responses, there are various methods
reported in the literature like Box Behnken method, CCD
method, neural networking and Taguchi’s orthogonal
array based methodology. Singh et al., 2008 [16] used
artificial intelligence based optimization method for en-
hancement of exocellular glucansucrase production from
Leuconostoc dextranicum NRRL B-1146. Purama and
Goyal, 2008 [17] employed response surface methodol-
ogy for maximizing dextransucrase production from
Leuconostoc mesenteroides NRRL B-640 in a bioreactor.
Recently Patel et al., 2011 [18] optimized the medium
component for a new isolate Pediococcus pentosaceus
(SPAm) using response surface methodology.
Taguchi’s orthogonal methodology appends planning
of the experiments, their conductivity and finally evalua-
tion of the results of matrix experiments to find out the
best level of the factors for the optimization process. In
this method the best levels of the factors maximize the
Signal-to-Noise ratios which are log functions of desired
output characteristics. Weissella confusa is a lactic acid
bacterial strain which produces dextransucrase. There are
very few reports where the dextran production capacity
of this strain have been explored. However Weissella
confusa is a very potent candidate for dextran production
in the sense that it has high dextran yield and the dextran
produced by this strain is more linear in nature thus finds
its place in food industries. In our earlier reports, the
dextransucrase and dextran production capacity by a
fermented cabbage isolate Weissella confusa Cab3 were
studied [19]. Weissella confusa Cab3 produced dextran-
sucrase (6.0 U/ml) in medium described by Tsuchiya et
al., 1952. Further the effects of various carbon sources,
nitrogen sources and buffering agents were investigated
for their effects on dextransucrase and dextran produc-
tion from Weissella confusa Cab3 [20]. In the present
study the medium components were optimized for the
enhanced dextransucrase production by Weissella con-
fusa Cab3 using Taguchi’s orthogonal method. Subse-
quently, the dextransucrase production by this strain was
scaled up to lab scale bioreactor using the statistically
designed medium. As of my knowledge this is the first
report of enhancement of the dextransucrase activity by
Weissella confusa using statistically methods.
2. Materials and Methods
2.1. Microorganism, Maintenance and
Preparation of Seed Culture
The bacterial strain Weissella confusa Cab3 (Genbank
Accession Number JX649223) isolated from fermented
cabbage [19] was used for optimization of medium
composition for dextransucrase production. The organ-
ism was maintained in MRS medium [21] stabs incu-
bated at 25˚C, stored at 4˚C and subcultured every two
weeks. Fermentation experiments were carried out using
Weissella confusa Cab3, subcultured and grown (12 - 14
h old) in medium described by Tsuchiya et al., 1952 [22]
at optimised culture conditions i.e. 25˚C and 180 rpm
[19]. The pH of the medium was adjusted to 7.0 using
2M HCl solution.
2.2. Production of Dextransucrase
The production of dextransucrase was carried out in 250
ml Erlenmeyer flasks containing 100 ml medium as per
the design inoculated with 1% culture inoculum. The
inoculated flasks were incubated under orbital shaking at
180 rpm and 25˚C for 12 - 15 h. The samples (1 ml) were
withdrawn at indicated time intervals and centrifuged at
8000 g for 10 min at 4˚C to separate the cells. The cell
free supernatant was analyzed for enzyme activity.
2.3. Dextransucrase Activity Assay
The enzyme assay was carried out in 1 ml reaction mix-
ture containing 5% (w/v) sucrose, 20 mM sodium acetate
buffer (pH 5.4) and 20 μl cell free supernatant. The en-
zymatic reaction was performed at 30˚C for 15 min. 100
μl aliquot from the reaction mixture was taken for reduc-
ing sugar estimation. The enzyme activity was deter-
mined by estimating the released reducing sugar by Nel-
son, 1944 [23] and Somogyi, 1945 [24] method. The
absorbance of the color developed was measured by
spectrophotometer (Varian, Carry 100) at 500 nm. Fruc-
tose was used to plot the standard graph.
2.4. Taguchi Methodology
An L16 orthogonal array in four levels was used consist-
ing of 16 different experimental trials for the medium
optimization for dextransucrase production by Weissella
confusa Cab3. The design for the L16 OA was developed
and analyzed using “MINITAB 15” software. All four
Copyright © 2012 SciRes. AiM
Copyright © 2012 SciRes. AiM
selected factors, their assigned levels and the experimen-
tal design along with dextransucrase production data are
listed in Tables 1 and 2, respectively. To achieve the
maximum dextransucrase production by Weissella con-
fusa Cab3, sucrose (%, w/v), Yeast extract (%, w/v), di-
potassium hydrogen orthophosphate (K2HPO4; %, w/v)
and Tween80 (%, v/v) were selected for medium optimi-
zation for enhanced dextransucrase production because
they had significant impact on dextransucrase production
as screened in our earlier findings [20]. Sucrose was the
most effective medium component for dextransucrase
production. However yeast extract, K2HPO4, and Tween80,
displayed moderate effect on dextransucrase production
from Weissella confusa Cab3. Experimental results were
fitted in Taguchi software to analyze further for predicted
values, individual and interactive influences, ANOVA,
optimum conditions and to know the contribution of each
selected fermentation factor in the production of dex-
transucrase by this bacterial strain. Validation experi-
ments were performed using optimized parameters of
fermentation medium components and levels by software.
The dextransucrase production medium was validated at
shake flask level. The optimized medium for dextransu-
crase production from Weissella confusa Cab3 were in-
oculated with 1% of fresh seed culture of Weissella con-
fusa Cab3 and various fermentation parameters like op-
tical density of cell (OD600), enzyme activity, protein
concentration [25], and sucrose concentration were de-
The dextransucrase production using optimized me-
dium was scaled up in 1L volume of culture medium in a
3L bioreactor (Applikon, model Bio Console ADI 1025).
The bioreactor is equipped with pH probe, oxygen probe,
foam sensor, and stirrer of two-six bladed Rushton tur-
bines. For controlled pH cultivations, the pH was main-
tained at 7.0 by addition of 2 M NaOH and 2 M HCl so-
lution. During the experiments, temperature and aeration
rate were controlled at 25˚C and 2 vv1min 1, respec-
tively. The Dissolved Oxygen (DO) was calibrated to
100% before inoculation. The initial agitation rate was
set to 200 rpm and it was changed accordingly to main-
tain the DO above 30%. 1% inoculum from 12 h grown
culture was inoculated in the bioreactor. The parameters
like dextransucrase activity, sucrose concentration, cell
optical density and dry cell weight were analyzed at
regular interval. The cell optical density was taken at 600
nm. The sucrose concentration was determined by esti-
mating the reducing sugars by the method of Sumner and
Sisler (1944) [26]. Sterile Soybean oil was simultane-
ously employed as anti-foam agent.
3. Results and Discussion
The optimum temperature, pH and shaking condition
Table 1. Selected factors and their assigned levels for dex-
transucrase production by Weissella confusa Cab3.
Factor Level 1 Level 2 Level 3Level 4
Sucrose (%, w/v) 2 3 4 5
Yeast extract (%, w/v)0.1 0.5 1.0 2.0
K2HPO4 (%, w/v) 0.1 0.5 1.0 2.0
Tween80 (%, v/v) 0.01 0.1 0.5 1.0
Table 2. Fractional factorial design of L-16 orthogonal array used for dextransucrase production optimization by Weissella
confusa Cab3.
S. No. Sucrose YEP K2HPO4 T80 U/ml FITS (U/ml)
1 2.0 0.1 0.1 0.01 1.60 1.19
2 2.0 0.5 0.5 0.1 1.67 0.85
3 2.0 1.0 1.0 0.5 2.87 3.37
4 2.0 2.0 2.0 1.0 4.00 4.73
5 3.0 0.1 0.5 0.5 5.80 6.53
6 3.0 0.5 0.1 1.0 3.99 4.49
7 3.0 1.0 2.0 0.01 4.60 3.78
8 3.0 2.0 1.0 0.1 6.68 6.26
9 4.0 0.1 1.0 1.0 12.00 11.18
10 4.0 0.5 2.0 0.5 10.42 10.01
11 4.0 1.0 0.1 0.1 6.40 7.13
12 4.0 2.0 0.5 0.01 11.29 11.79
13 5.0 0.1 2.0 0.1 11.85 12.35
14 5.0 0.5 1.0 0.01 12.87 13.61
15 5.0 1.0 0.5 1.0 14.96 14.55
16 5.0 2.0 0.1 0.5 17.00 16.18
were 25˚C, 7.0 and 180 rpm, respectively for dextransu-
crase production from Weissella confusa Cab3 as re-
ported earlier [19]. Weissella confusa Cab3 grew well
within the range of pH 5.0 to 7.0, and temperature 20˚C
to 45˚C however Weissella confusa Cab3 produced
maximum dextransucrase (6.0 U/ml) at pH 7.0 and 25˚C
in the enzyme production medium [19]. As reported ear-
lier, the effects of several medium components were in-
vestigated and sucrose, Tween80, yeast extract and
K2HPO4 were effective nutrients which displayed higher
dextransucrase production [20]. Based on the earlier re-
sults, L-16 Taguchi’s orthogonal array method was de-
signed, where the factors were varied in four levels for
determining optimal medium components for dextransu-
crase production from Weissella confusa Cab3. Level 1
for each factor was fixed at negative side, considering the
factors’ role in dextransucrase production whereas, level
2 and 3 were considered as intermediate level for the
production of dextransucrase. Level 4 of each factor was
selected at relatively higher concentration range. Table 1
indicates the selected fermentation factors and their lev-
els for optimization of dextransucrase production by this
bacterial strain. The design matrix and dextransucrase
production data are represented in Table 2. A little varia-
tion was noted between software-predicted and experi-
mental values in dextransucrase production. The dex-
transucrase production by Weissella confusa Cab3 varied
widely from 1.6 - 17.0 U/ml. The variation in dextransu-
crase production is shown in Figure 1 and Table 2.
The difference between average value of each factor at
higher level and lower level indicated the relative influ-
ence of the effect at their individual capacities. The order
in which the individual components selected in the pre-
sent study affected the dextransucrase production can be
ranked as sucrose > yeast extract > Tween80 > K2HPO4,
suggesting that sucrose, yeast extract and Tween80 had
substantial effect and K2HPO4 had least effect on dex-
transucrase production by Weissella confusa (Table 3).
Highest delta value of sucrose (11.635) reflected it to be
most significant factor for the dextransucrase production.
As the concentration was increased to 5% (level 4),
maximum dextransucrase production occurred. Yeast
extract and Tween80 were the next important nutrients
for dextransucrase production, 2% (w/v) and 0.5% (v/v)
being optimum for dextransucrase production. The sig-
nificance of each factor on dextransucrase production can
be seen from the corresponding t and P values listed in
Table 4. The ANOVA table demonstrates that the su-
crose was the significant factor for the dextransucrase
production, as is evident from the Fisher’s F-test with a
very low probability value (p > F) = 0.005 (Table 4).
Table 5 represents the optimum conditions required for
the production of maximum dextransucrase production.
The experimental data revealed that selected level 4
Sucrose Yeast extract
Enzyme activity (U/ml)
Enzyme activity (U/ml)
Enzyme activity (U/ml)
Enzyme activity (U/ml)
Factor assigned levels Factor assigned levels
Factor assigned levels Factor assigned levels
2.0 2.5 3.0 3.5 4.0 4.5 5.00.5 1.0 1.5 2.0
0.5 1.0 1.5 2.00.2 0.4 0.6 0.8 1.0
Figure 1. Multiple graphs of main effects on dextransucrase production by Weissella confusa Cab3.
Copyright © 2012 SciRes. AiM
Table 3. Impact of fermentation factors and their assigned
levels on dextransucrase production by Weissella confusa
Factor Level 1 Level 2 Level 3 Level 4 Delta Rank
Sucrose 2.536 5.267 10.028 14.172 11.6351
Yeast extract 7.814 7.239 7.208 9.742 2.5342
K2HPO4 7.248 8.431 8.605 7.718 1.3584
Tween80 7.592 6.649 9.024 8.738 2.3753
Table 4. Analysis of variance of experimental data on dex-
transucrase production by Weissella confusa Cab3.
Factors DF SS MS F- value Prob. (P) > F
Sucrose 3 318.095 106.032 48.88 0.005
Yeast extract 3 17.107 5.702 2.63 0.224
K2HPO4 3 4.790 1.597 0.74 0.596
Tween80 3 14.338 4.779 2.20 0.267
Residual error 3 6.507 2.169
Others 15 360.837
Table 5. Optimum conditions and levels of factors.
Factor Optimum Concentration Level
Sucrose (% w/v) 5 4
Yeast extract (% w/v) 2.0 4
K2HPO4 (% w/v) 1.0 3
Tween80 (% v/v) 0.5 3
value of sucrose and yeast extract of the medium were
observed to be optimum for dextransucrase production,
whereas for K2HPO4 and Tween80, selected level 3 val-
ues, were observed to be good for optimal enzyme pro-
duction. The model was highly significant considering to
its 99.1% R2 value.
Taguchi’s orthogonal methodology predicted the
maximum dextransucrase production of 17.54 U/ml, in a
medium containing (g/L) sucrose, 50.0; K2HPO4, 10;
yeast extract, 2.0; Tween80, 5 (ml/L); MgSO4·7H2O, 0.2;
MnSO4·4H2O, 0.01; FeSO4·7H2O, 0.01; CaCl2·2H2O,
0.01 and NaCl 0.01. The dextransucrase production using
statistically optimized medium was validated at shake
flask level and scaled up in a 3l lab scale bioreactor using
1l of statistically designed medium. The fermentation
profile of dextransucrase production from Weissella
confusa Cab3 at shake flask and bioreactor level is
shown in Figures 2(a) and (b), respectively. Table 6
shows comparison of maximum attained fermentation
parameters using unoptimised medium and optimized
medium from Weissella confusa Cab3. For the bioreactor
the online data such as dissolved oxygen, pH, tempera-
ture and agitation were monitored and the offline data
like enzyme activity, sucrose concentration and cell op-
tical density were plotted with time (Figure 2(b)). The
enzyme activity and the cell optical density reached
maximum at 10 - 12 h of fermentation at both shake
flask and bioreactor level. The experimentally calculated
maximum dextransucrase activity at shake flask level
was 17.9 U/ml which was in agreement with the pre-
dicted values. The increase in dextransucrase activity of
the Weissella confusa Cab3 after medium optimization
(17.9 U/ml) was about 3.0 fold higher as compared to
unoptimized medium (6.0 U/ml). The dextransucrase
activity at bioreactor level after 10 - 12 h was 22.0 U/ml
(3.5 U/mg) which was more than that observed at shake
flask level using the optimized medium (Table 6). Oxy-
gen is known to have positive effects on the growth of
certain strains of L. mesenteroides [27]. Thus the higher
production of dextransucrase in bioreactor as compared
to flask culture is possibly be due to the effect of oxygen
mass transfer rates on biosynthesis of dextransucrase. In
both the cases (shake flask and bioreactor level) sucrose
concentration profiles showed maximum consumption of
sucrose during first 10 - 15 h, with the maximum produc-
tion of dextransucrase using the optimized medium. The
dextransucrase production (22.0 U/ml, 3.5 U/mg) using
statistically optimized medium by Weissella confusa
Cab3 at lab scale bioreactor level is higher than that ob-
served with other lactic acid strains in their respective
optimized medium. There is no literature available about
the optimization of medium composition for dextransu-
crase production from Weissella confusa. Leuconostoc
mesenteroides NRRL B-640 produced 10.7 U/ml dex-
transucrase activity in the optimised medium [17]. How-
ever according to a recent study by Patel et al., 2011 [18]
a mutant Pediococcus pentosaceus (SPAm) showed sub-
stantially higher dextransucrase activity (15.6 U/ml). A
Leuconostoc mesenteroides strain isolated from idli bat-
ter, an Indian fermented food was used for the optimisa-
tion for enhanced dextransucrase yield using response
surface methodology [28]. After optimization 489.12
DSU/ml (23.8 U/ml) activity was reported which was 5.5
fold higher than that in basic medium, however, they
used very high concentration of sucrose (13.75%, w/v).
In contrast to their findings, in present study lesser con-
centration of sucrose (5.0%, w/v) was used which re-
sulted in substantial high dextransucrase production.
4. Conclusion
Dextransucrase activity of Weissella confusa Cab3 was
Copyright © 2012 SciRes. AiM
Figure 2. Fermentation profile of Weissella confusa Cab3 using statistically designed medium for dextr ansucrase production.
The cell grow th, dry cell weight, de xtransucr ase production and suc rose conc entration changes ar e show n at: (A) Shake flask;
(B) Lab scale bioreactor level.
Table 6. Comparison of fermentation parameters for dextransucrase production using unoptimized and optimised medium
from Weissella confusa Cab3.
Tsuchiya medium (18)
Levels Enzyme activity (U/ml) Specific activity (U/mg) Cell OD600 Dry cell wt (mg/ml)
Shake Flask (100 ml) 6.0 1.0 5.4 4.1
Optimized medium for dextransucrase production
Shake Flask (100 ml) 17.9 3.0 7.6 7.1
Bioreactor (1000 ml) 22.0 3.5 8.4 9.9
6.0 U/ml with unoptimized medium. Using statistical
methods the medium composition for Weissella confusa
Cab3 was optimized. The optimization by Taguchi’s Or-
thogonal array method gave optimized medium consist-
ing of 5.0% sucrose; 2.0% yeast extract; 1.0% K2HPO4
and 0.5% Tween80 resulting in enhanced dextransucrase
production. The predicted value of dextransucrase (17.54
U/ml) was in good agreement with the experimental val-
ues from shake flask culture (17.9 U/ml). After scaling
up the dextransucrase production in a lab scale bioreactor
22.0 U/ml enzyme activity was obtained. The optimized
medium gave 3.0 and 3.7 fold higher dextransucrase
production at shake flask and bioreactor level, respec-
tively from Weissella confusa Cab3 as compared to un-
optimized medium. This research article is first effort to
understand the effects of various medium components on
Weissella confusa Cab3, which is very potent microor-
ganism for the production of dextransucrase at industrial
5. Acknowledgements
The research work was financially supported by a project
grant from an Indo-Finland joint project, Department of
Biotechnology, Ministry of Science and Technology,
New Delhi, India to AG.
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