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Sunantha Ketnawa
Phanuphong Chaiwut
Purdue University
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Mae Fah Luang University
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Article
Abstract
Large amount of pineapple peels (by-products) is left over after processing and they are a potential source for
bromelain extraction. Distilled water (DI), DI containing cysteine and ethylenediaminetetraacetic acid (EDTA)
(DI-CE), sodium phosphate buffer pH 7.0 (PB) and PB containing cysteine and EDTA (PB-CE) were used as
extractants for bromelain from the pineapple peels. The highest bromelain activity was obtained when it was
extracted with PB-CE (867 and 1032 units for Nang Lae and Phu Lae cultv, respectively). The PB could
maintain the pH of the extract (pH 5.15.7) when compared with others. Under sodium dodecyl sulfate
polyacrylamide gel electrophoresis, the extract showed protein bands in the range 2428 kDa. The protein
band with a molecular weight of 28 kDa exposed the clear zone on blue background under the caseinsubstrate gel electrophoresis. The effects of the bromelain extract on the protein patterns of beef, chicken and
squid muscles were also determined. Trichloroacetic acid soluble peptide content of all the treated muscles
increased when the amount of bromelain extract increased. Decrease in myosin heavy chains and actin was
observed in all the muscle types when bromelain extract was used. The best extractant for bromelain from
pineapple peels was PB-CE. Moreover, bromelain extract could be used as a muscle food tenderizing agent
in food industries.
Keywords
Extractant, bromelain, pineapple peel, TCA-soluble peptides, meat tenderization
Date received: 3 June 2010; revised: 17 August 2010
INTRODUCTION
Bromelain, the proteolytic enzyme, is found in the tissues of the plant family Bromeliaceae of which pineapple (Ananas comosus L. Merryl) is a well-known source.
It is also found in pineapple wastes such as cores, peels
and leaves in relatively smaller quantities as compared
to stems and fruits (Maurer, 2001; Sriwatanapongse
et al., 2000; Umesh et al., 2008). The stem bromelain
(EC 3.4.22.32) and fruit bromelain (EC 3.4.22.33)
obtained from the pineapple stem and pulp are commercially available. Pineapple wastes, especially peels
and cores, are left in large amounts (4050%) after
fresh-cut processing in Chiang Rai, Thailand
(24 tons per year; DOAE, 2009). Normally, these
Food Science and Technology International 17(4) 395402
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DOI: 10.1177/1082013210387817
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Corresponding author:
S. Rawdkuen, Food Technology Program, School of AgroIndustry, Mae Fah Luang University, Muang, Chiang Rai 57100,
Thailand
Email: saroat@mfu.ac.th
Ketnawa et al.
polyacrylamide gel (15% running and 4% stacking
gels) and subjected to electrophoresis at a constant
current of 15 mA/gel. After separation was achieved,
the protein was stained with 0.02% Coomassie
Brilliant Blue R-250 and destained with a mixture of
aceticmethanol solution.
Activity staining. The separated protein by electrophoresis was veried for bromelain by using activity
staining (casein-substrate gel electrophoresis) as
described by Garcia-Carreno et al. (1993). The gel
was immersed in 50 mL of 2% (w/v) casein 50 mM of
PB, pH 7.0 for 45 min with constant agitation at 4 C.
The gel was then incubated at 37 C for 30 min with
constant agitation. The development of a clear zone
on the blue background indicated bromelain activity.
Effect of crude bromelain
proteins degradation
extract
on
muscle
Table 1. Characteristics of crude bromelain extracts from 100g of pineapple Nang Lae (NL) and Phu Lae (PL) peels using
different extractants
143.67 1.00
148.67 0.50
143.67 2.78
146.00 0.87
Protein
content
(mg/mL)
pH
Total
protein (mg)
Activity
(unit/mL)
Specific
activity
(unit/mg
protein)
Total activity
(unit)
a
c
a
b
3.64 0.18
3.58 0.12
5.15 0.01
5.14 0.02
b
a
c
d
0.337 0.02
0.335 0.01
0.702 0.01
0.564 0.02
a
a
c
b
48.48 2.85
49.81 2.19
100.79 1.18
82.41 3.55
a
a
c
b
2.28 0.02
2.16 0.04
2.76 0.04
5.74 0.06
b
a
c
d
327.71 3.51
321.01 6.07
396.44 7.53
866.88 3.73
b 6.77 0.35
a
6.45 0.34
c
3.93 0.05
d 10.54 0.47
155.67 1.73 c
152.00 1.73 a
153.00 2.29 b
161.6 2.50 d
3.75 0.02
3.75 0.03
5.83 0.03
5.74 0.03
a
a
c
b
0.215 0.01
0.257 0.01
0.574 0.02
0.625 0.02
a
b
c
d
33.47 1.46
40.89 1.86
87.79 2.89
101.00 4.56
a
b
c
d
2.85 0.08
3.15 0.12
5.97 0.19
6.98 0.11
a
b
c
d
443.66 16.47
478.90 13.68
913.77 20.84
1031.94 21.51
a
b
c
d
b
b
a
c
13.26 0.34 d
11.79 0.89 c
10.41 0.27 a
10.23 0.26 b
Means SD (n 3). Different letters in the same column indicate the significant differences (p < 0.05).
DI, distilled water; DI-CE, distilled water with 15 mM cysteine and 2 mM EDTA; PB, 100 mM phosphate buffer pH 7.0; and PB-CE, 100 mM
phosphate buffer pH 7.0 with 15 mM cysteine and 2 mM EDTA.
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prevents the disulde bonds of the protein/enzyme cysteine molecules from reducing (Sluyterman, 1967). EDTA,
can trap heavy metals that enhance the oxidation of
thiols with molecular oxygen and they can form complexes with specic groups, which may cause problems
like lowering enzyme activity (Janson and Ryden, 1998).
Between NL and PL peel extracts, the latter provided
higher total activity than the former. This is because
PL peels gave a high volume of the extract when using
the same amount of the starting material. According to
the results, the highest total activity of the extract was
found when PB-CE was used for both NL and PL peels.
Characteristics of bromelain from
pineapple peels
Protein patterns. The protein patterns of BE from NL
and PL with dierent extractants are shown in Figure 1.
In the reducing condition, the migrations of proteins in
BE were quite similar in all the extractants used. BE
from both cultivars showed the protein composition
MW to be in the range 2428 kDa. The main protein
component found in BE of NL and PL has an MW of
around 28 kDa. However, the smear protein bands with
the MW lower than 18.3 kDa were also observed in all
samples. The reducing agent (bME) could split the protein molecule which stabilized with a disulde bond
into a dierent low MW protein (Walsh, 2002). The
commercial stem bromelain (lane ST) exhibited the protein band at an MW of 28 kDa. For the non-reducing
condition, the migrations of proteins in BE were quite
the same as that of the reducing condition. Dierent
protein patterns using DI (numbers 1 and 2) and PB
(numbers 3 and 4) were observed in the non-reducing
condition. The two major protein bands in the nonreducing condition of the samples extracted with DI
(numbers 1 and 2) were found at around 28 kDa and
below 18.3 kDa, while only a single protein band
(MW 28 kDa) was observed when using PB
(number 3 and 4) as an extractant. Nonetheless, the
BE of NL extracted by PB and PB-CE showed other
major protein bands at around 30 and also below
18.3 kDa in some samples. Dierences in protein components among those BEs are probably due to the differences in the amount and characteristics of interfering
proteins obtained from using dierent extractants.
Activity staining. To verify the band of bromelain,
activity staining was performed (data not showed).
No clear zones were observed for the protein band in the
reducing condition. The presence of a reducing agent,
such as bME or dithiothreitol, breaks the disulde
bonds in the protein structure, making the protein less
conformationally stable and forcing it to lose functional and/or structurally important elements of the
398
Ketnawa et al.
Reducing
Non-reducing
Figure 1. SDSPAGE patterns of crude bromelain extract from Nang Lae (NL) and Phu Lae (PL) pineapple peels
extracted by (1) DI; (2) DI-CE; (3) PB; and (4) PB-CE in reducing and non-reducing conditions.
Protein (6 mg) was loaded into the gel for protein staining. ST: stem bromelain. M: MW markers.
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Muscle
BE (mL)
DI
NL
Beef
1000
2000
1000
2000
1000
2000
1.11 0.01
1.50 0.02
1.27 0.02
1.52 0.01
1.64 0.01
1.97 0.01
b, A
b. A
b. B
c, A
a, C
a, B
1.37 0.01
1.79 0.01
1.38 0.01
1.60 0.01
2.04 0.00
2.21 0.01
c, A
c, B
c, A
d, A
b, B
b, C
1.09 0.01
1.32 0.01
1.10 0.01
1.32 0.01
2.55 0.01
2.86 0.01
a,
a,
a,
a,
c,
c,
A
A
B
A
C
B
1.86 0.01
2.02 0.01
1.37 0.01
1.61 0.01
2.68 0.01
2.77 0.07
d, B
d, B
c, A
d, A
d, C
d, C
1000
2000
1000
2000
1000
2000
0.97 0.00
1.30 0.00
1.12 0.01
1.32 0.01
1.88 0.00
1.92 0.01
a,
a,
a,
a,
a,
a,
1.21 0.01
1.58 0.02
1.32 0.01
1.65 0.15
2.03 0.02
2.39 0.01
b, A
b, A
c, B
c, B
b, C
b, C
1.76 0.01
2.07 0.01
1.55 0.01
1.77 0.02
2.42 0.11
2.50 0.01
d, B
d, B
d, A
d, A
c, C
c, C
1.55 0.01
1.78 0.01
1.17 0.01
1.59 0.01
2.45 0.01
2.87 0.01
c, B
c, B
b, A
b, A
d, C
d, C
Chicken
Squid
PL
Beef
Chicken
Squid
DI-CE
A
A
B
B
C
C
PB
PB-CE
Means SD from triplicate determinations. Mean values followed by different small letters in the same row indicate significant differences
(p < 0.05). Mean values followed by capital letters in the same column indicate significant differences (p < 0.05).
DI
PB
DI-CE
PB-CE
Figure 2. SDSPAGE patterns of muscles treated with crude bromelain extract from NL pineapple peels extracted by
different extractants.
BC, CC and SC, controls without extract; B, beef; C, chicken; and S, squid. The numbers 1 and 2 indicate the amount of
extract, 1 and 2 mL, respectively. MHC, myosin heavy chain; AC, actin; TM, tropomyosin; TN-T, troponin-T; and MLC,
myosin light chain.
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Ketnawa et al.
DI
DI-CE
PB-CE
PB
Figure 3. SDSPAGE patterns of muscles treated with crude bromelain extract from PL pineapple peels extracted by
different extractants.
BC, CC, and SC, control without extract; B, beef; C, chicken; and S, squid. The numbers 1 and 2 indicate the amount of
extract, 1 and 2 mL, respectively. MHC, myosin heavy chain; AC, actin; TM, tropomyosin; TN-T, troponin-T; and MLC,
myosin light chain.
CONCLUSION
The extractants played an important role in maintaining bromelain activity from the pineapple peel. PB-CE
showed to be the most ecient in bromelain extraction
provided by the BE with the highest activity for both
cultivars. TCA-soluble peptide contents and hydrolytic
patterns of muscle proteins conrmed that the extractant containing cysteine and EDTA could better
401
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FUNDING
This work was supported by Mae Fah Luang University and
the National Research Council of Thailand [grant number:
PK/2553-40].
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