Concentration by Membrane Separation Processes of a Medicinal Product Obtained from Pineapple Pulp

457
Vol. 52, n. 2 : pp. 457-464, March-April 2009

BRAZILIAN ARCHIVES OF
ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY


A N I N T E R N A T I O N A L J O U R N A L



Concentration by Membrane Separation Processes of a
Medicinal Product Obtained from Pineapple Pulp

Francisco Luiz Gumes Lopes1, João Baptista Severo Júnior2, Roberto Rodrigues de Souza2,
Daniela Diniz Ehrhardt3, José Carlos Curvelo Santana4 and Elias Basile Tambourgi5*
1Departamento de Administração e Processos Industriais e Químicos; Centro Federal de Educação Tecnológica da
Bahia, Salvador - BA - Brasil. 2Departamento de Engenharia Química; Universidade Federal de Sergipe; São
Cristóvão - SE - Brasil. 3Faculdade de Ciências Farmacêuticas; Pontifícia Universidade Católica de Campinas;
Campinas - SP - Brasil. 4Departamento de Ciência Exatas; Universidade Nove de Julho; São Paulo - SP - Brasil.
5Faculdade de Engenharia Química; Universidade Estadual de Campinas; Av. Albert Einstein, 500; C. P.: 6066;
13083-970; Barão Geraldo; Campinas - SP - Brasil


ABSTRACT

The concentration of pineapple juice is needed to retain the bromelain activity and to standardize the composition
and proteolytic activity. Thus, this work aimed to obtain a pure bromelain extract from the Ananas comosus L.
Merril juice by membrane separation process. A 22 experimental planning was used to study the influence of pH and
transmembrane pressure on the activity recovery by micro-filtration using a plain membrane. In second step, this
enzyme was purified by the ultra-filtration using a 10 kDa millipore kit. The best operation condition to bromelain
concentration using the plain membrane was at pH 7.5 and transmembrane pressure of 0.05 bar, while 85% of
bromelain activity was recovered. Ultra-filtration retained 100% of proteolytic activity and concentrated in 10 fold
the bromelain extract. SDS-PAGE electrophoresis showed that the ultra-filtrated had high purity and the bromelain
from A. comosus pulp had a molecular weight of 24.5 kDa.

Key words: Ananas comosus, bromelain, membrane separation processes, purification, medicinal product


INTRODUCTION
and 2005b; Secor et al., 2005; Wen et al. 2006). It

has been applied in the anticancer activity
Bromelain is a mixture of proteinases derived from
(Harrach et al., 1994), in the immunization of
pineapple stem, which is sold as a nutritional
influenza virus (Vareniková, et al. 1995) and in the
supplement to “promote digestive health” and as
treatment of allergic airway disease (Secor et al.,
an
anti-inflammatory
medication
in
some
2005). It is also used in cosmetic compositions
developed countries (Hale et al., 2005a and 2005b;
(Chatsworth, 1996). Adverse results had been
Wen et al., 2006). Bromelain is clinically used
reported on an allergic activity of bromelain
from the pineapple extract and the natural product.
caused by the inhalations of beer or wheat snack
Studies have reported, anti-inflammatory and
(Anton et al., 2005).
immunomodulatory activities (Hale et al., 2005a
Wen et al. (2006) showed that oral administration
of bromelain improves decrease in defecation in

* Author for correspondence: eliastam@feq.unicamp.br
Braz. Arch. Biol. Technol. v.52 n.2: pp. 457-464, Mar/Apr 2009

---

458
Lopes, F. L. G. et al.
abdominal postoperative rats. Results showed that
copolymers aqueous solutions (Rabelo et al.,
bromelain increased the wet weight, dry weight,
2004) and using adsorption on the magnetic nano-
water content and number of fecal pellets in
particles of polyacrylic acid (PAA)-bound iron
laparotomized plus mechanically manipulated rats,
oxide (Chen and Huang, 2004).
suggesting improvement of postoperative ileums.
The membrane Technologies e.g. micro-filtration
Hale et al. (2005a) studied the treatment of the
(MF) and ultra-filtration (UF), as recent method
colonic inflammation with oral bromelain in
used to separate the components of a solution
patients. They observed that the bromelain had
based on molecular size differences, are much
anti-inflammation activity, but is need additional
required for liquid foods, in juice processing, as
studies of this complementary biologically based
well as protein isolate and concentrate production
approach
to
the
treatment
of
colonic
(Cassano et al., 2003; Howell et al., 1993; Sá et
inflammation.However, bromelain is instable and
al., 2003). They are used in filtration, which long
frequently is deactivated spontaneously, due the
for concentrates and purifies enzymes and
action of the components of the medium.
supports hydraulic pressure among 0.07 and 7 bar
Concentrated bromelain solutions are more
(Howell
et
al.,
1993).
Clarification
and
resistant to spontaneous inactivation of their
concentration of cajá, citrus and carrot juices and
proteolytic activity than are the dilute solutions,
maize malt by integrated membrane processes
with the proteinase stability in the order of stem
have been reported which besides reducing the
bromelain>fruit
bromelain ananain.
The
production costs, show several other advantages
proteolytic activity of concentrated bromelain
for quality and production yield (Cassano et al.,
solutions remains relatively stable for at least one
2003; Sá et al., 2003; Severo Junior et al., 2007a,
week at room temperature, with minimal
2007b).
inactivation by multiple freeze–thaw cycles or
The purpose of this work was to study the
exposure to the digestive enzyme trypsin. The
bromelain recovery, extracted from the pineapple
relative stability of concentrated versus dilute
(A. comosus L. Merril) juice, using flat membrane
bromelain solutions to inactivation under the
module and a millipore kit, aiming to retain the
physiologically relevant conditions suggests that
bromelain activity and to standardize the
delivery of bromelain as a concentrated bolus
composition and proteolytic activity in a
would be the preferred method to maximize its
concentrated extract. For this, a 22 experimental
proteolytic activity in vivo (Arroyo-Reyna and
planning was studied for the influence of pH (7.0
Hernández-Arana, 1995; Hale et al., 2005b). Other
and 7.5) and transmembrane pressure (0.05 and
problem in vivo studies using bromelain is the
0.15 bar) on the activity recovery in micro-
limitation by the lack of assays to control for
filtration process, as well as process optimization
potential differences in the composition and
by RSM methodology and in optimal condition the
proteolytic activity of this naturally derived
purification was done by ultra-filtration process.
proteinase mixture (Hale et al., 2005b).

Thus, the concentration of pineapple juice is

needed to retain the bromelain activity and to
MATERIALS AND METHODS
standardize the composition and proteolytic

activity. According to Sigma (1996), bromelain
Obtainment of pineapple juice
enzyme had price of U$ 300 per gram of protein
The pineapple (A. comosus L. Merril) fruit was
purified. These factors have been stimulated the
provided by EMBRAPA (Aracaju, SE, Brazil), its
researches aiming to concentrate, recover and/or
juice was prepared at room temperature and
purify the bromelain from pineapple. The
pressure, using a pulp mass of 650 g that initially
purification of bromelain from pineapple juice was
was passed to simple filtration through cotton to
studied
using
sequential
batch
membrane
remove the dispersed solids. Phosphate buffer at
processing systems which included micro-
pH 7.0 and 7.5 were used. The solution volume
filtration (MF) and ultra-filtration (UF), followed
was adjusted to 1.0 L.
by
ammonium
sulfate
extraction,

ultracentrifugation and freeze drying (Doko et al.,
Enzyme assays
1991);
using
the
thermo-separation
by
The enzymatic activity was measured in permeate
poly(ethylene oxide) (PEO)– poly(propylene
and in the concentrate using to method described
oxide) (PPO)– poly(ethylene oxide) (PEO) block
by Murachi (1976) and Baldini (1993). One unit of
Braz. Arch. Biol. Technol. v.52 n.2: pp. 457-464, Mar/Apr 2009

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Concentration by Membrane Separation Processes of a Medicinal Product
459
enzymatic activity was defined as the variation of
equal to 0.1 µm was used. The experiments were
one absorbance unit at 280 nm during 10 minutes
conducted in a membrane module composed of
at 35oC. Permeate and concentrate total protein
two flat props and between them: one membrane
concentration were determined by the modified
between two spacers. The feed flow circulation
Bradford method (1976) using BSA the standard
was tangential to the membrane surface, with a
protein.
discontinuous
operation
and
a
concentrate

recirculation. Figure 1 shows the scheme of
Membrane module
experimental micro-filtration unit (Lopes, 2005,
A polyvinyl fluorite membrane (TECH-SEP-6501
Severo Junior et al., 2007a, 2007b).
model) with an area of 0.0225 m2 and a pore size



Figure 1 - Scheme of the micro-filtration unit used in experiments. While: 1 - iron plates, 2 -
acrylic plates, 3 - membrane, 4 - feed flow, 5 - permeate, 6 - concentrate, 7 -
manometer, 8 - pump


In a second step, the pre-concentrated extract was
used as response. Equation 1 is the form to obtain
put into a 10 kDa millipore kit and the ultra-
the yield of activity recovery. The pineapple
filtration (UF) was carried out at 4 ºC and 7000
extract was termed as crude and the filtered global
rpm per 20 min. Salts, glycosides and other
permeate. Models evaluated were made by
substances of small molecular weight were
variance analyses (ANOVA) that was statistical
eliminated. Total volume was reduced to 10 times
analyses to authenticate the models based in the
after UF process (Doko et al., 1991).
Gauss curve deviation. Process optimization was

evaluated by the response surface methodology
Experimental design and statistical analysis
(Barros Neto et al., 1995 and 2001; Biazus et al.,
The experiments were made using a factory design
2005; Ferreira et al., 2007; Rodriguez-Nogales et
of the type 22, with two factors: pH (x1) and
al., 2005; Severo Junior et al., 2007b).
transmembrane pressure, P (x

2). Table 1 shows the
?
?
coded valor of factors. Yield of activity recovery
?
?
specific
activity
of
global
permeate
(1)
Y (%) = ?
? * 100
(Y, %) was given in percent form (%) and was
?
specific
activity
of
crude
?
?
?

Table 1 - Coded valor used in the experiment.
Levels
Variables
-1
+1
pH, x1
7.0
7.5
P (bar), x2
0.05
0.15


Polyacrylamide gel electrophoresis
separated on 0.8 mm thick homogeneous 12%
SDS-PAGE was performed on mini-PROTEAN II
(w/v) acrylamide resolving gels and 4.8% (w/v)
cell (Bio-Rad, USA) with 12 % acrylamide gel,
acrylamide stacking gels with the buffer systems
using protein standard for molecular weight
described by Laemmli (1970), using the Bio-Rad
marker (Dermirkan et al., 2005; Nirmala and
Protean II apparatus. Equal volume of sample
Muralikrishna, 2003). Proteins of UF extract were
buffer that contained 25mM Tris/HCl, pH 6.8,
Braz. Arch. Biol. Technol. v.52 n.2: pp. 457-464, Mar/Apr 2009

---

460
Lopes, F. L. G. et al.
20% (v/v) glycerol, 8% w/v) SDS and 0.04%
condition of bromelain purification, the activity
(w/v) brome-phenol blue, was added to the protein
recovery was approximated of 90%.
sample
and
mixed
with
2.5%
(v/v)
2-
Chen and Huang (2004) retained 87.4% activity
mercapttoethanol. This mixture was boiled for 10
after adsorption/desorption of bromelain from an
min prior to loading on the gels. The proteins were
aqueous solution by polyacrylic acid (PAA)-bound
separated at constant amperage of 20 mA using the
iron oxide magnetic nano-particles. Rabelo et al
running buffer contained 25mM Tris, 192mM
(2004) obtained an enzyme activity recovery
glycine, and 0.1% (w/v) SDS, pH 8.3. Separated
around 79.5%, using aqueous two-phase system
proteins were visualized after fixation with
with thermo-separated polymers. Doko et al.
coomassie brilliant blue G-250 (staining solution:
(1994),
using
semi-permeable
membrane,
10% (v/v) phosphoric acid and 0.02 (w/v)
ammonium sulfate extraction, centrifugation and
coomassie).
freeze-drying processes achieved low-moisture

freeze-dried, and light-colored extracts, free of

non-protein constituents, which accounted for
RESULTS AND DISCUSSION
about 50% yielded extracts containing 98%

protein. The extracts assayed for bromelain and
Results obtained are shown in Table 2, which
proteolytic activity resulted in almost 100%
describes the experimental conditions realized to
potential recovered at completion. However,
bromelain enzymes recovery process. Table 3
bromelain and proteolytic activity decay during
shows the experimental and predicted results for
the processes described above is essentially caused
activity recovery. In enzymes purification, the
by the losses through adsorption on the UF
percent of activity recovery (Y, %) is commonly
membrane relative to the level of concentration
used to show the purification method is efficient in
reached.
the capturing the target enzyme. In the best

Table 2 - Results of microfiltration of bromelain from pineapple juice in plain membrane.
pH
Assay
Pressure (bar)
Total protein (mg/L)
Activity (U/mL)
SA (U/mg)
Y (%)
Crude
16.7780
131.322
0.127762
100.000
1
0.05
10.2152
96.6475
0.105696
82.7284
2
0.05
10.5557
98.2759
0.109218
85.4857
3
0.05
10.4574
93.9655
0.108201
84.6897
7
Crude
5.00435
154.885
0.032310
100.000
4
0.15
2.95270
107.567
0.027450
84.9575
5
0.15
3.03091
106.034
0.028177
87.2080
6
0.15
2.92785
109.770
0.027219
84.2425
Crude
6.13410
146.168
0.041966
100.000
7
0.05
2.66832
93.6782
0.028484
67.8739
8
0.05
2.89733
106.034
0.027325
65.1111
9
0.05
2.47699
85.9195
0.028829
68.6965
7.5
Crude
2.86401
92.6724
0.030905
100.000
10
0.15
0.62701
72.7012
0.008625
27.9069
11
0.15
0.64766
70.4023
0.009199
29.7669
12
0.15
0.73984
74.4253
0.009941
32.1657
While: SA is the specific activity and Y is the yield of activity recovery.


Table 4 shows the results of statistical analysis to
empirical model introduced larger correlation and
model validity by variance analysis methodology
larger rate between Fcal/Ftab (~ 155). According to
(ANOVA) (Barros Neto et al., 1995 and 2001;
Barros Neto et al., (2001) this rate could be above
Biazus et al., 2005; Ferreira et al., 2007; Severo
10 times for that empirical model been fitting.
Junior et al., 2007b). Table 3 showed that
Variance and correlation are approximated of
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Concentration by Membrane Separation Processes of a Medicinal Product
461
optimal values that are 100% and 1.0, respectively.
predict the activity recovery of bromelain enzyme
Thus, it indicated that hyper-plain empirical model
by plain membrane process.
is fitted and was the best empirical model to

Table 3 - Planning matrix with experimental and predict data.
Assays
pH
P (bar)
Yexp (%)
Ypred (%)
1
7.0
0.05
82.7284
84.2221
2
7.0
0.05
85.4857
84.2221
3
7.0
0.05
84.6897
84.2221
4
7.5
0.05
84.9575
85.6379
5
7.5
0.05
87.2080
85.6379
6
7.5
0.05
84.2425
85.6379
7
7.0
0.15
67.8739
67.3957
8
7.0
0.15
65.1111
67.3957
9
7.0
0.15
68.6965
67.3957
10
7.5
0.15
27.9069
29.7779
11
7.5
0.15
29.7669
29.7779
12
7.5
0.15
32.1657
29.7779
While: P is the transmebrane pressure, Yexp and Ypred are experimental and predict recovery of bromelain.

Table 4 - Variance analysis of the empirical model.
Variation Source
Square Sum
Free Degree
Square mean
Fcalc
Ftab
Regression
5483.112
3
1827.704


Residual
19.885
8
2.486
735.32
4.07
Total
5522.024
11



% Explainable Variance =
99.29
Multiple Correlation (R2) =
0.9929


Equation 2 gives activity recovery of bromelain
2005b). Results showed that the transmembrane
enzyme (Y, %) as a function of factors x1 (pH) and
pressure effect was more than pH effect, hence the
x2 (transmembrane pressure) as a hyperplain
best operation region to bromelain enzymes
empirical model obtained by quadratic regression.
recovery by plain membrane separation process

was at 0.05 bar and pH 7.0 or 7.5. It was possible
Y (%) = ?200.421 + 41.8652 * pH + 5296.44 * P ? 780.672 * pH * P (2)
to obtain a concentrated bromelain solution, which

Figures 2 and 3 show the response surface and
was more resistant to spontaneous inactivation due
level curves to facilitate the understanding of
to proteolytic activity than the diluted solutions.
effects of the factors on bromelain activity
This facilitates it medicinal application and
recovery by surface response methodology. It
aggregate value.
showed above 70% of yield of activity recovery for
In a second step, the pre-concentrated extract was
pH 7.0. Lima et al. (2001) and Lopes (2005) also
purified by ultra-filtration using a 10 kDa millipore
reported that the optimal pH of bromelain was 7.0.
kit. This process eliminated the salts, glycosides,
The transmembrane pressure effect showed that an
other substances of small molecular weight (below
increase of operation pressure carried out a loss of
of 10 kDa) and part of water content. Thus, the
the enzymatic activity, because of the enzyme
total volume was reduced by 10 times after UF
inactivation by the rupture or modification of its
process, but activity was 100% recovered and
structure while passing through the membrane
enzyme was concentrated. In similar purification
pores or due to rude contact with fouling (see Fig.
process, Doko et al. (1991) working at 27,000 × g
2 and 3).
at 2–3°C observed that bromelain and proteolytic
Bromelain is much instable and frequently is
activity decayed during the processes caused by
deactivated spontaneously due to the action of
losses through the adsorption on the UF membrane
temperature, pressure, pH or inhibitors (Arroyo-
relative to the level of concentration reached. In
Reyna and Hernández-Arana, 1995; Hale et al.,
present study, apparently, the UF condition (4ºC
Braz. Arch. Biol. Technol. v.52 n.2: pp. 457-464, Mar/Apr 2009

---

462
Lopes, F. L. G. et al.
and 7,000 rpm per 20 min) retained the more
the multiple freeze–thaw cycles.
activity than Doko et al. (1991). Activity recovery
SDS-PAGE was used for observation of bromelain
obtained in this work was more than in ATPS
purity and it molecular weight determination (Fig.
using PEO–PPO–PEO block copolymers (Rabelo
4), which showed that the total protein into
et al., 2004) and adsorption chromatography in
purified extract was only the enzyme and that the
magnetic nanoparticles (Chen and Huang, 2004).
bromelain from A. comosus pulp had a molecular
According to Hale et al. (2005b), the proteolytic
weight of 24.5 kDa. This molecular weight was
activity of the concentrated bromelain solutions
approximated to the 31 kDa of the bromelain
remained relatively stable for at least one week at
reported in Martins et al (1992).
room temperature, with minimal inactivation by


Figure 2 - RSM for describes to influence of the transmembrane pressure and pH on bromelain activity recovery (Y).


Figure 3 - Levels curves for describing to influence of the transmembrane pressure and pH on bromelain activity recovery.



Figure 4 - Molecular weight determination by SDS-PAGE electrophoreses. SMW is standard of molecular
weight, it is compound of following proteins: phosphorylase b (94kDa), bovine serum albumin
(67kDa), ovalbumin (43kDa), carbonic anhydrase (30kDa), trypsin inhibitor (20.1kDa) and ?-
lactoalbumin (14.4kDa). UFB is the ultra-filtrated bromelain.
Braz. Arch. Biol. Technol. v.52 n.2: pp. 457-464, Mar/Apr 2009

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Concentration by Membrane Separation Processes of a Medicinal Product
463
CONCLUSION
Structure and Molecular Enzymology 1248 (2), 123-

128.
The yields of activity recoveries were high
Baldini, V. L. S. et al. (1993), Ocorrência da bromelina
e cultivares de abacaxizeiro. Colet. ITAL, 23(1), 44-
showing that it was possible to recovery the
55.
bromelain enzymes by plain membrane separation
Barros Neto, B.; Scarminio, I. S.; Bruns, R. E. (1995),
process and a hyper-plain was the best empirical
Planejamento e Otimização de Experimentos. v.1, 1ª
model to predict the experimental data of activity
edição, EDUNICAMP, Campinas – SP, Brazil, 1995,
recovery.
p 302.
It could concluded that pH 7.0 or 7.5 at 0.05 bar
Barros Neto, B.; Scarminio, I. S.; Bruns, R. E. (2001)
pressure were best for the activity recovery of
Como
Fazer
Experimentos:
Pesquisa
e
bromelain
enzyme
(approximately
90%).
Desenvolvimento na Ciência e na Indústria. v1, 2ª
Bromelain had a molecular weight of 24.5 kDa.
edição, EDUNICAMP, Campinas – SP, Brazil, 2001,
It could also be concluded that concentrated
406p.
Biazus, J. P. M.; Souza, A. G.; Santana, J. C. C.; Souza,
bromelain enzymes from pineapple juice, was
R. R.; Tambougi, E. B. (2005), Optimization Of
more resistant to spontaneous inactivation by their
Drying Process Of Zea Mays Malt To Use As
proteolytic activity than the diluted solutions. This
Alternative
Source
Of
Amylolytcs
Enzymes.
could facilitate their medicinal application.
Brazilian Archives of Biology and Technology, 48,

special, 185-190.

Bradford, M. M. (1976), A rapid and sensitive method
RESUMO
for the quantitation of microgram quantities of

protein. Utilizing the principle of protein-dye binding.
A concentração do suco de abacaxi é necessária
Anal. Biochem. 72, 248-254.
para manter a atividade da bromelina e padronizar
Cassano, A.; Drioli, E.; Galalaverna, G.; Marchelli, R.;
a composição e atividade proteolítica. Assim, este
Di Silvestro, R.; Cagnasso, P. (2003), Clarification
trabalho objetivou a obter um extrato de bromelina
and concentration of citrus and carrot juices by
integrated membrane processes. Journal of Food
pura do suco do Ananas comosus L. Merril por
Engineering, 57, 153-163.
processos de separação por membranas. Um
Chatsworth, O.
S. (1996), 5441740 Cosmetic
planejamento experimental do tipo 22 foi feito para
composition
containing
alpha
hydroxyacids,
estudar a influência do pH a da pressão
salicyclic acid, and enzyme mixture of bromelain and
transmembranar sobre a recuperação da atividade
papain. Biotechnology Advance, 14 (4), patent
por micro-filtração usando uma membrana plana.
abstract, 562.
Em uma segunda etapa, purificou-se a enzima alvo
Chen, D.-H. and Huang, S.-H. (2004), Fast separation
por ultra-filtração usando um “kit millipore” de 10
of bromelain by polyacrylic acid-bound iron oxide
kDa. A melhor condição para a concentração da
magnetic nanoparticles. Process Biochemistry, 39
bromelina foi a pH 7,5 e pressão transmembranar
(12), 2207-2211.
Dermirkan, E. S.; Mikami, B.; Adachi, M.; Higasa, T.;
de 0,05 bar, onde 85% da atividade da bromelina
Utsumi,
S.
(2005),
?-Amylase
from
B.
foi recuperado. A ultra-filtração manteve 100% da
amyloliquefacies: purification, characterization, raw
atividade proteolítica e concentrou em 10 vezes o
starch degradation and expression in E. col. Process
extrato de bromelina. A eletroforese via SDS-
Biochemistry, 40, 2629-2636.
PAGE mostrou que o ultra-filtrado teve alta pureza
Doko, M. B.; Bassani V.; Casadebaig J.;Cavailles L.
e a bromelina da polpa do Ananas comosus tem
and Jacob, M. (1991), Preparation of proteolytic
um peso molecular de 24.5 kDa.
enzyme extracts from Ananas comosus L., Merry

fruit
juice
using
semipermeable
membrane,

ammonium sulfate extraction, centrifugation and
REFERENCES
freeze-drying processes. International Journal of

Pharmaceutics, 76, 199-206.
Antón, M.; Diéguez, M.; García, A.; Rubio, G.; Díez,
Ferreira, G. B.; Evangelista, A. F.; Severo Junior, J. B.;
M. (2005), A case of beer and wheat allergy. Journal
Souza, R. R.; Santana, J. C. C.; Tambourgi, E. B. and
of Allergy and Clinical Immunology. 113 (2),
Jordão, E. (2007), Partitioning Optimization of
Supplement 1, S316.
Proteins from Zea mays Malt in ATPS PEG
Arroyo-Reyna, A. and Hernández-Arana, A. (1995).
6000/CaCl2. Brazilian Archives of Biology and
The thermal denaturation of stem bromelain is
Technology, 50 (3), 557-564.
consistent with an irreversible two-state model.
Hale, L. P.; Greer, P. K.; Trinh, C. T.; Gottfried, M. R.
Biochimica et Biophysica Acta (BBA) - Protein
(2005a), Treatment with oral bromelain decreases
Braz. Arch. Biol. Technol. v.52 n.2: pp. , Mar/Apr 2009

---

464
Lopes, F. L. G. et al.
colonic inflammation in the IL-10-deficient murine
Rodriguez-Nogales, J. R.; Roura, E.; Contreras, E.
model of inflammatory bowel disease. Clinical
(2005), Biosynthesis of ethyl butyrate using
Immunology, 116 (2), 135-142.
immobilized lipase: a statistical approach. Process
Hale, L. P.; Greer, P. K.; Trinh, C. T.; James, C. L.
Biochemistry, 40, 63–68.
(2005b), Proteinase activity and stability of natural
Sá, I. S; Cabral, L. M. C.; Matta, V. M. (2003),
bromelain
preparations.
International
Concentration of Pineapple Juice by Membrane
Immunopharmacology, 5(4), 783-793.
Separation Processes. Brazilian Journal of Food
Harrach, T; Garbin, F.; Munzig, E.; Eckert, K.; and
Technology, 6 (1), 53-62.
Maurer, H. R. (1994), P182 bromelain: An
Secor, Jr., E. R.; Carson Iv, W. F.; Cloutier, M. M.;
immunomodulator with anticancer activity. European
Guernsey, L. A.; Schramm, C.M.; Wu, C.A.; Thrall,
Journal of Pharmaceutical Sciences, 2 (1/2), 164.
R.S. (2005), Bromelain exerts anti-inflammatory
Howell, J. A.; Sanchez, V.; Field, R. W. (1993),
effects in an ovalbumin-induced murine model of
Membranes
in
bioprocessing:
theory
and
allergic airway disease. Cellular Immunology, 237
applications. 1st edition, Chapmam and Hall.
(1), 68-75.
Laemmile, U. K. (1970), Cleavage of structural proteins
Severo Junior, J. B.; Almeida, S. S.; Naraim, N.; Souza,
during the assembly of head of Bacteriophage T4.
R. R.; Santana, J. C. C.; Tambourgi, E. B. (2007a),
Nature, 227 (5259), 680-685.
Wine Clarification from Spondias lutea L. Pulp by
Lima, U. A.; Aquarone, E.; Borzani, W.; Schimidell,
Hollow
Fiber
Membrane
System,
Process
W. (2001), Biotecnologia industrial, processos
Biochemistry, 42 (11), 1516-1520.
fermentativos e enzimáticos. v.3, Edgard Blucher, São
Severo Júnior, J. B.; Oliveira, L. S. S.; Sardeiro, F.
Paulo-SP, Brazil, 595p.
S.;Souza, R. R.; Lopes, F. L. G.; Santana, J. C. C and
Lopes, F. L. G. (2005), Bromelain recovery from
Tambourgi, E. B. (2007b), Response surface
Ananas comosus L. Merril pulp, using the membrane
methodology to evaluation the recovery of amylases
separation processes. DSc Thesis, Campinas, Sp,
by hollow fiber membrane. Brazilian Archives of
Brazil. FEQ/UNICAMP.
Biology and Technology, 50 (4), 713-718.
Martin, Z. J. de et al. (1992), Processamento: produtos e
Sigma Chemical Co., Catalog. (1996), Biochemicals
subprodutos, características e utilização. In: Instituto
organic compounds and diagnostic reagents.
de Tecnologia de Alimentos. Abacaxi, cultura,
Vareniková, E., Betáková, T.; Mucha, V.; Soláriková,
matéria
prima,
processamento
e
aspectos
Kostolanský, L.F.; Waris, M.; Russ, G. (1995),
econômicos. 2 ed., Vol.2, Série Frutas Tropicais,
Preparation of monoclonal antibodies for the
Campinas: ITAL, p.165-219.
diagnosis of influenza A infection using different
Murachi, T. (1976), Bromelain enzyme. In: Methods in
immunization protocols. Journal of Immunological
Enzymology, ed. L. Lorand, Academic Press, New
Methods, 180 (1), 107-116.
York, 45, pp.475-585.
Wen, S.; Huang, T. H. W.; Li, G. Q.; Yamahara, J.;
Nirmala, M. and Muralikrishna, G. (2003), Three ?-
Roufogalis, B. D.; Li, Y. (2006),