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Science and Technology
Optimization of enzymatic liquefaction of papaya (Carica papaya L.) and jackfruit
(Artocarpus heterophyllus Lam.) pulp using response surface methodology
Attar Singh Chauhan1*, Sadia Gul Afroze1, Mysore Nagaraj Rao Ramesh2, Ramesh Yadav Avula1, Mysore
Narayan Rekha1 and Ramesh Shyam Ramteke1
1Department of Fruit And Vegetable Technology, Central Food Technological Research Institute, Mysore570 013, Karnataka,
India, 2Department of Food Engineering, Central Food Technological Research Institute, Mysore570 013, Karnataka, India.
*email: attar2k@hotmail.com
Received 12 January 2004, accepted 21 April 2004.
Abstract
Enzymatic liquefaction of jackfruit and papaya pulp, using a commercial enzyme (pectinase) was investigated and evaluated by response surface
methodology. The effect of enzyme concentration, incubation time and hydrolysis temperature were found to be significant in case of jackfruit
whereas juice yield in case of papaya was influenced by enzyme concentration and incubation time. In the present study, polynomial equations were
derived using multivariate analysis, for predicting the reduction in alcohol insoluble solids and increase in juice yield for jackfruit and papaya
respectively. The models were verified experimentally against predicted response and were found to be suitable.
Key words: Enzymatic liquefaction, optimization, jackfruit, Artocarpus heterophyllus Lam, papaya, Carica papaya, response surface methodology.
Introduction
Use of enzymes as processing aids has been practiced in the fruit
concentration (X ) were found to be the two independent variables
2
and vegetable processing industry to obtain low viscosity pulp,
for papaya, and therefore a central composite rotatable design
high yield of juices and solids, clarity and filterability of juices13.
(CCRD) to fit a second order polynomial by a least square
Recently, a mixture of cellulolytic and pectinolytic enzyme
regression technique was adopted. Preliminary studies on jackfruit
preparations has been increasingly employed in fruit and
revealed that the interaction of enzyme concentration (X ),
1
vegetable liquefaction due to their synergistic effects 46. Few
incubation time (X ) and temperature (X ) had significant effect.
2
3
research papers on enzymatic liquefaction of banana79 and
Therefore, a shell design was adopted for its optimization. Shell
mango10, 11 have been published. World demand for fruit juices,
design allows the selection of different levels (3, 5 and 7) of
including exotic or tropical juices is increasing. Value added
individual variables. The factor that has higher influences is studied
products from underutilized fruits like papaya (Carica papaya L.)
at more number of levels and that with least effect at lower levels.
and jackfruit (Artocarpus heterophyllus Lam.) can be commercially
An equation is used to describe the influence of variables on the
exploited due to their high nutritive value. The fruit juices of papaya
response to determine the interrelationship among the variables
and jackfruit can also compete either as individual juice or as
and the response.
blends.
A new product has been developed in this work, namely jackfruit
Although there are reports on enzymatic liquefaction of jackfruit
and papaya juice concentrates. The enzymatic liquefaction of
and papaya4, little information is available on their optimization
jackfruit and papaya pulp is exploited for the reduction in the
with respect to operating parameters. In order to obtain juice from
viscosity and AIS, in order to increase flux during microfiltration.
jackfruit and papaya pulp, pectinase enzyme is added which
The juice thus obtained can be concentrated by reverse osmosis
reduces the viscosity of the pulp and facilitates separation of the
without any phase change, retaining the important constituents
juice from the pulp. The enzymatic liquefaction is reported to be
intact, and later the juice is concentrated to obtain jackfruit and
influenced by the enzyme concentration, incubation time and
papaya juice concentrate. The enzymatic liquefaction step is the
hydrolysis temperature12. The effect of enzyme concentration and
most critical one, which will influence the yield and clarity of the
incubation time and temperature has been investigated in the
juice and the presence of AIS.
present study. A graphical optimization technique was used to
The objective of the present work is to 1) study the effect of
minimize alcohol insoluble solids (AIS) and to increase juice yield
enzyme concentration, incubation time and hydrolysis temperature
of jackfruit and papaya respectively.
on i) reduction in AIS during liquefaction of jackfruit ii) increase in
When many factors and interactions influence the desired
juice yield in case of papaya and 2) optimize the enzymatic
response, response surface methodology (RSM) is an effective
liquefaction of papaya and jackfruit pulp.
tool for optimizing the process. RSM is a statistical method that
uses quantitative data from appropriate experimental designs to
Materials and methods
determine and simultaneously evaluate multivariate equations.
Materials: Jackfruit and papaya pulp were prepared using a pulper
Based on the preliminary studies, incubation time (X ) and enzyme
1
(Bsen and Barry, India) having a sieve size of 0.80 mm. The pulp
108
Food, Agriculture & Environment, Vol.2 (2), April 2004
was pasteurized at the temperature of 80±1°C for 20 minutes,
Y
= 45.1828+0.2212x +36.5733x 0.0003x 20.125x1x 6.5733x 2
YIELD
1
2
1
2
2
cooled to room temperature and stored at –18°C. The pulp was
(2)
thawed and used for the experiments. The chemical and physical
properties of the jackfruit and papaya pulp are given in Table 1.
where Y
predicted responses for AIS (%), x is enzyme
AIS
1
Pectinase enzyme was procured from Biocon, India. The
concentration, x is time of incubation and x is temperature of
2
3
polygalacturonase activity of the enzyme was estimated by the
incubation for jackfruit. In case of papaya, Y
is the predicted
YIELD
method of Collmer et al.13, and was found to be 1590 U ml1.
response for yield (%), x is the time of incubation and x is the
1
2
enzyme concentration.
Experimental procedure
Optimization of the fitted polynomials was done graphically.
Papaya liquefaction: Papaya pulp was homogenized and 250 g
The optimum condition was verified by conducting experiments
was taken for each experiment. The samples were kept in a
at that condition. Responses were monitored and results were
temperature controlled water bath (29±1°C) with intermittent
compared with the model predictions.
stirring and subjected to treatments as given in Table 2. Later the
The fitted polynomial equation was expressed as surface and
samples were kept in boiling water bath for 10 minutes to inactivate
contour plots using the Microsoft EXCEL program (Microsoft
the enzyme.
Inc. 2000) in order to visualize the relation between the response
and experimental levels of each factor and to deduce the optimum
Jackfruit liquefaction: Jackfruit pulp was homogenized and 250
conditions.
g was taken for each experiment. The samples were kept in a
temperature controlled water bath (29±1°C, 37±1°C, 45±1°C) with
Other statistical analysis: Three statistical parameters were chosen
intermittent stirring and subjected to the treatments (Table 3).
to evaluate the comparative results 17.
Later the samples were kept in boiling water bath for 10 minutes to
inactivate the enzyme.
a. Root mean square difference (RSMD); (Equation3)
(k ?k
exp
prd ) 2
Alcohol insoluble solids: Alcohol insoluble solids were determined
RSMD
=
by AOAC procedure14.
N
Experimental design:
b. Standard deviation of difference (S ); (Equation4)
Papaya: A central composite rotatable design with two variables
D
was used to study the response pattern and to determine the
2
(
?
2
exp
prd
k
k
?
?
exp
) [ (k k )]
optimum combination of the variables15. The variables optimized
prd
N
SD=
were enzyme concentration (0.10.85%) and time of reaction (20
1

N
180 min) each at five levels (Table 2).
c. The average percent error; (Equation5)
The CCRD was arranged to allow for fitting an appropriate
regression model using a multiple regression program (Table 2).
k
k
100
exp ?
E =
prd
The CCRD combines the vertices of the hypercubes whose co
N
k
exp
ordinates are given by 2n factorial design to provide for the
estimation of the curvature of the model. Five replicates (treatments
where k
and k are experimental and predicted values
exp
prd
913) at the center of the design were used to allow for the
respectively, and N denotes the number of data points.
estimation of the pure error sum of squares. Experiments were
randomized in order to maximize the effects of unexplained
variability in the observed responses owing to extraneous factors.
Results and Discussion
Softening of fruit flesh occurred as indicated by the rapid decrease
Jackfruit: A shell design as described by Doehlert16 was planned
in the amount of AIS and increase in juice yield, in both fruits
for the optimization of jackfruit liquefaction process with three
investigated. The action of the enzyme leads to degradation and
variables to study the response pattern and to determine the
solubilisation of otherwise insoluble materials (pectin,
optimum combination of the variables (Table 2). The variables
hemicelluloses and some cellulosic materials) from the fruit pulp
optimized were enzyme concentration (0.10.85%), time of reaction
cell wall, resulting in increased juice yield 18. The fruit processing
(20180 min) and temperature of incubation (2945°C). A total of 17
industry takes advantage not only of the ability of enzyme
experiments were carried out, out of which the ninth experimental
preparations to facilitate juice release, but also of the increase in
domain was repeated five times in order to determine the residual
the juice yield.
variance value for the polynomial equations derived (Table 3).
Diagnostic checking of the models: Response was measured
Statistical analysis: A second order polynomial equation was
when the enzyme treated pulp was centrifuged at 10,000 rpm that
used to fit the experimental data (Table 2 and 3). The model
is % of AIS (Y
) in case of jackfruit and % of juice yield (Y
) in
AIS
Yield
proposed for the responses Y
and Y
for jackfruit and papaya
case of papaya. The response under different combinations as
AIS
YIELD
respectively are:
defined in the design (Tables 2 and 3) was analyzed using the
ANOVA appropriate to the experimental design. The ANOVA for
Y = 37.7326313.28017x 0.09465x 1.406192x +2.617284x 2
AIS
1
2
3
1
the data obtained for jackfruit and papaya are presented in Tables
+0.011944x x +0.20810x x +0.0000563x 2+0.002003x x +0.01553
1 2
1 3
2
2 3
4 and 5. It is evident from the data presented that the second order
(1)
terms were found to be significant and the lack of fit was not
significant. The lack of fit measures the failure of the model to
Food, Agriculture & Environment, Vol.2 (2), April 2004
109
Table 1. Physicochemical properties of jackfruit and papaya.
Properties
Fruit
*
Jackfruit
Papaya
TSS
°Brix
28.86
± 0.03
10.21 ± 0.04
Acidity (as % anhydrous citric acid)
0.23 ± 0.02
0.12 ± 0.01
pH
5.20
± 0.03
4.88 ± 0.01
Total carotenoids (mg/100g)
0.31 ± 0.01
1.89 ± 0.01
Total sugars (%)
26.0 ± 0.2
9.2 ± 0.1
* Values are the mean of triplicate determinations (n ± SE).
Table 2. Treatment schedule for 2factor CCRD for papaya and the response in terms of yield.
Run Coded parameter
Actual parameter
X1 X2
x1
x2
(YYield)
1
1 1
180
0.3
77
2
1
1
180
0.7
81
3
1
1
240
0.3
81
4
1
1
240
0.7
82
5
0 1.414
210
0.1
81
6
0 1.414
210
0.9
83
7
1.414 0
150
0.5
79
8
1.414 0
270
0.5
83
9 0 0
210
0.5
83
10 0
0
210
0.5
83
11
0
0
210
0.5
83
12
0
0
210
0.5
83
13 0
0
210
0.5
83
X1 and X2, represent the coded variables for incubation time and pectinase concentration; x1, x2, represent the actual variables for incubation time (h), and pectinase concentration
(g/100g pulp), YYield represents response in terms of % juice yield.
Table 3. Treatment schedule for 3factor shell design for jackfruit and the response in terms of alcohol
insoluble solids.
Run Coded parameter
Actual parameter
X1
X2 X3
x1
x2
x3
(YAIS)
1
0
0 0
0.55 100 37
4.45
2
0
1
0
0.55
180
37
3.9
3
0.866 0.5 0
1.0
140
37
5.62
4
0.289 0.5
0.816
0.7
140
45
3.97
5
0
1
0
0.55
20
37
5.47
6 0.866 0.5
0
0.1
60
37
6.29
7 0.289 0.5
0.816
0.4
60
29
4.78
8
0.866
0.5 0
0.1
140
37
3.77
9
0.289
0.5
0.816
0.4
140
29
3.62
10
0.577
0
0.816
0.85
100
29
3.73
11
0.866
0.5
0
1.0
60
37
5.29
12
0.289
0.5
0.816
0.7
60
45
5.54
13 0.577 0
0.816
0.25
100
45
3.79
14
0
0
0
0.55
100
37
4.26
15
0
0
0
0.55
100
37
3.72
16
0
0
0
0.55
100
37
3.73
17
0
0
0
0.55
100
37
3.75
X1, X2, X3 represent the coded variables for pectinase concentration, incubation time and temperature; x1, x2, x3 represent the actual variables for pectinase concentration (g/100g
pulp), incubation time (h), and temperature (°C); Y1 represents response in terms of % AIS.
represent data in the experimental domain at points, which are not
concentration, incubation time) on the response (AIS) is reported
included in the regression. The high values of the coefficient of
(Table 3) by the coefficients of the secondorder polynomials.
determination (R2) (0.94 in case of jackfruit and 0.93 for papaya)
The figure indicates the responses at room temperature. The trend
also suggest that the model is a good fit. The R2 is the proportion
was similar for other two temperatures also. The response surfaces
of variability in the response values explained or accounted for
based on these coefficients are shown in Fig.2.
by the model19.
Effect of enzyme concentration and incubation time on % juice
Response surface plotting: The effect of the conditions of the
yield of papaya: The juice yield of papaya was found to be a
enzymatic liquefaction of papaya (enzyme concentration and
function of the linear and quadratic effects of enzyme
incubation time) on the response (% yield) is reported (Table 2)
concentration. The linear effect (P?0.01) is positive, whereas the
by the coefficients of the secondorder polynomials. The response
quadratic effect (P?0.01) is negative, which results in a curvilinear
surfaces based on these coefficients are shown in Fig.1. The effect
increase in yield for all the incubation times. The yield is linearly
of the conditions of the enzymatic liquefaction of jackfruit (enzyme
related (P ? 0.05) to the incubation time, and the quadratic term is
110
Food, Agriculture & Environment, Vol.2 (2), April 2004
Jack fruit
6.50
6.00
5.50
5.00
%AIS 4.50
4.00
0.7
%Enzyme
3.50
0.1
concentratio
10
30
50
70
90
110 130 150 170
n
Time (min)
Figure 2. Response surface graph showing the effect of enzyme concentration and incubation time on % AIS of
jackfruit at 29±1°C.
Papaya
84
82
)
80
78
(
%
0.9
76
74
Yeild
72
0.5
70
%Enzyme
68
0.1
concentration
150
170
190
210
230
250
270
Time (mins)
Figure 1. Response surface graph showing the effect of enzyme concentration and incubation time on % juice yield of
papaya.
Optimization: The graphical optimization technique was adopted
not found to be significant, which results in a linear increase in
to determine the workable optimum conditions for the enzymatic
yield with the incubation time for all the enzyme concentrations
liquefaction of jackfruit and papaya pulp. This technique reduces
(P?0.01). It is interesting to note that the interaction is not
the amount of time and effort required for the investigation of
significant (P ? 0.01). The yield increases with enzyme
multifactor, multiresponse systems. It also provides
concentration and incubation time up to a certain level and then
comprehensive and informative insight into the system, leading
remains constant (Fig.1). Based on optimized conditions, the
to the process optimization. The specifications necessary for the
process for the extraction of papaya juice was scaled up at pilot
response was set and these also served as constraints on
plant level 20.
optimization. An acceptable compromise was made based on the
Effect of enzyme concentration and incubation time on the %
following criteria: in case of jackfruit: Y
? 3.91%, whereas in
AIS
reduction of AIS of jackfruit: The AIS is the measure of the
case of papaya Y
? 75%. These constraints serve as a basis
YIELD
remaining crude pectin present in the jackfruit pulp. It depends on
for further economically feasible option for commercialization of
the enzyme concentration as well as the incubation time. The
the process.
linear effects are negative and quadratic terms are positive for
Verification of results: The suitability of the model equation for
both the variables of enzyme concentration and incubation time;
predicting the optimum response values was tested using the
the interaction term is not significant. The AIS decreases with
recommended optimum conditions. The set of conditions were
enzyme concentration and incubation time up to a certain level
used to predict the value of the responses using model equations.
and then remains constant (Fig. 2).
The predicted experimental values were found to be in excellent
Food, Agriculture & Environment, Vol.2 (2), April 2004
111
Table 4. Analysis of variance (ANOVA) for the fitted secondorder polynomial model
and lack of fit for AIS as per shell design.
Degree of Sum of Mean square FValue Significance F
freedom squares
Regression
9 10.60
1.18 5.84 0.015
Residual
7 1.41
0.20
Total
16 12.01
Table 5. Analysis of variance (ANOVA) for the fitted secondorder polynomial
model and lack of fit for % yield as per CCRD.
Degree of Sum of Mean Square FValue Significance F
freedom
squares
Regression
5 25.55 5.11
1.61
0.27
Residual
7 22.22 3.17
Total
12 47.77
Table 6. Actual versus predicted values for the increase in %yield of papaya juice.
Sl No. Time (min) Concentration (%)
Predicted value (%) Experimental value (%)*
1
230
0.65
83
82.5±0.5
2
160
0.5
80
79.3±0.58
3
160
0.3
77
77.5±0.87
4
150
0.2
75
74.3±0.58
5
170
0.85
82
81.8±0.29
*Values are the mean of triplicate determinations (n ± SE).
Table 7. Experimental versus predicted values for the %AIS in jackfruit pulp.
Sl No Time (min) Concentration (%) Predicted value (%) Experimental value (%)*
1
170
0.6
3.91
3.88±0.01
2
165
0.7
3.86
3.83±0.01
3
155
0.8
3.82
3.78±0.06
4
90
0.75
3.66
3.64±0.02
5
80
0.7
3.73
3.73±0.00
* Values are the mean of triplicate determinations (n ±.SE).
Table 8. Statistical analysis of the comparison
Conclusions
of experimental values*.
The effect of the enzyme concentration, incubation time and
incubation temperature seems to be significant in case of
Fruit
RSMD
SD
E (%)
liquefaction of jackfruit and papaya using response surface
Papaya
0.551
0.679
0.76
methodology. The different conditions (time, temperature and
Jackfruit
0.018 0.021
0.74
enzyme concentration) for enzyme treatment revealed that, all these
*Values are the mean of triplicate determinations.
variables markedly affect the parameter under study. However,
enzyme liquefaction does not have any significant effect on the
agreement with the experimental ones (Tables 6 and 7). The values
nutritive properties of the juice. To explore whether the predictive
of statistical analysis are given in Table 8, which defines the degree
model from RSM could be applied to scale up the process, the
of fit of the model.
study was carried out at pilot plant level. The model equation
Enzymatic liquefaction of jackfruit and papaya pulp was found
developed for yield and AIS could predict values close to the
to be significantly influenced by the enzyme concentration and
experimental ones. The response surface methodology provides
incubation time. The model developed for AIS could predict values
insight into the interaction and identifies the optimum combination
very close to the experimental ones. Based on the set constraints,
of variables. The methodology developed in this research can
the optimum condition for enzymatic liquefaction of jackfruit and
also be extended to other fruits as well.
papaya have been determined.
112
Food, Agriculture & Environment, Vol.2 (2), April 2004
Acknowledgements
19Myers, R. H. 1971. Response surface methodology. Allyn and Bacon,
Authors are thankful to the Department of Food Processing
Boston.
20
Industries, New Delhi, India for funding the project. Thanks are
Ramteke, R. S., Chauhan, A. S., Rekha, M. N., Avula, R. Y. and Singh,
also due to the support and encouragement forwarded by Dr. V.
N. I. 2002. A process for the preparation of clarified papaya
juicebased products. Indian patent application no. NF94/02.
Prakash, Director, CFTRI during the course of this study.
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