Indian Journal of Fibre & Textile Research

Vol. 37, June 2012, pp. 183-190

Prediction and process optimization of pectinolytic reaction on organic cotton

fabrics for bioscouring with alkaline pectinase
C Vigneswaran1, N Anbumani 2, M Ananthasubramanian 3 & R Rajendran4
1
Department of Fashion Technology, 2Department of Textile Technology, 3Department of Biotechnology, PSG College of Technology,
Coimbatore 641 004, India
4
Department of Microbiology, PSG College of Arts and Science, Coimbatore 641 014, India

Received 7 December 2010; revised received and accepted 27 May 2011

An improved enzymatic cotton scouring process has been developed on the basis of a fast enzyme reaction to efficiently
remove the pectin and wax compounds from the organic cotton fabrics. An attempt has been made to study the pectinolytic
activity of degrading rate of pectin on the organic cotton fabric using alkaline pectinase enzyme with various process
parameters such as enzyme concentration, temperature and reaction time. These process variables are selected based on the
box-behnken design of experiment and the output of experiment results for weight loss, wax content and pectin degradation
on the fabric. All the results are optimized with minimum error. The test results are also analyzed using design-expert
software 8.0 to predict the optimum process parameters for achieving the required pectin degrading rate and then compared
with actual trial results. The performance of alkaline pectinase enzyme during bioscouring process has also been assessed by
Ruthenium Red dye test and FTIR results confirm the degradation of pectin on the bioscouring of organic cotton fabrics.

Keywords: Cotton, Enzyme kinetics, Enzyme scouring, Fabric weight loss, Pectin, Pectinase, Wax removal

1 Introduction the natural component present in the outer layer of

The organic cotton fabric for its wet processing
needs the use of minimum safe chemicals to health1-3
and for this the alternative way is to go into enzyme
technology, because enzymes are substrate specific
bio-catalysts and they operate best at an ambient
pressure, mild temperature and often at a neutral pH
range4,5. Enzymes are gaining an increasingly
important role as a tool in various wet textile pre-
treatment and finishing processes6-12. Biocatalysts
have proven to be a flexible and reliable tool in wet
textile processing and found to be a promising
technology to fulfill the expected future requirements.
Enzymatic scouring has been investigated extensively
by various researchers for nearly one decade13-18.
Different enzymes like pectinases such as lyases (EC
4.2.2.2); polygalacturonase endo-acting type (EC
3.2.1.15) and polygalacturonase exo-acting type (EC
3.2.1.67), proteases (EC 3.4.21-25), cellulases such as
endo-glucanases (EC 3.3.1.4); cellobiohydrolases (EC
3.2.1.91), xylanases (EC 3.2.1.8), lipases (EC
3.1.1.3); and recently cutinases (EC 3.1.1.74) have
been examined to degrade and subsequently remove
___________
a
To whom all the correspondence should be addressed.
E-mail: vignes_waran@rediffmail.com
cotton fibres19,20. These studies incorporated staining
tests, scanning electron microscopy (SEM), weight
loss analysis, cotton wax residue and nitrogen
content analysis.
Pectins are acidic polysaccharides, which are found
in fruits, fibres and vegetables21. Pectin being a non-
cellulosic material in cotton fibres plays several
important roles. It contributes to the firmness and
structure of cotton fibre, both as a part of the primary
cell wall and as a component of the winding layer22.
Pectin acts as cementing material for the cellulosic
network in the primary wall23,24. Pectin has a
complex structure and comprises α-(1, 4)-linked
D-galacturonan backbone, occasionally interrupted by
α-(1, 2)-linked α-L-rhamnopyranose residue. In cotton
fibres, up to 60% of the galacturonic acid residues of
the backbone are methyl esterified25.
Scouring is related to hydrophilicity and can be
achieved by uncovering the pores present in the
fibres, by removing waxes and other non-cellulosic
materials in the primary wall. The technical feasibility
of enzymatic scouring has been recognized by many
researchers over the last decade. However, continuous
enzymatic scouring process has not yet been widely
implemented by textile industries, due to inability to
184 INDIAN J. FIBRE TEXT. RES., JUNE 2012
remove waxes from cotton fibre during enzymatic
scouring.
Much of the work in the area of cotton bioscouring
has been focused on investigating the utility of
various enzymes. Although several types of enzymes
including pectinases14,26-29, cellulases12,15,30-32,
12,17,33,34
proteases , cutinases , xylanases35, and
11
lipases35,36 have been studied, pectinases have proved
to be the most effective and suitable enzymes for
cotton bioscouring. The mechanism of pectinase
scouring reportedly assumes that the degradation and
elimination of pectins makes the loosened waxes
more easily removable with the help of mechanical
agitation. This allows the cotton to achieve superior
hydrophilicity without fibre deterioration14. A
rationale approach is adopted to design a new
efficient enzymatic scouring process. Several aspects
were considered such as the specificity of enzymes,
the complexity of the cotton fibre substrate and mass
transfer. Different commercial as well as specially
produced pectinases were tested for bioscouring
performance. Alkaline pectinases (PL and Bioprep
3000L) work better than acidic pectinases (PGs). The
pectin removal efficiency of specially produced PL
was comparable to commercial Bioprep 3000L. The
most important parameters, such as enzyme
concentration, pH, temperature, ionic strength and
chelators, for the bioscouring process have been
evaluated6,10. Hardin27 postulated that pectin acts as a
cementing material in the primary wall of cotton
fibres. After enzymatic destabilization of the pectin
structure, the different components present in the
primary wall layer can be removed easily in
subsequent rinsing steps. The aim of this research was
to study the potential of enzyme technology to design
an efficient and low-temperature scouring process for
100% organic cotton fabric using alkaline pectinases
treated at various concentrations, temperatures and
time. The enzyme treated organic fabrics were
analysed for the percentage of fabric weight loss,
pectin removal, and wax removal; and their results
were optimized using Design Expert 8.0 software to
achieve required level of bioscouring process.
2 Materials and Methods
2.1 Grey Organic Cotton Fabric
The grey organic cotton yarns of 2/40s Ne for warp
and 40s Ne for weft yarns were procured from
M/s. Arm Strong Mills (P) Limited, Tirupur, India.
These yarns were taken into weaving of plain organic
cotton fabric using powerloom. The 100% organic
cotton fabric was produced having the specifications
64 ends/inch, 60 picks/inch, 19.0 fabric cover factor
and 120.4 g/m2 average fabric mass.
2.2 Warp Yarn Sizing and Fabrication
The sizing of warp yarns of 100% organic
cotton was carried out on laboratory model yarn
sizing machine using polyvinyl alcohol starch size
(PVA) and the average size add-on of the warp
yarn was 12.21%. The sized warp yarns were then
taken into warp beam preparation for weaving. The
average size add-on on the organic cotton fabric was
measured with respect to warp and weft yarn mass; it
was found to be 8.27%. The aerial density of the
organic cotton grey fabric after weaving was found to
be 130.2 g/m2.
2.3 Enzymatic Desizing
The PVA sized 100% organic cotton sized fabrics
were treated with alpha-amylase using various
process variables such as enzyme concentration,
temperature and reaction time. The process variables
were chosen according to the Box-Behnken method of
statistical tool for process optimization37,38. In this
study, a systematic statistical approach has been
adopted to obtain optimum weight loss of the sized
fabric with different process conditions of alpha
amylase enzyme concentration, temperature and
treatment time for achieving required level (8.2%) of
size removal. The enzymatic desizing process was
carried out at pH 6-7 and then the fabrics were
thoroughly rinsed with hot water followed by cold
water and dried at 80°C to constant weight.
2.4 Enzymatic Scouring with Alkaline Pectinase
Alkaline pectinase and purified pectate lyase were
selected for the degradation of cotton pectin. Scouring
experiments were performed in 1 L beaker in which
three fabric samples of 10 × 10 cm were treated using
enzyme solution of different concentrations of 2-6%,
non-ionic wetting agent of 1-2%, treatment time of
30, 45 & 60min, and pH of 8.5-9.0. The fabric
samples thereafter were rinsed in 500 mL of water at
90°C for 15 min to inactivate the enzymes followed
by drying at 80°C to constant weight.
2.5 Design of Experiment
Experiments were conducted based on the Box-
Behnken second order design for three variables
 VIGNESWARAN et al.: PREDICTION & PROCESS OPTIMIZATION OF PECTINOLYTIC REACTION 185

Table 1 − Box-Behnken experimental design

Coded Variables
values Enzyme Temperature Time
concentration, % °C min
-1 2 50 30
0 4 55 45
+1 6 60 60

(Table 1). The response (Y) is given by a second

order polynomial, as shown below:
k k k k
2
Y = bo + ∑ bi X i + ∑ bii X i + ∑∑ bij X i X j
i =1 i =1 i ≥ j i =1
…(1)
where Y is the predicted response; bo, the offset term;
bi, the linear effect; bii, the squared effect; bij, the
interactive effect. The above equation was solved
using the design expert (State-Ease Statistics Made-
Easy, version 8.0.2, 2010) to estimate the response of
the independent variables.

2.6 Testing
FTIR Analysis
The organic cotton fabric with and without alkaline
pectinase treatment were analyzed using FTIR
Spectrophotometer (Model: 8400S, Make:
Schimadzu).
Pectin Determination
The pectin removal of organic cotton fabrics was
carried out as per the procedure reported earlier39. K/S
values were calculated using the following equation:
K/S = (1-R)2 / 2R …(2)
The lower the K/S values the lesser is the pectic and
proteinic substance present in cotton fibre40. Fig. 1 − Effect of enzyme concentration and temperature on
weight loss of bioscoured organic cotton fabric at various time
Fabric Water Absorbency intervals of (a) 45 min and (b) 60 min
Water absorbency of organic cotton fabric treated
with alkaline pectinase was evaluated according to %WL = (W1-W2) *100 / W1 .. …(3)
AATCC test method 79-2000.
where W1 and W2 are the weights of fabric before and
Wax Content after enzymatic treatment.
The wax content of the grey organic cotton fabric
and alkaline pectinase treated fabrics were measured 3 Results and Discussion
as per AATCC test method 97-2009 (revised) by 3.1 Effect of Enzymatic Process Variables
solvent extraction using Soxhlet apparatus. Enzymatic treatments were carried out based on the
experimental design given in Table 1. The effect of
Weight Loss enzyme concentration and temperature on weight loss
After the enzymatic treatments, the weight loss of organic cotton fabric at various time intervals of (i)
(WL) of the treated fabrics was calculated using the 45 min, and (ii) 60 min are shown as 3D surface plot
following formula: (Fig.1). The empirical model was fitted to the
186 INDIAN J. FIBRE TEXT. RES., JUNE 2012

response and lack of fit test was carried out. The temperature ranges in the scouring process. With
polynomial equation derived from the experimental respect to reaction time in the scouring process, it is
design is given below taking into account the found that significant differences occur at Factual
significant interaction effects as given in Table 2. >Fcritical (F2,14 values of 2770.93>66.15) at 95%
confidence level. This may be due to the enzyme
Final Equation in Terms of Coded Factors reaction time on the degradation of pectin to achieve
required weight loss of the cotton fabric.
Fabric weight loss = +2.42 +0.23 * X1+0.072 *
X2+0.74 * C-0.023* X1* B-0.050* 3.2 Predicted vs Actual
X1* X3-7.5E-003* X2* X3+0.075* The predicted and actual fabric weight losses of the
2 2
X1 +0.088* X2 -0.22* X3
2 scoured 100% organic cotton fabric with alkaline
pectinase are shown in Table 3 and Fig. 2. From the
Final Equation in Terms of Actual Factors test results, it is observed that the error % of the
predicted and actual value of the experimental design
Fabric weight loss = +6.82+0.163* X1-0.35700 * Table 3 − Design model and error of experiment
X2+0.149* X3-2.25E-003* X1*
X2-1.66E-003* X1* X3-1.0E-004* Run Enzyme Temp. Time Weight loss , % Error
2 conc., % (X2) (X3) Predicted Actual %
X2* +0.018* X1 +3.50E-003* (X1) °C min
2 2
X2 -9.77E-004* X3
1 4.0 60 30 1.64 1.61 1.83
2 4.0 55 45 2.42 2.48 -2.48
where X1 is the enzyme concentration (%); X2 , the 3 2.0 55 60 2.8 2.74 2.14
temperature (°C); and X3, the time (min). 4 2.0 50 45 2.3 2.25 2.17
The ‘Model F-value’ was 66.15 which implies that 5 2.0 60 45 2.45 2.38 2.86
6 6.0 55 60 3.2 3.15 1.56
the model is significant and there is only 0.01% 7 6.0 50 45 2.76 2.85 -3.26
chance that a ‘Model F-value’ of this large value 8 4.0 50 30 1.44 1.42 1.39
could occur due to noise. The predicted R2 value is 9 4.0 50 60 2.95 3.04 -3.05
found to be 0.94 and is in reasonable agreement with 10 4.0 55 45 2.42 2.43 -0.41
the adjusted R2 value of 1.0. Adequate precision 11 2.0 55 30 1.25 1.22 2.40
12 6.0 55 30 1.85 1.87 -1.08
which measures the signal to noise ratio is found to be 13 6.0 60 45 2.82 2.81 0.35
16.821, which is greater than 4, indicating that the 14 4.0 60 60 3.12 3.18 -1.92
model can be used to navigate the design space. From 15 4.0 55 45 2.42 2.43 -0.41
Table 2, it is observed that the enzyme concentration
affects the degradation of pectin in the scouring
process, showing significant differences at Factual
>Fcritical (F2,14 values of 267.48>66.15) at 95%
confidence level. With respect to the design of
experiments, there is no significant difference found
between the temperature levels of 50 °C and 60 °C at
Factual <Fcritical (F2,14 values of 26.87<66.15) at
95% confidence level. This may be due to better
stability and kinetics of enzyme at selected
Table 2 − Analysis of variance for response surface

Source F P (Prob > F)

Enzyme conc. (X1), % 267.48 0.0001

Temp. (X2), oC 26.87 0.0035
Time (X3), min 2770.93 0.0003
X1*X2 1.29 0.3069
X1*X3 6.39 0.0527 Fig. 2 − Relationship between actual and predicted weight loss of
X2 * X3 0.14 0.7201 PVA desized organic cotton fabric
 VIGNESWARAN et al.: PREDICTION & PROCESS OPTIMIZATION OF PECTINOLYTIC REACTION 187

is below 3% and not found to be statistically

significant at 5%.

3.3 Effect of Enzyme Concentration and Temperature

Figures 3 and 4 clearly indicate, as expected, a
progressive weight loss with increasing enzyme
concentration due to the hydrolysis of pectin. At
higher time duration, there is higher rate of pectin and
wax hydrolysis observed with the increase in enzyme
concentration. The organic cotton fabric shows higher
water absorbency rate at higher enzyme concentration
of 6%, treatment time of 60 min and temperature of
60oC with maximum weight loss of 3.2% and above.
Figure 4 represents the interaction of alkaline
pectinase enzyme concentration and temperature for
60 min reaction time which shows the higher rate of
pectin removal by the reaction of alkaline pectinase
enzyme at 60oC on the organic cotton fibre to break
the pectin components in the fibre structure. The
water absorbency of fabric is in the range of 12-14 s
when treated with 2-3% alkaline pectinase at 55°C for
30min. The better water absorbency is observed for
5-6% alkaline pectinase concentration at 55-60°C for
60min treatment time. The lower the water
absorbency time the better scouring can be done.

3.4 FTIR Spectroscopic Analysis

The FTIR spectra of the desized organic cotton
fabric and 2, 4 and 6% pectinase enzyme treated
cotton fabrics are analysed and it mainly highlights
the changes in the non-cellulosic impurities by
characterizing the carboxyl acids and esters that are

Fig. 3 − Effect of enzyme concentration and temperature at

various reaction time (a) 30 min (b) 45 min (c) 60 min Fig. 4 − Interaction of enzyme concentration and temperature
188 INDIAN J. FIBRE TEXT. RES., JUNE 2012

Table 4 − FTIR test results of organic cotton fabrics and their

transmittance values

Wavelength Transparency, % Difference

cm-1 Desized 6% alkaline %
fabric pectinase treated
fabric

559 45.8 48.2 5.24

617 45.2 47.2 4.42
667 45.8 47.8 4.37
898 53.4 57.4 7.49
1058 34.8 35.3 1.44
1112 35.3 37.4 5.95
1165 37.5 38.2 1.87 Fig. 5 – Wax content of alkaline pectinase treated organic cotton
1371 25.1 27.6 9.96 fabric at 60°C for 45 min reaction time
1431 44.8 46.2 3.13
1617 52.2 57.2 9.58
1736 54.3 62.3 14.73
2362 46.8 47.8 2.14
2917 36.3 37.8 4.13
3415 22.7 23.2 2.20

Table 5 – Regression equation of pectin degradation of organic

cotton fabrics

Enzyme Regression equation Correlation

conc. % coefficient
(R2)

2 Y1=2.465X12-0.515X1+33.69 1
4 Y2=2.691X22-2.315X2+46.20 1 Fig. 6 – Pectin degradation of alkaline pectinase treated organic
6 Y3=2.465X32-0.515X3+33.69 1 cotton fabric enzyme kinetics – interaction of pectin degradation
X ─ Reaction time and Y ─ Pectin removal (%).
present in pectin and waxes. The hydrolysis of the
pectin at 6% enzyme concentration for 45 min
reaction time indicates the maximum removal of
pectin and waxes, as indicated by the peak at 3315
cm-1, responsible for the –OH group stretching, the
CH stretching at 2917 cm-1, the asymmetrical
COO- stretching at 1617 cm-1, and the CH wagging at
1316 cm-1. The absorbance intensity of the
characteristics peaks at around 1736 cm-1 varies in the
following order: desized fabric > 2% pectinase > 4%
pectinase > 6% pectinase fabrics. The residual non-
cellulosic components were analyzed after enzyme
treatment using FTIR reports (Table 4).
3.5 Wax and Pectin Removal
The wax content of alkaline pectinase treated
cotton fabric is shown in Fig. 5. It is observed that the
wax present in the grey organic cotton fabric is 0.81%
and the subsequent alkaline pectinase treated fabric
with 2, 4 and 6% enzyme concentrations at 60°C
rate at 60°C
temperature and 45 min reaction time shows 35.80,
48.15 and 54.32% loss in wax content respectively. It
may be due to the fact that the alkaline pectinase
enzyme degrades the pectin component in the organic
cotton fibre which hydrolysis the wax component in
the fibre. Figure 6 shows the pectin degradation level
of organic cotton fabric treated with various process
conditions of time and temperature with 2-6%
alkaline enzyme concentration. It is observed that the
rate of pectin removal increases with increase in
enzyme concentration and at higher time and
temperature. The enzyme kinetics of alkaline
pectinase at various concentrations with time interval
of 30, 45 and 60 min show that the pectin removal
rate is 1.30 and 1.32 times higher in case of 2-4% and
4-6% pectinase concentration respectively at 60°C.
The higher pectin removal (82.41%) is observed at
60°C and 60 min treatment time and in addition the
efficient wax removal step improves the performance
of pectinase in terms of pectin removal and
hydrophilicity. The regression equation for evaluating
 VIGNESWARAN et al.: PREDICTION & PROCESS OPTIMIZATION OF PECTINOLYTIC REACTION 189

Table 6 − Output results of design-expert software

Solution Enzyme Temperature, °C Time, min Fabric weight Pectin Water

no. concentration, % loss, % degradation, % absorbency, s

1 5.65 60.00 45.00 2.80079 80.21 5.999

2 5.67 59.94 45.00 2.80114 80.42 5.999
3 5.70 59.84 45.00 2.80173 79.34 6
4 5.72 59.78 45.00 2.80215 78.40 6
5 5.68 60.00 44.82 2.79741 81.07 5.999
6 5.53 60.00 45.79 2.81662 79.38 5.999
7 5.41 60.00 45.00 2.76276 80.32 6.270
8 6.00 58.20 45.00 2.79149 78.92 6.140
9 5.42 59.76 47.04 2.84409 79.43 5.999

the pectin degradation rate on the organic cotton
fabrics is given in Table 5.
3.6 Process Optimization
The process variables such as alkaline pectinase
enzyme concentration, temperature and reaction time
have been optimized using box-behnken experimental
design and their output values are executed using
design-expert software 8.0. The software of design-
expert was executed to get various options/predicted
process parameters to achieve required pectin
degradation range of 75-82% and weight loss of
2.80%. The output result of the design-expert
software to achieve the desired weight loss in the
alkaline pectinase process for predicted process
variables of 9 solutions is shown in Table 6. The
software shows the best process conditions, such as
alpha-amylase enzyme concentration 5.65%,
temperature 60°C, and reaction time 45 min with
1.0% desirability. The best opted test results are the
loss of 78.40% pectin with 2.80% weight loss at an
error of 1.21%.
4 Conclusion
It is possible to employ alkaline pectinase to scour
the organic cotton fabric to achieve better water
absorbency rate of < 5s and 52.5% wax removal by
higher pectin degradation of 75-80% using 6%
enzyme concentration, 60°C temperature, and 60 min
reaction time with 3.2% fabric weight loss. The
higher concentration of alkaline pectinase enzymes
shows better activity and catalyzes the degradation of
pectin groups at 55-60 °C and 8.5-9.0 pH, taking
lesser time to achieve required pectin hydrolysis. This
study will be helpful to the organic cotton processors
for the ecofriendly and sustainable textile wet
processing using alkaline pectinase enzyme in
bioscouring processes and also will pave the way to
find better scouring with mixed enzymatic systems, to
predict required alkaline pectinase and to bring better
scouring performance of cotton fabrics.
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