Vol.4, No.5B, 65-72 (2013) Agricultural Sciences
doi:10.4236/as.2013.45B013
Optimisation of beef tenderisation treated with
bromelain using response surface methodology
(RSM)
S. Zainal, K. Z. Nadzirah*, A. Noriham, I. Normah
Department of Food Science and Technology, Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor,
Malaysia; *Corresponding Author:
Received 2013
ABSTRACT
The purpose of this study is to determine the op-
timum condition for the tenderization of beef by
bromelain using Response Surface Methodology
(RSM). Initially, bromelain powder was produced
from pineapple crown of variety N36. Production
of the bromelain powder involves several proc-
ess steps such as extraction, purification, desalt-
ing and freeze drying. The cube s ize beef of round
part was treated with bromelain at different pHs
of beef, immersion temperatures, bromelain so-
lution concentrations, and immersion times ac-
cording to the experimental design which was
recommended by RSM of MINITAB softw are ver-
sion 15. Beef tenderness was then measured by
Texture Analyser. The MINITAB software Ver-
sion 15 was used to optimise the tenderisation
of beef by bromelain. The determination coeffi-
cient R2 was 99.97% meaning that the experi-
mental data were acceptable. It was found that
beef could be optimize tenderised 89.907% at
the optimum condition at pH of beef of 5.4, im-
mersion temperature of 60℃, bromelain solution
concentration of 0.1682% and immersion time of
10 minutes. The verification value of beef ten-
derisation at the feasible optimum condition
which was determined by experiment was
89.571%. Since the difference between the veri-
fication and predicted values was less than 5%,
therefore, the optimum condition for the tender-
isation of beef predicted by MINITAB software
Version 15 could be accepted.
Keywords: Beef; Bromelain; Tenderisation;
Response Surface Methodology (RSM )
1. INTRODUCTION
Beef from Brahman breed is known for its toughness
[1]. Thus, it is unsatisfactory to the consumer, if the meat
is so tough that it is difficult to eat [2]. Therefore, numerous
attempts have been made to encounter this problem and
tenderising beef using bromelain becomes one of the
widely used methods to improve the beef tenderness.
This is because bromelain showed hydrolytic activity on
the connective tissue leading to the better tenderisation
of the tough meat [3].
Bromelain is a proteolytic enzyme found in pineapple
plant [4-5] where it is accumulated in the entire part with
different extents and properties depending on its source
[6]. Production of bromelain powder involves extraction,
purification, desalting and drying processes which can be
performed using varieties of procedure. In the present
study, production of the bromelain powder from pineapple
crown of variety N36 was carried out according to the
method by [7] with slight modification.
The action of bromelain in tenderising beef is affected
by pHs of beef [8-9] immersion temperatures, bromelain
solution concentrations and immersion times [10,11]. Re-
search on the optimisation of beef tenderisation treated
with bromelain has not been carried. Therefore, this study
was conducted to determine the optimum condition for
the tenderisation of beef by bromelain using Response
Surface Methodology (RSM).
RSM is a collection of statistical techniques for design-
ing experiments, building models, evaluating the effects
of factors on the response and searching for the optimum
conditions [12]. The principal advantage of using RSM is
the number of experimental runs required to evaluate
multiple factors and their interactions can be reduced
[13-16]. Consequently, less time-consuming used to op-
timise a process compared to other approaches [17].
2. MATERIALS AND METHODS
2.1. Materials
Pineapple crown of variety N36 from maturity index 2
was obtained from Peninsula Plantations Sdn Bhd at
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S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72
66
Simpang Renggam, Johor, Malaysia. Beef of round part
from three years old bull of Brahman breed was pur-
chased from a butcher at Kota Kemuning, Shah Alam,
Selangor, Malaysia. Analytical grade of methanol and
acetonitrile and food grade of acetic acid, sodium hy-
droxide, hydrochloric acid and sodium chloride were
purchased from Merck Sdn Bhd (Petaling Jaya, Selangor,
Malaysia). All other chemicals of analytical grade in-
cluding standard bromelain were purchased from Sigma
Technologies Sdn Bhd (Petaling Jaya, Selangor, Malay-
sia). Custom-made cation exchange resin and diafiltrator
were purchased from IT Tech Research (M) Sdn Bhd
(Subang Jaya, Malaysia) and Isetake Enterprise (Kajang,
Selangor, Malaysia), respectively.
2.2. Methods
2.2.1. Production of Bromelain Po wder
The pineapple crowns were extracted by crushing their
small pieces using fruit juice processor with ratio of pine-
apple crown to purified water 1:1. The extract was fil-
tered through a muslin cloth. Then, the pineapple crown
extract was centrifuged at 360 x g for 10 min at 4℃. The
clear supernatant was collected followed by purification
process by Preparative High Performance Liquid Chro-
matography (HPLC) using cation exchange resin column
of 21.2 mm internal diameter and 250 mm length. The
eluents used were acetate buffer (25 mm, pH 4.0) and
1M sodium chloride (NaCl) solution. Removal of salt in
the purified bromelain samples was carried out by con-
tinuous diafiltrator with hollow fiber membrane using
purified water for exchange. Finally, the desalted brome-
lain solution sample was converted to powder form using
freeze dryer (Christ alpha 1-4LD Plus model).
2.2.2. Optimisation of Beef Tenderisat ion Treated
with Bromelain Using RSM
2.2.2.1. Tenderisation of Beef Usi n g Bromelain
Beef of round part was cut into cube size of approxi-
mately 2 cm3. Then, the beef cube was treated with bro-
melain at different pHs of beef, immersion temperatures,
bromelain solution concentrations, and immersion times
according to the experimental design which was recom-
mended by RSM of MINITAB software Version 15 as
shown in Table 2. Non-immersed or untreated beef cube
was served as a control.
2.2.2.2. Texture measurement
Beef tenderness was measured by Texture Analyser
TAX-T2i (Stable Micro Systems, Ltd., England, UK)
using P2N needle probe with a load cell of 10 kg cross
head speed equipped with a 0.5 in diameter. The depth of
beef was 5 mm.
2.2.2.3. Optimisation using RSM of MINITAB
Software Version 15
The range of four selected factors namely pH of beef,
immersion temperature, bromelain solution concentration
and immersion time is as shown in Table 1.
The factors listed in Table 1 were then applied into
MINITAB software Version 15 whereby full factorial
Central Composite Design (CCD) is employed to obtain
the experimental design as shown in Table 2.
Table 1. Coded and uncoded factors for the design of experi-
ment.
-2.000 (-)-1 0 1 2.000 ()
X1 5.0 5.2 5.4 5.6 5.8
X2 55 60 65 70 75
X3 0.10 0.15 0.20 0.25 0.30
X4 5 10 15 20 25
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min).
Table 2. Experimental design recommended by MINITAB
software Version 15.
No X1 X
2 X 3 X 4
1 5.2 60 0.15 10
2 5.6 60 0.15 10
3 5.2 70 0.15 10
4 5.6 70 0.15 10
5 5.2 60 0.25 10
6 5.6 60 0.25 10
7 5.2 70 0.25 10
8 5.6 70 0.25 10
9 5.2 60 0.15 20
10 5.6 60 0.15 20
11 5.2 70 0.15 20
12 5.6 70 0.15 20
13 5.2 60 0.25 20
14 5.6 60 0.25 20
15 5.2 70 0.25 20
16 5.6 70 0.25 20
17 5.0 65 0.20 15
18 5.8 65 0.20 15
19 5.4 55 0.20 15
20 5.4 75 0.20 15
21 5.4 65 0.10 15
22 5.4 65 0.30 15
23 5.4 65 0.20 5
24 5.4 65 0.20 25
25 5.4 65 0.20 15
26 5.4 65 0.20 15
27 5.4 65 0.20 15
28 5.4 65 0.20 15
29 5.4 65 0.20 15
30 5.4 65 0.20 15
31 5.4 65 0.20 15
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min).
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S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72 67
Response surface regression analysis was performed
to obtain a second-order polynomial equation or model.
Statistical analysis of the model was represented in the
form of Analysis of Variance (ANOVA). The MINITAB
software Version 15 was also used to generate response
contour and surface plots.
3. RESULTS AND DISCUSSION
Results in Table 3 show that the highest actual and
predicted responses were 89.907% and 89.813%, respec-
tively at factors whereby pH of beef was 5.6, immersion
temperature at 60℃, bromelain solution concentration of
0.15% and immersion time of 10 minutes. The lowest
actual and predicted responses were 58.267% and
58.608%, respectively at factors whereby pH of beef was
5.4, immersion temperature at 65℃, bromelain solution
concentration of 0.20% and immersion time of 15 min-
utes.
Table 3. Factors and comparison between actual (Y) and pre-
dicted (FITS) responses.
Test variables Responses
No X1 X
2 X 3 X 4 Y (%) FITS (%)
1 5.2 60 0.15 10 81.530 81.663
2 5.6 60 0.15 10 89.907 89.813
3 5.2 70 0.15 10 86.442 86.429
4 5.6 70 0.15 10 87.551 87.543
5 5.2 60 0.25 10 85.521 85.314
6 5.6 60 0.25 10 86.931 86.971
7 5.2 70 0.25 10 71.343 71.352
8 5.6 70 0.25 10 66.053 65.974
9 5.2 60 0.15 20 85.458 85.263
10 5.6 60 0.15 20 89.147 89.439
11 5.2 70 0.15 20 85.807 86.068
12 5.6 70 0.15 20 83.273 83.208
13 5.2 60 0.25 20 86.146 86.455
14 5.6 60 0.25 20 84.399 84.139
15 5.2 70 0.25 20 68.713 68.534
16 5.6 70 0.25 20 59.013 59.181
17 5.0 65 0.20 15 64.929 64.810
18 5.8 65 0.20 15 64.205 64.209
19 5.4 55 0.20 15 73.532 73.513
20 5.4 75 0.20 15 63.511 63.417
21 5.4 65 0.10 15 62.901 62.589
22 5.4 65 0.30 15 52.202 52.401
23 5.4 65 0.20 5 67.019 67.237
24 5.4 65 0.20 25 65.972 65.640
25 5.4 65 0.20 15 58.663 58.608
26 5.4 65 0.20 15 58.574 58.608
27 5.4 65 0.20 15 58.309 58.608
28 5.4 65 0.20 15 58.267 58.608
29 5.4 65 0.20 15 58.881 58.608
30 5.4 65 0.20 15 58.894 58.608
31 5.4 65 0.20 15 58.328 58.608
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min).
Table 4. Estimated regression coefficients of second-order
polynomial model for optimisation of beef tenderisation treated
with bromelain.
Term CoefficientSE Coefficient t p
Constant5013.43 122.781 40.832 0.000
X1 -1433.30 47.267 -30.324 0.000
X2 -38.43 0.984 -39.051 0.000
X3 2206.78 50.616 43.599 0.000
X4 -1.13 0.473 -2.380 0.030
X1X1 147.55 4.353 33.895 0.000
X2X2 0.39 0.007 56.609 0.000
X3X3 -445.22 69.648 -6.392 0.000
X4X4 0.31 0.007 44.973 0.000
X1X2 -1.76 0.070 -25.083 0.000
X1X3 -162.31 7.012 -23.146 0.000
X1X4 -0.99 0.070 -14.168 0.000
X2X3 -18.73 0.280 -66.762 0.000
X2X4 -0.04 0.003 -14.119 0.000
X3X4 -2.46 0.280 -8.764 0.000
R² = 99.97 % R² (adj) = 99.95 %
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min), SE = standard
error, t = student test, p = probability, R2 = R – squared, R2 (adj) = adjusted
R – squared.
Response surface regression analysis was performed
and results of estimated regression coefficients of sec-
ond-order polynomial model for optimisation of beef
tenderisation treated with bromelain are as shown in Ta-
ble 4.
Based on Table 4, the second-order polynomial model
equation for optimisation of beef tenderisation treated
with bromelain is as given in equation 2:
Y = 5013.43 – 1433.30X1 – 38.43X2 + 2206.78X3
– 1.13X4 + 147.55X1
2 + 0.39X2 2 – 445.22X3 2
+ 0.31X4
2 – 1.76X1X2 – 162.31X1X3
– 0.99X1X4 – 18.73X2X3 – 0.04X2X4
– 2.46X3X4 (1)
Where: X1 = pH of beef, X2 = immersion temperature
(℃), X3 = bromelain solution concentration (w/v%), X4
= immersion time (min)
The significant second-order polynomial model equa-
tion at the 5% level for the optimisation of beef tender-
isation treated with bromelain is same as in equation (1).
By referring to Table 4, it was found that linear factors
such as pH of beef (X1), immersion temperature (X2) and
immersion time (X4) showed negative coefficients, re-
spectively while bromelain solution concentration (X3)
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S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72
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showed positive coefficient. Square factors such as pH of
beef (X1X1), immersion temperature (X2X2) and immer-
sion time (X4X4) showed positive coefficients, respec-
tively while bromelain solution concentration (X3X3)
showed negative coefficient. Quadratic or interaction
factors such as pH of beef and bromelain solution con-
centration (X1X3), pH of beef and immersion time (X1X4),
immersion temperature and bromelain solution concen-
tration (X2X3), immersion temperature and immersion
time (X2X4) and bromelain solution concentration and
immersion time (X3X4) showed negative coefficients,
respectively.
Student t and p tests were performed and it was found
that linear effect factors namely X1, X2 and X4 showed
negative t values, respectively while X3 showed positive
t value. p value for X1, X2, X3 and X4 were 0.000, re-
spectively. Square effect factors namely X1X1, X2X2 and
X4X4 showed positive t values, respectively while X3X3
showed negative t value. p value for X1X1, X2X2, X3X3
and X4X4 were 0.000, respectively. Quadratic or interac-
tion effect factors namely X1X3, X1X4, X2X3, X2X4 and
X3X4 showed negative t values, respectively and the re-
spective p values were 0.000. It means that all linear,
square and quadratic or interaction factors gave signifi-
cant (p  0.05) effect on beef tenderisation.
Student t test was used to determine the significance of
the estimated coefficient of the regression model equa-
tion (equation 1). The student t test value can be obtained
by dividing each coefficient by its SE [18]. p values were
used as a tool to evaluate the significance and contribu-
tion of each factor and the statistical polynomial model
equation [19]. [20,21] reported that the larger the magni-
tude of the t value and the smaller the p value, the more
significant is the corresponding coefficient. Based on those
reported by [20,21], results of the present study showed
that linear factors (X1, X2, X3, X4), square factors (X1
2,
X2
2, X3 2, X4 2) and quadratic or interaction factors (X1X2,
X1X3, X1X4, X2X3, X2X4, X3X4) terms were highly sig-
nificant.
The goodness of fit of the regression model was de-
termined by determination coefficient R2 which provides
a measure of how much variability in the observed re-
sponse values can be explained by the experimental fac-
tors and their interactions [22]. Results in Table 4 showed
that R2 value was 99.97% which signified 99.97% of
variability in the observed response values could be ex-
plained by the model while only 0.03% of variability in
the observed response values cannot be explained by the
model. The remaining R2 value of 0.03% of the total
variations would be due to other factors which were not
included in the model.
The adjusted R2 was a corrected value for R2 after the
elimination of unnecessary model terms. The adjusted R2
would be remarkably smaller than the R2 if there were
many non-significant terms have been included in the
model [23]. In this study, it was found that the adjusted
R2 was high and very close to the R2 and the respective
values of adjusted R2 and R2 were 99.95% and 99.97%.
The high adjusted R2 value was attributed to the absence
of non–significant terms in the model. The high adjusted
R2 and R2 values thus, indicated a high dependence and
correlation between the observed and predicted value
responses. This is based on [24], who also found the high
adjusted R2 (98.81) and R2 (97.96) values in their study
on the optimisation of enzymatic detection of cadmium
in aqueous solution using immobilized urease from
vegetable waste.
ANOVA was performed to test for the significance and
adequacy of the second-order polynomial model [25].
The results are as summarised in Table 5.
The significance of regression was evaluated by f and
p values using Fischer's and null-hypothesis tests. The f
value predicts the quality of the entire model considering
all design factors at a time. The p value is the probability
of the factors having very little or insignificant effect on
the response. Larger f value signifies better fit of the
RSM model to the experimental data [26]. [27] stated
that f value with low p value indicates the high signifi-
cance of the regression model. However, the p value
should be lower than 0.05 for the model to be statistically
significant [28]. Based on those reported by [26-28], the
regression model found in this study was highly signifi-
cant as denoted by the large f and low p values with
4185.84 and 0.000, respectively. The linear, square and
quadratic factors were also highly significant as denoted
by the large f values of 1073.06, 10142.46 and 1016.48,
respectively and low p values of 0.000, respectively.
Lack of fit test was also performed. It describes the
variation in the data around the fitted model [29]. [28]
testified that insignificant lack of fit indicates a good
model. Insignificant lack of fit is desired as significant
lack of fit indicates that there might be contributions in
the regresses-response relationship that are not accounted
for by the model. The f value for the lack of fit can be
Table 5. ANOVA for optimisation of beef tenderisation treated
with bromelain.
Source DFSeq SSAdj SS Adj MS f P
Regression 144610.77 4610.77 329.341 4185.840.000
Linear 4938.89337.71 84.428 1073.060.000
Square 43192.023192.02 798.005 10142.460.000
Interaction 6479.86479.86 79.976 1016.480.000
Residual Error161.26 1.26 0.079
Lack of fit 100.83 0.83 0.083 1.16 0.445
Pure Error 60.43 0.43 0.071
Total 304612.03
Where: DF = degree of freedom, Seq SS = sequential sum of square, Adj SS
= adjusted sum of square, Adj MS = adjusted mean square, f = fischer, p =
probability.

S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72 69
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/
obtained by dividing the lack of fit mean square by its
pure error mean square. Results of the lack of fit are
shown in Table 5 and it was found that the f and p values
for the lack of fit were 1.16 and 0.445, respectively. The
insignificant p value thus indicates that the model was
good and fitted well to the experimental data.
Response optimiser was performed and the results at
optimum condition for target, maximum and minimum
goals are shown in Figures 1, 2 and 3, respectively.
The feasibility of experiment for target, maximum and
minimum goals was determined from the overlaid con-
tour plot and the results are shown in Figures 4, 5 and 6,
respectively.
Results of optimum conditions for different goals of
actual and predicted responses and the feasibility of ex-
periments obtained from response optimiser of MINI-
TAB software Version 15 are shown in Table 6.
Based on Table 6, it was found that optimum conditions
of target goal with pH of beef of 5.6, immersion tem-
perature of 60℃, bromelain solution concentration of
0.1864% and immersion time of 10 minutes, and maximum
goal with pH of beef of 5.6, immersion temperature of 60
℃, bromelain solution concentration of 0.1682% and
Cur
Hi g h
Low
0.99867
D
Optimal
d = 0.99743
Targ: 89.9071
% Tender
y = 89.8102
d = 0.99992
Targ: 89.8133
FITS1
y = 89.8102
0.99867
Desirability
Composite
10.0
20.0
0.150
0.250
60.0
70.0
5.20
5.60
[5.60][60.0] [0.1864] [10.0]
Temp ConcTimepH
Figure 1. Response optimiser at optimum condition for target
goal.
Cur
High
Low
1.0000
D
Optimal
d = 1.0000
Maximum
% Tender
y = 89. 95 90
d = 1.0000
Maximum
FITS 1
y = 89. 95 90
1.0000
Desirability
Composite
10.0
20.0
0.150
0.250
60.0
70.0
5.20
5.60
[5.60][60.0][0.1682][10.0]
Temp ConcTimepH
Figure 2. Response optimiser at optimum condition for maxi-
mum goal.
Cur
Hi g h
Low
1.00 00
D
Opti m al
d = 1.0000
Mini mum
% Tender
y = 49 .5157
d = 1.0000
Mini mum
FITS1
y = 49 .5157
1.0000
Desirability
Composite
10.0
20.0
0.15 0
0.25 0
60. 0
70. 0
5.20
5.60
[5.4505] [67.5758][0.250][15.6566]
Temp ConcTimepH
Figure 3. Response optimiser at optimum condition for mini-
mum goal.
pH
Temp
5.585.525.465.405.345.285.22
70
68
66
64
62
60
Conc 0.1864
Time 10
Hold Values
52.2025
89.9072
% Tenderis ation
52.4008
89.8134
FITS1
O ve rla id C ont o ur P lo t (T a r g e t )
pH = 5.59861
Temp = 60.0099
% Tenderisation = 89.6596
FITS1 = 89.6596
Figure 4. Overlaid contour plot at optimum condition for target
goal; pH of beef of 5.6, immersion temperature of 60˚C, bro-
melain solution concentration of 0.1864% and immersion time
of 10 minutes.
pH
Temp
5.585. 525.465.405.345.285.22
70
68
66
64
62
60
Conc 0.1682
Time 10
Hold Values
52. 2025
89. 9072
% Tenderisation
52. 4008
89. 8134
FITS1
Ove rlaid C on to ur Plot ( Ma x imum)
pH = 5.59861
Temp = 60.0459
% Tenderisation = 89.6470
FITS1 = 89.6470
Figure 5. Overlaid contour plot at optimum condition for maxi-
mum goal; pH of beef of 5.6, immersion temperature of 60˚C,
bromelain solution concentration of 0.1682% and immersion
time of 10 minutes.

S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72
70
pH
Temp
5.585.525.465.405.345.285.22
70
68
66
64
62
60
Conc 0.25
Time 16
Hold Values
52.2025
89.9072
% Tenderisation
52.4008
89.8134
FITS1
Overlaid Con tour Plot ( Minimu m)
pH = 5.45055
Temp = 67.5562
% Tenderisat ion = 49.54 56
FITS1 = 49.5456
Figure 6. Overlaid contour plot at optimum condition for mini-
mum goal; pH of beef of 5.45, immersion temperature of 68℃,
bromelain solution concentration of 0.25% and immersion time
of 16 minutes.
Table 6. Comparison values of target and predicted responses
for different optimum conditions and feasibilities of experiment.
Goal Lower Target Upper
Target Y 52.2025 89.9071 89.9072
FITS 52.4008 89.8133 89.8134
Max Y 52.2025 89.9072 89.9072
FITS 52.4008 89.8134 89.8134
Min Y 52.2025 52.2025 89.9072
FITS 52.4008 52.4008 89.8134
Optimum Condition
Goal X1 X
2 X 3 X 4 FITS (%)F/NF
Target 5.6 60 0.186410 89.8102 F
Max 5.6 60 0.168210 89.9590 F
Min 5.5 68 0.250016 49.5157 NF
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min), Y = actual re-
sponse (%), FITS = predicted response (%), F = feasible, NF = not feasible.
immersion time of 10 minutes were feasible to be carried
out. Meanwhile, optimum condition for minimum goal
with pH of beef of 5.5, immersion temperature of 68℃,
bromelain solution concentration of 0.25% and immersion
time of 16 minutes was not feasible to be carried out.
This is because according to the overlaid contour plots
for target and maximum goals as shown in Figures 4 and
5, respectively, the optimum conditions of target and
maximum goals located in white or feasible region.
Meanwhile, the overlaid contour plot of minimum goal as
shown in Figure 6 located in grey or not-feasible region.
However, optimum condition from maximum goal was
chosen because the target and FITS values was closer.
Contour and surface plots for beef tenderisation treated
with bromelain at feasible optimum condition are shown
in Figures 7 and 8, respectively.
2D contour and 3D surface plots are the graphical
representatives of the regression equation illustrating the
function of two factors at a time while holding other
factors at a fixed level [27]. The 2D contour and 3D surface
plots as demonstrated in Figures 7 and 8, respectively
showed the effect of pH of beef, immersion temperature
(℃), bromelain solution concentration (%) and immersion
time (min) on beef tenderisation. The plots illustrating
the values for pH of beef and immersion temperature while
holding the values of bromelain solution concentration
and immersion time at 0.1682% and 10 minutes, respec-
tively.
pH
Temp
5.585.525.465.405.345.285.22
70
68
66
64
62
60
Conc 0.1682
Time 10
Hold Values
>
–
–
–
–
< 72
72 76
76 80
80 84
84 88
88
Tenderisation
%
Contour Plot
Figure 7. Contour plot of beef tenderisation treated with bromelain
at feasible optimum condition; pH 5.6, immersion temperature
of 60℃, bromelain solution concentration of 0.1682% and im-
mersion time of 10 minutes (holding value:bromelain solution
concentration and immersion time fixed at 0.1682% and 10
minutes, respectively).
70
70 65
80
5.2
90
5.4 60
5.6
% T e nd e risation
Temp
pH
Conc0.1682
Time 10
Hold Values
Surfac e Pl ot
Figure 8. Surface plot of beef tenderisation treated with bro-
melain at feasible optimum condition; pH 5.6, immersion tem-
perature of 60℃, bromelain solution concentration of 0.1682%
and immersion time of 10 minutes (holding value : bromelain
solution concentration and immersion time fixed at 0.1682%
and 10 minutes, respectively).
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/

S. Zainal et al. / Agricultural Sciences 4 (2013) 65-72 71
Table 7. Comparison of verification and predicted values of
beef tenderisation treated with bromelain at feasible optimum
condition.
Optimum condition
X1 X2 X3 X4
V
(%) P (%)
5.6 60 0.1682 10 89.571 89.813
Where: X1 = pH of beef, X2 = immersion temperature (℃), X3 = bromelain
solution concentration (w/v%), X4 = immersion time (min), V = verification
value, P = predicted value.
Circular or elliptical shapes of contour plot indicate
whether the reciprocal interactions between the factors
are significant or not. Circular contour plot indicates the
interactions between corresponding factors are negligible,
while elliptical contour plot indicates the interactions
between corresponding factors are significant [30][20].
Results of the present study showed that the contour plot
was elliptical shape thus indicates significant interaction
effect between pH of beef and immersion temperature on
beef tenderisation.
The surface plot showed that the beef tenderisation
increased at the lower and higher levels of pH of beef and
immersion temperatures while at the middle level of pH
of beef and immersion temperature, the beef tenderisation
decreased.
Verification of beef tenderisation treated with brome-
lain at the feasible optimum condition was performed
and the result is shown in Table 7.
The verification value of the beef tenderisation treated
with bromelain at the feasible optimum condition was
89.571% which was very close to the predicted value
with 89.813%. Since the difference between the verifica-
tion and predicted values was less than 5%, therefore the
feasible optimum condition of the beef tenderisation pre-
dicted by MINITAB Software Version 15 was accept-
able.
4. CONCLUSIONS
The determination coefficient R2 (99.97%) was high,
thus the experimental data was acceptable. Optimum con-
dition for the tenderisation of beef by bromelain using
RSM had been determined. It was found that beef could
be optimise tenderized 89.907% at the optimum condi-
tion pH of beef of 5, immersion temperature of 60℃,
bromelain solution concentration of 0.1682% and im-
mersion time of 10 minutes. It aws also found that the
difference between the verification and predicted values
was less than 5%, therefore, the optimum condition for
the beef tenderisation predicted by MINITAB Software
Version 15 could be accepted.
5. ACKNOWLEDGEMENTS
Special thanks to Ministry of Higher Education, Malaysia for fund-
ing this project under Fundamental Research Grant Scheme [600-
RMI/ST/FRGS/Fst(32/2010)] and also Peninsula Plantation Sdn Bhd,
Simpang Renggam, Johor, Malaysia for supplying pineapple for this
project.
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