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Aqueous Two-phase Extraction of Bromelain

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Biochemical Properties

Article in Food science and biotechnology · November 2011

DOI: 10.1007/s10068-011-0168-5

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Food Sci. Biotechnol. 20(5): 1219-1226 (2011)
DOI 10.1007/s10068-011-0168-5
RESEARCH ARTICLE
Aqueous Two-phase Extraction of Bromelain from Pineapple Peels
(‘Phu Lae’ cultv.) and Its Biochemical Properties
Sunantha Ketnawa, Phanuphong Chaiwut, and Saroat Rawdkuen
Received: 29 November 2010 / Revised: 27 June 2011 / Accepted: 4 July 2011 / Published Online: 31 October 2011
© KoSFoST and Springer 2011
Abstract This study examines the extraction of bromelain
from pineapple peels by using an aqueous two-phase system
(ATPS). Bromelain from the crude extract predominantly
partitioned to the polyethylene glycol rich phase. The best
condition for bromelain partitioning was found to be 18%
PEG6000-17% MgSO4, which increased purity 3.44-fold
with an activity recovery of 206%. Protein patterns and
activity staining showed the molecular weight of bromelain
to be about 29 kDa. The bromelain showed the highest
relative activity at pH 8.0 and at 60oC. When increasing
NaCl concentrations (up to 1.5%, w/v), its activity
continuously decreased. The bromelain (0-0.3 units) was
applied to hydrolyze collagen. The β, α1, α2 components of
collagen extensively degraded into small peptides when
treated with the bromelain. This study showed that the
ATPS can be employed to isolate the bromelain from
pineapple peels and the bromelain extract could be used as
a meat tenderizing agent.
Keywords: aqueous two-phase system, bromelain, pineapple
peel, collagen
Introduction
Thailand is one of the world’s largest pineapple producers
(aboout 2.0 million tons in 2009) and the biggest exporter
of canned and frozen pineapples around the world. ‘Nang
Sunantha Ketnawa, Saroat Rawdkuen ( )
Food Technology Program, School of Agro-Industry, Mae Fah Luang
University, Muang, Chiang Rai 57100, Thailand
Tel: +66-5391-6752; Fax: +66-5391-6739
E-mail: saroat@mfu.ac.th
Phanuphong Chaiwut
School of Cosmetic Science, Mae Fah Luang University,
Muang, Chiang Rai 57100, Thailand
Lae’ and ‘Phu Lae’ are well known cultivars in Chiang
Rai, Thailand. Pineapple peels and cores account for about
40% of the whole fruit, and they are largely wasted after
fresh-cut processing (1). These wastes are occasionally
utilized as fertilizers or animal feed, yet they have very low
economic value. Pineapple wastes are found to have
potential uses as raw materials that could be converted into
value-added products, especially as sources for bioactive
compounds extraction; for example, citric acid (2),
antioxidants (3), and bioprotein (4).
Bromelain, a proteolytic enzyme, has been reported to
be present in pineapple wastes such as in cores, peels, and
leaves (5). It has been used for meat tenderization (6), for
protein hydrolyzates production (7), for softening and
bating in leather industries (8), and for decomposing or
partially solubilizing protein fibers from silk and wool (9).
Furthermore, novel uses for bromelain as hydrolyzing
agents to release antimicrobial peptides of leatherjacket
insoluble proteins have been discovered (10). In addition,
bromelain is well known for clinical and therapeutic
applications (11). There is much research involved in the
extraction and purification of bromelain from stems and
crowns (5,12). Bromelain has been isolated by many
techniques: precipitation with acetone (13), or ammonium
sulfate (12,14), chromatography techniques (5,14), and
lyophilization to finally obtain purified bromelain powder
(5).
The aqueous two-phase system (ATPS) is an effective
and economically viable method for separating and purifying
mixtures of proteins and enzymes. The ATPS is composed
of either 2 incompatible polymers [e.g., polyethylene
glycol (PEG) and dextran] or of 1 polymer and of 1 salt
(e.g., PEG and a phosphate salt) in an aqueous solution. If
these phase-forming compounds are solubilized above a
critical concentration in an aqueous solution, the two-
phases separate. It can remove undesirable byproducts
1220
present in the system such as unidentified polysaccharides,
pigments, and interfering proteins that lower the activity of
the enzymes. The mild process conditions as well as the
fast phase separation together allow for the purification of
biomolecules by providing a biocompatible environment,
reducing protein denaturation, and deactivating enzymes
(15). ATPS has been used for partitioning and recovering
various proteases such as papain (16) and protease from
Calotropis procera latex (17).
Practically, there are only a few current reports available
about the extraction of bromelain from pineapple wastes.
Hence, in this study, an attempt has been made to isolate
the bromelain from pineapple peels by using an ATPS. The
aim of this study is to investigate the feasibility of using the
pineapple peel from ‘Phu Lae’ cultivars as a raw material
for bromelain extraction by using the aqueous two-phase
technique. The biochemical properties and applications of
the extract were also investigated.
Materials and Methods
Chemicals Polyethylene glycols (PEG2000, 4000, and
6000), casein, cystein, and bovine serum albumin (BSA)
were purchased from Fluka (Buchs, Switzerland).
Ammonium sulfate ((NH4)2SO4), magnesium sulphate
(MgSO4), potassium hydrogen phosphate (K2HPO4),
ethylenediamine tetraacetic acid (EDTA), and trichloroacetic
acid (TCA) were procured from Merck (Darmstadt,
Germany). β-Mercaptoethanol (β-ME), Coomassie brilliant
blue G-250, sodium dodecyl sulfate (SDS), stem bromelain
(EC 253-387-5), and collagen from bovine achilles tendon
(C9879) were purchased from Sigma-Aldrich (St. Louis,
MO, USA).
Raw material The pineapple peels (‘Phu Lae’ cultv.)
were obtained from a shop in Nang Lae subdistrict, in
Chiang Rai, Thailand. The peel was washed, air-dried, and
then stored at 4oC for the experiments.
Bromelain extraction
Crude extract preparation: The pineapple peel was
blended in a cold extraction buffer (0.1 M sodium
phosphate buffer (SPB) with 15 mM cysteine and 2 mM
EDTA, pH 7.0) at a 1:1 ratio (w/v) for 3 min and then
filtered through a cheese cloth. The filtrate was centrifuged
at 10,000×g for 20 min at 4oC. The obtained supernatant
was referred to as the ‘crude extract (CE)’ and was used for
further experiments.
Aqueous two-phase system (ATPS): The ATPS was
performed in 50-mL centrifuge tubes according to the
method of Ketnawa et al. (1) with slight modification. To
study the effect of PEG on the partitioning of bromelain,
Ketnawa et al.
the concentrations (12, 15, and 18%, w/w) of PEG2000,
4000, and 6000 Da were added into the system with 17%
MgSO4. Different salts including (NH4)2SO4, MgSO4, and
K2HPO4 at different concentrations (14, 17, and 20%, w/w)
were also investigated by mixing them with the PEG that
gave the highest yield. Fifty % of the crude extract were
used in the ATPS. The mixture was mixed thoroughly for
3 min using a vortex mixer. The phases were separated by
centrifuge at 7,000×g for 20 min at 4ºC. Each fraction
obtained was determined for protein content and protease
activity. The following partitioning parameters were
calculated: partitioning coefficient of protein (KP),
partitioning coefficient of bromelain (KE), specific activity
(SA), purification factor (PF), and bromelain recovery
(%yield) as reported by Ketnawa et al. (18). The phase that
gave the highest bromelain recovery was chosen for
characterization.
Characterization of isolated bromelain
Determination of protease activity: The bromelain activity
was determined by using the Murachi method (19). Casein
was used as a substrate in the presence of an activator (0.03
M cysteine and 0.006 M EDTA in a 0.05 M phosphate
buffer pH 7.0). The reaction mixture consisted of 0.1 mL
of enzyme solution, 0.8 mL of sodium phosphate buffer pH
7.0, and 0.2 mL of the activator. After the mixture was
incubated at exactly 37oC for 10 min, the reaction was then
stopped by adding 3.0 mL of 5%(w/v) TCA. Precipitated
protein was removed by centrifuge at 10,000×g for 10 min.
The absorbance of the clear supernatant was measured at
275 nm. One unit of bromelain activity is defined as the
amount of enzyme-releasing product equivalent to 1 µmol
of tyrosine 1/min·mL under assay conditions.
Protein determination: The protein content of the sample
was determined by using the Bradford method (20). BSA
was used as a standard.
SDS-PAGE: The distribution of molecular weight was
determined by using SDS-PAGE according to the Laemmli
method (21). The sample was mixed with the sample
buffer [0.5 M Tris-HCl, pH 6.8, containing 4%(w/v) SDS
and 20%(v/v) glycerol] with and without βME. The
proteins were loaded into the polyacrylamide gel (15%
running and 4% stacking gels) and then subjected to
electrophoresis at a constant current of 15 mA/gel. After
separation, the protein was stained with 0.02% Coomassie
brilliant blue R-250 and then destained with a mixture of
acetic-methanol solution.
Activity staining: The separated protein was verified for
bromelain by using activity staining (casein-substrate gel
electrophoresis) as used in Garcia-Carreno et al. (22). The
gel was immersed in 50 mL of 2%(w/v) casein and
dissolved with 50 mM SPB, pH 7.0 for 45 min of constant
agitation at 4ºC. The gel was then incubated at 37oC for 30
Bromelain Extraction by Aqueous Two Phase System 1221

min to activate the bromelain activity. The development of
a clear zone on the blue background indicated hydrolytic
activity of the bromelain.
Determination of pH profile and stability: The pH profile
was tested by assaying the protease activity in the different
pH (3-12). The buffers of 0.05 M glycine-HCl (pH 3.0),
0.05 M Na-acetate (pH 4.0-5.0), 0.05 M Na-phosphate (pH
6.0-7.0), 0.05 M Tris-HCl (pH 8.0-10.0), and 0.05 M Na-
carbonate (pH 11.0-12.0) were all used. The residual
activity was measured and expressed as the relative
bromelain activity.
The pH stability was determined by incubating the
isolated bromelain at different pH buffers (as mentioned
above) for 20 min at room temperature. The casenolytic
activity was expressed as the relative activity when
compared with that of the control.
Determination of thermal profile and stability: The
isolated bromelain (200 µL) was used to assay the
casenolytic activity at different temperatures (30, 40, 50,
60, 70, 80, 90, and 100oC) for 15 min. The residual activity
was measured and expressed as the relative bromelain
activity.
The effect of temperature on the activity of the isolated
bromelain was investigated by incubation the enzyme
sample (200 µL) at a temperature of 90ºC for 0-60 min.
The casenolytic activity was expressed as the relative
activity when compared with that of the control.
Effect of salt on bromelain activity: The isolated
bromelain (200 µL) was incubated at room temperature for
20 min in the presence of NaCl at different concentrations
(0, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50%(w/v) with a ratio
of 1:1 (v/v). The residual activity of the bromelain was
measured as previously described and expressed as the
relative activity when compared with that of the control.
Effect of bromelain on collagen hydrolysis Saito et al.
(23) method was used with slight modifications to
investigate the degradation of collagens from bovine
achilles tendon and from farmed giant catfish skins through
the use of bromelain extract. The collagen was dissolved
with a buffer solution [0.02 M sodium phosphate buffer,
pH 7.2, containing 1%(w/v) SDS and 3.5 M urea]. Then,
the isolated bromelain (0-0.3 units) was added to the
collagen solutions. The mixture was incubated at 37ºC for
10 min and then stopped by submerging the mixture in
boiling water for 3 min. Newly generated peptides were
determined by SDS-PAGE, using 7.5% separating and 4%
stacking gels as previously mentioned.
Statistical analysis Analysis of variance (ANOVA) was
employed to analyze the data from 3 replications.
Differences between means were evaluated by Duncan’s
multiple range test and by using the SPSS statistics
program (version 11.5; SPSS, Chicago, IL, USA).
Results and Discussion
Extraction of bromelain by ATPS
Effects of PEG on bromelain partitioning: Bromelain
partitioning in an ATPS of different molecular weights
(Mw; 2,000, 4,000, and 6,000 Da) and concentrations of
PEG (12, 15, and 18%,w/w) with 17% MgSO4 was studied.
As shown in Table 1, the partitioned bromelain strongly
depended on the Mw and the concentration of PEG. The
highest bromelain recovery at each Mw of PEG was found
when the highest concentration (18%,w/w) of PEG was
applied. In addition, increasing the PEG concentration
resulted in a higher recovery of bromelain (p<0.05). High
KP values indicated that most of the proteins were
partitioned to the top phase. Meanwhile, high KE values
implied that only the target enzymes were partitioning to
the top phase. The KP obtained from most of the systems
was higher than 1, indicating that the proteins preferentially
partitioned to the top phase. High protein content in the top
phase is probably due to the effect of volume exclusion

Table 1. Effect of PEG molecular weights and concentrations on partitioning of bromelain from ‘Phu Lae’ pineapple peel
Phase composition (%, w/w) KP1) KE SA PF Yield (%)
cd2) bc e e
12% PEG2000–17% MgSO4 1.23±0.01 6.76±0.08 19.02±0.13 2.08±0.03 118.54±1.27cd
15% PEG2000–17% MgSO4 1.13±0.03b 7.90±0.54c 16.62±0.29cd 1.82±0.03cd 115.11±1.69c
18% PEG2000–17% MgSO4 1.15±0.01bc 8.01±0.38c 17.32±0.33d 1.90±0.04d 128.11±2.26f
12% PEG4000–17% MgSO4 1.16±0.03bc 7.20±0.20c 16.49±0.34cd 1.81±0.05cd 101.62±2.51b
15% PEG4000–17% MgSO4 1.20±0.03bc 6.28±0.48bc 15.74±0.36c 1.72±0.03c 122.97±2.85e
18% PEG4000–17% MgSO4 1.41±0.05e 7.88±0.45c 14.60±0.78b 1.60±0.02b 134.12±1.30g
12% PEG6000–17% MgSO4 1.27±0.02d 7.62±0.42c 16.11±0.37c 1.76±0.05c 120.53±2.37de
15% PEG6000–17% MgSO4 0.90±0.00a 4.84±0.19a 12.60±0.19a 1.38±0.03a 086.29±1.71a
18% PEG6000–17% MgSO4 0.87±0.06a 10.17±0.41d 31.39±1.52f 3.44±0.19f 205.78±2.97h
1)
KP, partition coefficient of protein in the upper phase; KE, partition coefficient of bromelain in the upper phase; SA, specific activity (unit/mg
protein); PF, purification factor (fold); yield, activity recovery (%)
2)
Means±SD from triplicate determinations; Different superscripts in the same column indicate the significant differences (p<0.05).
1222 Ketnawa et al.

Table 2. Effect of salts and concentrations on partitioning of bromelain from ‘Phu Lae’ pineapple peel
Phase composition (%, w/w) Kp1) KE SA PF Yield (%)
a2) a g f
18% PEG6000–14% MgSO4 00.60±0.01 05.05±0.15 25.93±0.24 2.85±0.03 162.88±0.12h
18% PEG6000–17% MgSO4 0.87±0.06b 08.30±0.12b 31.39±1.52h 3.44±0.19g 205.78±2.97i
18% PEG6000–20% MgSO4 0.92±0.03b 05.94±0.11a 21.41±0.26e 2.34±0.03e 117.42±0.64c
18% PEG6000–14% (NH4)2SO4 1.67±0.06c 12.51±0.65d 18.00±0.52d 1.97±0.11d 101.69±0.90a
18% PEG6000–17% (NH4)2SO4 2.82±0.15e 15.27±0.61e 22.08±0.20f 2.43±0.15e 112.76±0.70b
18% PEG6000–20% (NH4)2SO4 7.23±0.46g 23.73±1.69g 18.61±0.12d 2.04±0.15d 158.06±1.54g
18% PEG6000–14% K2HPO4 2.48±0.15d 10.52±0.18c 12.49±0.02a 1.38±0.01a 133.88±0.48e
18% PEG6000–17% K2HPO4 2.80±0.06e 17.14±0.65f 16.59±0.06c 1.82±0.02c 141.83±1.14f
18% PEG6000–20% K2HPO4 6.46±0.28f 22.83±1.73g 15.59±0.01b 1.73±0.01b 0125.39±13.45d
1)
KP, partition coefficient of protein in the upper phase; KE, partition coefficient of bromelain in the upper phase; SA, specific activity (unit/mg
protein); PF, purification factor; yield, activity recovery
2)
Means±SD from triplicate determinations; Different superscripts (%) in the same column indicate the significant differences (p<0.05).

over salting out (15). The KE obtained from all of the
systems were also higher than 1, indicating that the
bromelain preferentially partitioned to the top phase. The
highest KE was obtained from 18% PEG6000−17% MgSO4.
It has been stated that an increase in polymer concentration
or Mw resulted in the reduction of biomolecule partitioning
to the bottom phase (17). Among all of the ATPS tested,
the system that comprised of 18% PEG6000 and 17%
MgSO4 effectively partitioned the bromelain to the top
PEG-rich phase with a recovery of 205.78%, and it
provided an approximate PF of 3.44-folds. The bromelain
activity recovery was very high (>>100%) for the system
studied. This was probably due to the structural alteration
of the enzyme active sites in the presence of PEG. The
increase in PF was due to the increased partitioning of
bromelain to the top phase when compared to that of other
proteins partitioned in the bottom phase of the system. It
has been reported that the system with high concentrations
of polymer or of high Mw polymer, and with high salt
concentrations resulted in the partitioning of biomolecules
at the interphase. This was due to the influence of both
volume exclusion and the salting out effect (24). Nalinanon
et al. (24) reported that the Mw of PEG4000 produced a
higher yield of protease from the stomach of albacore tuna
than that of PEG1000 when 15% MgSO4 was used. Based
on the highest activity recovery (206%) of ATPS, the 18%
PEG6000 was selected for investigating the effect of salts
on bromelain partitioning.
Effect of salts on bromelain partitioning: The effects of
salts at different concentrations on partitioning (Kp, KE,
SA, PF, and yield) are shown in Table 2. Salts are
frequently used in ATPS to improve the partitioning of the
target molecules between the phases (24,25). The addition
of salts to the aqueous PEG solution led to an arrangement
of ordered water molecules around the PEG molecule
because of the water-structure-breaking effects (24). From
the results of this study, the KP and KE of all ATPS systems
were in the range of 0.60 to 7.23 and 5-24, respectively.
The KP and KE increased with higher additions of salt
(except in MgSO4). This phenomenon can be explained by
the salting out effect, whereby both the bromelain and
impure proteins partitioned more to the PEG-rich top phase
and resulted in decreases of SA and PF. The shift of
protease as well as the undesirable proteins to the top phase
was probably due to similar hydrophobic properties on
their surface. This phenomenon could be due to the ‘salting
out effect’, which resulted in increased bromelain
partitioning to the top phase. The highest KP (7.23) and the
highest KE (23.73) were found in the system of 18%
PEG6000−20%(NH4)2SO4. In contrast, the phase
composition of 18% PEG6000−14% MgSO4 showed the
lowest KP (0.60) and KE (5.05), indicating that both desired
protease and contaminant proteins were preferable to the
lower phase. A phase containing 18% PEG6000 and 17%
MgSO4 gave the highest SA (31.39) and PF (3.44). The
activity yield by using ATPS was in the range of 101 to
206%. The maximal yield of 206% was obtained from the
system containing 18% PEG6000−17% MgSO4. It
provided the highest bromelain recovery when compared to
the other 2 salts. The effectiveness of the salt is mainly
determined by the nature of the anion. Multi-charged
anions are the most effective in this order (from most to
least): SO42− >HPO42− >acetate>Cl-1. The order of cations
is usually given as NH4+ >K+ >Na+ >Li+ >Mg2+ >Ca2+ (26).
Increasing the salt concentration in the range of 14 to 20%
could not produce good bromelain recovery. It may have
needed a wider range of salt concentrations. The decrease
in bromelain purity at the top phase is due to the increased
partitioning of proteins. Babu et al. (15) reported that the
system of 18% PEG1500 and 20% K2HPO4 provided the
highest activity recovery of bromelain from pineapple
juice. Klomklao et al. (25) reported that using 20%
PEG1000 with 20% MgSO4 gave the highest SA and PF in
partitioning protease from tuna spleen. Besides, Nalinanon
et al. (24) reported the same result in partitioning protease
from the stomach of albacore tuna. For this study, the
Bromelain Extraction by Aqueous Two Phase System 1223

system comprising of 18% PEG6000−17% MgSO4 was

chosen and the top phase of this system was used as the
‘isolated bromelain’ for further characterization.

Characteristics of isolated bromelain

Protein patterns and activity staining: The protein patterns
of the crude extract and the isolated bromelain from the
system 18% PEG6000-17% MgSO4 are shown in Fig. 1.
The migration of the proteins (Fig. 1A) in the crude extract
(lane 1, 5) top phase (lane 2, 6) and bottom phase (lane 3,
7) fractions were quite different. In the non-reducing
condition, the crude extract (lane 1) showed a major
protein composition with Mw of 28 kDa. In addition, the
proteins with the Mw range of 28-30 kDa clearly
partitioned to the top phase (lane 2), while others did not
clearly appear in the bottom phase (lane 3). The
commercial stem bromelain exhibited a protein band at
Mw of 28 and 30 kDa (lane 4). The differences in the
protein component fractions are probably due to the
differences in the amount and characteristics of the
interfering proteins of each phase. In reducing conditions
(with βME), the protein migration in the crude extract, top,
and bottom phase fractions were also quite different. The
crude extract (lane 5) and the bottom phase (lane 7)
showed 2 small protein bands with a Mw range of 18.3-
28 kDa, while the protein in the top phase (lane 6) did not
appear. Disappearance of the major components in the top
phase may be because this protein was reduced to a low
Mw by the reducing agent. The presence of a reducing
agent, such as βME or dithiothreitol, breaks the disulfide
bonds in the protein structure, making the protein less
stable and losing functional and/or structurally important Fig. 1. Protein patterns (A) and activity staining (B) of
bromelain from ‘Phu Lae’ pineapple peel partitioned with
elements of the domain tertiary structure (8). Umesh et al. 18% PEG6000−17% MgSO4 ATPS. (6 and 4 µg protein were
(5) reported that the bromelain extracted from pineapple loaded in to the gel for protein patterns and activity staining,
cores showed a Mw of around 26 kDa. respectively); lane 1, 5: crude extract; lane 2, 6: top phase; lane 3,
7: bottom phase; lane 4, 8: stem bromelain; M: molecular weight
To verify the bromelain band, activity staining was protein makers
performed and is shown in Fig. 1B. The clear zone caused
by hydrolytic activity (on the blue background) was clearly
observed in the crude extract (lane 1) and the top phase kDa) (11). There was no clear zone in the crude extract, top
(lane 2). Not only was the crude extract and the top phase phase, or bottom phase fractions under the reducing
found to have proteolytic activity, but the bottom phase condition. This is probably because the stabilizing disulfide
(lane 3) was also found to have a clear band. The clear bonds in the bromelain structure were broken, resulting in
zone was observed in different positions due to the shifting a loss of enzyme structure, so hydrolytic activity could not
of the protease band in each fraction. This means that each be observed.
fraction contained different types of protease that could pH profile and stability: The effect of pH (3-12) on
hydrolyze the casein in the SDS-PAGE gel. A clear zone of bromelain activity was measured and reported as a relative
this sample appeared at Mw of 28 kDa (lane 4). The protease activity to the control (Fig. 2A). The bromelain
pineapple plant was shown to contain at least 4 distinct exhibited a broad pH activity profile, as indicated by
cysteine proteinases. The major proteinase present in the relatively high activity (>60%) at all tested pHs. The
plant stem extracts was from stem bromelain (23.8 kDa), maximum activity was found at pH 8.0. The bromelain
while fruit bromelain (23 kDa) was the major proteinase in activity gradually decreased at an acidic pH of 3-4 and also
the fruit. Two additional cysteine proteinases were detected at an alkaline pH of 10-12. In the case of the other plant
only in the stem: ananain (23.46 kDa) and comosain (24.50 proteases, Vallés et al. (26) reported that proteolytic
1224
Fig. 2. Effect of pH (A) and pH stability (B) on the activity of
bromelain from ‘Phu Lae’ pineapple peel partitioned with
18% PEG6000−17% MgSO4 ATPS.
enzymes from ripe fruits such as Bromelia antiacantha
Bertol. exhibited high caseinolytic activity (higher than
80%) in a broad pH range (5-9) as well. In addition,
enzymes from unripe fruits like Bromelia hieronymi Mez.
and Asclepain cI from the Latex of Asclepias curassavica
L. exposed optimum pH activity on casein between pH 7.5
and 9.0 (27). For the result in this experiment, relatively
high activity (>80%) was found in the pH range of 5-11. In
this regard, the isolated enzyme is unique, and therefore
might be useful for application in the food and pharmaceutical
industry.
The stability of bromelain incubated with various pH
buffers is shown in Fig. 2B. Bromelain was able to retain
most of its activity (>70%) at pH 9.0. The enzyme stability
slightly declined (60-70%) in an alkaline area, while it was
dramatically lost at a pH below 8.0 (<70%). The activity of
this enzyme sharply deceased (<70%) in an acidic pH
range, possibly due to the denaturation of the enzyme.
Under a very acidic and alkaline pH, there is a charge
repulsion, which decreases electrostatic bonds. It should be
pointed out that the bromelain molecule was stabilized by
the attached PEG6000. Polymers have been reported to
exhibit stabilizing effects on the biological activity and
structure of proteins and enzymes (28). For the present
result, it should be noticed that the enzyme seems to be
more stable in an alkaline rather than in an acidic
condition.
Thermal profile and stability: For the thermal profile, the
Ketnawa et al.
Fig. 3. Effect of temperature (A) and thermal stability (B) on
the activity of bromelain from ‘Phu Lae’ pineapple peel
partitioned with 18% PEG6000−17% MgSO4 ATPS. Enzyme
was incubated at 90°C for 0-120 min before determined its activity
at 37oC for pH 7.
isolated bromelain was assayed at a temperature ranging
from 30 to 100oC. The relative proteolytic activity against
the casein was calculated and is presented in Fig. 3A. The
highest relative bromelain activity (100%) was found at
50-60oC. As the incubation temperature increased higher
than 60oC, the bromelain activity dramatically decreased to
12% at 100oC. This result is similar to a previous report (9,
26). As the temperature increases, more molecules gain
enough kinetic energy to undergo the reaction. If the
temperature is raised above the optimum point, the kinetic
energy of the enzyme and water molecules is so great that
the structure of the enzyme molecule starts to be disrupted
(8). Therefore, a decrease in activity was detected when the
temperature increased. Knowing the optimum temperature
would help to explore the usefulness of the enzyme. As
reported, bromelain is remarkably heat stable, retaining
proteolytic activity (>80%) between 50 and 60oC where
most enzymes are destroyed or denatured.
The thermal stability of isolated bromelain was also
measured at 90oC at different times. The results are
presented in Fig. 3B. The relative activity of bromelain
decreased more than 40% after 5 min of incubation and
dropped drastically after that. However, some plant proteases
showed different losses of activity. Vallés et al. (26)
reported that proteolytic enzymes from ripe fruits of B.
antiacantha Bertol retained 80% of its initial activity after
Bromelain Extraction by Aqueous Two Phase System
Fig. 4. Effect of salt content on the activity of bromelain from
‘Phu Lae’ pineapple peel partitioned with 18% PEG6000−
17% MgSO4 ATPS.
incubation at 60oC for 30 min. High temperature is found
to be an irreversible-denaturant of proteins and enzymes.
Thus, the enzyme is deactivated at high temperatures due
to the partial unfolding of its molecule (8). The result
showed bromelain attached with PEG6000 was remarkably
heat stable, which expressed activity even at 100oC where
most of the enzymes were destroyed or denatured.
Effect of salt on bromelain activity: The ability of isolated
bromelain to retain its activity under growing ionic strength
was tested by exposing it at different concentrations of
NaCl (0-1.50%,w/v). Its activity decreased when NaCl
concentrations increased (Fig. 4). At 0.25% NaCl, the
enzyme activity decreased by about 20% when compared
with the control (without NaCl). However, when the salt
was added up to 1.5%, relative activity was still obtained
(>60%). The isolated bromelain showed less relative
activity than Asclepain cI, which declined only by 30%
when NaCl rose to 5.84% concentration. Loss of bromelain
activity might be due to the changes in the enzyme
structure induced by the salting out effect. This results
from the competition between the protein and the salt ions
for the water molecules necessary for their survivals. At
high concentrations, there is not enough water molecules
available for protein survival, since the majority of the
water molecules are strongly bound to the salts. Therefore,
protein-protein interactions become more powerful than
protein-water interactions, and this may lead to aggregation
followed by precipitation of protein molecules. In addition,
NaCl at higher concentrations possibly competed with the
enzyme in water binding; resulting in a stronger protein-
protein interaction. This was possibly associated with
precipitation (8). In this regard, the isolated bromelain can
be useful for further application in food and cosmetic, in
which the formulation might contain high salt
concentrations.
Effect of bromelain on collagen hydrolysis The protein
patterns of bovine achilles tendon and farmed giant catfish
1225
Fig. 5. Peptide mapping of bovine skin collagen (A) and giant
catfish skin collagen (B) digested with bromelain from ‘Phu
Lae’ pineapple peel partition with 18% PEG6000−17%
MgSO4 ATPS. M, molecular weight protein maker; C, control;
Numbers represent the activity units of bromelain; CE, crude
extract
skin collagens treated with isolated bromelain are shown in
Fig. 5. Similar hydrolyzed patterns were observed in both
collagen samples. Hydrolytic patterns of collagen were
clearly observed when the unit of the isolated bromelain
increased. Compared to giant catfish skin collagen, bovine
achilles tendon collagen was more resistant to bromelain
hydrolysis. A disappearance of major components (α and
β) of collagen were observed when the addition levels
were 0.3 and 0.12 units for bovine achilles tendon (Fig.
5A) and giant catfish skin (Fig. 5B) collagen, respectively.
Collagen hydrolysis was bromelain-concentration dependent.
The bromelain showed to be highly efficient at hydrolyzing
both collagens, while giant catfish skin collagen was more
susceptible to hydrolysis.
Collagen is the major determinate for the texture of
mature and older slaughtered animal meat. There is a
reasonable correlation between total collagen content and
the eating quality of meats (29). Tenderization of those
meats may be achieved by using protease for the
degradation of connective tissue and myofibrillar proteins
 1226
(8,29). This study demonstrates how the use of this enzyme
can be used as an effective a meat tenderizer.
In conclusion, an aqueous two-phase system could be
used successfully for the partial purification of bromelain
from ‘Phu Lae’ pineapple peels. PEG6000 and magnesium
sulphate gave the best bromelain recovery when compared
to others. Characteristics of the isolated bromelain in this
study provided new information about using this enzyme
in any system. Based on the model of collagen hydrolysis,
it can be said that bromelain extract from ‘Phu Lae’
pineapple peels could be effectively used for meat
tenderization and collagen hydrolyzate preparation.
Acknowledgments The authors would like to thank Mae
Fah Luang University and the National Research Council
of Thailand (NRCT) for financial support under the project
No. PK/2553-40. We also thank Prof. Matthew Robert
Ferguson, Naresuan University Language Center, for kindly
providing suggestions and corrections for the manuscript.
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