J Food Sci Technol (November 2015) 52(11):7103–7112

DOI 10.1007/s13197-015-1812-5

ORIGINAL ARTICLE

Characteristic properties of crude pineapple waste extract

for bromelain purification by membrane processing
M. Z. M. Nor 1,2 & L. Ramchandran 1 & M. Duke 3 & T. Vasiljevic 1

Revised: 5 March 2015 / Accepted: 12 March 2015 / Published online: 22 March 2015
# Association of Food Scientists & Technologists (India) 2015

Abstract Bromelain enzyme can be extracted from pineapple
fruit including its waste parts. It is a valuable ingredient for
various markets especially in the food and pharmaceutical
areas. This enzyme can be extracted by membrane-based tech-
nology, but there is a lack of understanding of the properties of
the crude pineapple extracts to link to functional membrane
properties for efficient purification operation. This study fo-
cused on establishing characteristic properties of the crude
pineapple waste mixture (CWM) extract, constituting of
57 % peel, 28 % crown and 15 % core, as a source of the
enzyme and relevant to membr'ane processing. Rheological
properties at different temperatures and pH levels were also
determined in order to propose appropriate processing condi-
tions for a practical and an efficient membrane operation. The
CWM extract contained appreciable specific enzyme activity
of 394.9 CDU/mg protein that would need to be purified at
least 2–4 fold to achieve a required market standard. Existence
of polysaccharides, particularly pectin, in the extract would be
expected to cause fouling issues in membrane process, thus
pre-treatment may be required to remove this compound from
L. Ramchandran holds a PhD., Victoria University.
M. Duke holds a PhD., Victoria University.
T. Vasiljevic holds a PhD., Victoria University.
* T. Vasiljevic
todor.vasiljevic@vu.edu.au
1
Advanced Food Systems Research Unit, College of Health and
Biomedicine, Victoria University, PO Box 14428, Melbourne 8001,
Australia
2
Department of Process and Food Engineering, Faculty of
Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor,
Malaysia
3
Institute for Sustainability and Innovation, College of Engineering and
Science, Victoria University, PO Box 14428, Melbourne 8001, Australia
the mixture. The protein molecular weight of the CWM ex-
tract ranged between 11.7 and 26.9 kDa. The selection of
membrane molecular weight cut-off (MWCO) should be
above or below this range to isolate the enzyme. The evalua-
tion on rheological properties of the CWM extract showed
that it demonstrated lower viscosity at higher temperature
and at neutral pH. Therefore, selection on temperature of
20–25 °C and pH 7 throughout the membrane purification
operation has been recommended.
Keywords Pineapple waste . Bromelain . Proteolytic
enzyme . Rheology . Membraneprocess . Enzyme purification
Introduction
Pineapple (Ananas comosus) is one of the most popular fruits
in the world and is the member of Bromeliaceae family. Ac-
cording to the FAO online database, the world’s production of
pineapple in 2012 increased to 23.3 million tonnes from 21.5
million and 19.7 million tons in 2011 and 2010, respectively
(FAOSTAT 2014). Such an increase in production of pineap-
ple leads to simultaneous increase in waste generation due to
selection and removal of fruit components unsuitable for hu-
man consumption during processing. While presenting an en-
vironmental burden, the pineapple waste has fortunately been
identified as a potential source of valuable components such
as bromelain enzyme.
Bromelain is a crude enzyme mixture obtained from pine-
apple plant with the major protease present in the mixture are
known as stem bromelain (EC 3.4.22.32) and fruit bromelain
(EC 3.4.22.33) (Bala et al. 2012). This enzyme is widely used
in the pharmaceutical industry as a drug for treatment of in-
flammatory ailments, intestinal disorders, blood-coagulation-
related diseases and improved absorption of antibiotics
7104
(Maurer 2001). Bromelain has also been successfully used in
the food industry for meat tenderization, baking process, beer
clarification and as a food supplement (Bala et al. 2012). The
commercial production of this enzyme from the pineapple
consists of several processes such as extraction, purification,
drying and packing in the powder form. Among the related
processes, the purification stage is the crucial process, which
affects purity levels of the end product and the overall pro-
cessing cost. Purification of bromelain from the pineapple can
be performed using ion-exchange chromatography, reverse
micellar system, gel filtration, ammonium sulphate fraction-
ation, aqueous two-phase system, metal affinity membranes as
well as membrane filtration process (Bala et al. 2012). Of
these, membrane filtration is considered as one of the most
practical approaches due to feasibility, ease to scale up, waste
reduction during purification, and relatively lower cost than
other techniques.
The application of membrane filtration process either with
or without combination of other techniques for purification of
bromelain from pineapple extracts has been reported to yield
the enzyme with relatively high specific activity and purity.
Doko et al. (1991) obtained bromelain powder with 2.8 fold
purification by employing sequential batch membrane pro-
cessing that involved microfiltration (MF) and ultrafiltration
(UF) followed by ammonium sulphate extraction, ultracentri-
fugation and freeze drying. An increment of bromelain purity
from 5.9 to 8.9-fold was reported by Hebbar et al. (2012),
when integrating reverse miceller extraction with the ultrafil-
tration process. Additionally, Lopes et al. (2009) has achieved
high bromelain purity and recovery from a pineapple pulp
extract by combination of MF and UF processes. All these
reports so far more focused on the effort of increasing the
specific activity and purity of the enzyme without paying
much attention to the conditions of the membrane processing
itself. However, the design of a high efficiency membrane
separation system would require a better understanding of
the properties of the components in the crude pineapple
extract.
Thus, a study on the membrane processing conditions
would first involve the characterization of the incoming feed,
which in this particular study is the crude pineapple extract.
Each component of the extract would influence membrane-
solute interactions differently in terms of solute shape and
size, conformation, hydrophobic interactions and solute solu-
bility (Cheryan 1986). The existence of complex sugars such
as polysaccharides (Bartolomé et al. 1995) in the pineapple
extract may also affect the efficiency of the membrane purifi-
cation process since these polymers have a tendency to ag-
glomerate with proteins under different conditions such as pH,
temperature and shear stress. These interactions can lead to
severe fouling and consequently substantial permeate flux de-
cline. The fouling problem would affect the overall bromelain
production line by increasing the maintenance and operating
J Food Sci Technol (November 2015) 52(11):7103–7112
costs, due to the need for pre-treatment, high cross flow ve-
locity, or frequent membrane cleaning.
In this study, the physicochemical and rheological proper-
ties of the crude extracts from commercial pineapple of the
Smooth Cayenne variety are reported. Characterisation was
carried out on a crude extract known as crude waste mixture
(CWM) from the key waste components of the pineapple ob-
tained after removal of the flesh at the processing plant. The
CWM extract consisted of a specific ratio of non-edible
crown, peel and core parts. The objectives of the present work
were to establish chemical and physical properties of the
CWM extract that relate to separation of the bromelain en-
zyme by membrane processing, and based on these the results
to propose appropriate processing conditions for practical and
efficient membrane operation.
Materials and methods
Materials
Commercial grade pineapples (Ananus comosus L.) of
Smooth Cayenne cultivars were provided by a local supplier
(Werribee, Victoria, Australia). The Smooth Cayenne cultivar
was selected since it is commonly grown worldwide and is
easily obtainable in Australia. Materials used in the study in-
cluded casein, L-tyrosine, L-cysteine HCl, D-glucose, brome-
lain from pineapple stem, pectin from citrus fruit, phenol,
ethylene diaminetetraacetic acid (EDTA), potassium bromide
(KBr) and trichloroacetic acid (TCA), and were obtained from
Sigma-Aldrich (St. Louis, Missouri, USA). Ethanol was pur-
chased from Chem-Supply (Gillman, South Australia, Austra-
lia). Agilent Protein 250 kit which contains chips and reagents
was purchased from Agilent Technologies (Santa Clara, Cal-
ifornia, USA). All chemicals used in the experiment were
analytical grade.
Determination of fruit parts proportion and preparation
of crude waste extract extracts
The whole fruits were washed, air dried and then manually
peeled with a knife. The different parts (flesh, crown, skin and
core) were separated, weighed and reported as a percentage of
the proportion of a pineapple. A mixture of the crown, peel
and core parts in a specific ratio was prepared and blended
(8011ES, Waring, Torrington, Connecticut, USA) for 3 min
with an equal weight of cold MiliQ water. The resulting blend
was filtered through a cheese cloth and then centrifuged
(Avanti J-26S XPI, Beckman Coulter, Pasadena, California,
USA) at 10,000×g at 4 °C for 20 min (Ketnawa et al. 2012).
The obtained supernatant, named crude waste mixture
(CWM) extract, was used for further characterization of its
physicochemical and rheological properties. Crude pineapple
J Food Sci Technol (November 2015) 52(11):7103–7112
extracts from each individual pineapple parts were also been
prepared by following the same procedure and used as refer-
ences during the characterization study. The sample prepara-
tion was performed at a room temperature except during the
centrifugation process.
Fourier transform infrared analysis
The FTIR was carried out on powders made from pineapple
parts to identify the specific components of the pineapple
waste, based on their functional groups. Pineapple powders
were prepared from different pineapple parts by freeze drying
(FD300, Dynavac Engineering, Bayswater, Victoria, Austra-
lia) and grinding (Waring, USA). The spectral analysis for all
samples was carried out using a diffuse reflectance accessory
(Valu-Line EasiDiff, Pike Technologies, Madison, Wisconsin,
USA) in an FTIR instrument (IRAffinity – 1, Shimadzu, Kyo-
to, KYT, Japan) to detect characteristic chemical functional
groups. All spectra recorded were an average of 20 scans in
the absorbance mode with a 4 cm−1 resolution in the range of
600–4000 cm−1. All samples were mixed with ground potas-
sium bromide and placed in the macro sampling cup accesso-
ry. The spectrum presented were smoothed (13–21 smoothing
points) using an IR Solution software (Shimadzu).
Determination of pH and total solid
The pH measurement of all extracts was accomplished by
using a pH meter (InoLab pH7110, WTW, Weilheim in
Oberbayern, Bavaria, Germany). The total solid (TS) in the
extracts was measured as reported previously (Ranganna
1986). Approximately 5 g of sample was evaporated on a
boiling water bath and dried in an oven at 105 °C until con-
stant weight was obtained. The total solids content was report-
ed as a percentage of the dried weight over the initial weight of
the sample.
Determination of total sugar
Total sugar in the crude pineapple extracts was determined
according to the method of Dubois et al. (1956). A known
quantity of aliquots of the extract was made up to 1 ml by
adding distilled water and 1 ml of 5 % phenol was added and
mixed thoroughly. Subsequently, 5 ml of a concentrated
sulphuric acid (98 %) was added and the mixture allowed to
stand for 10 min. The mixture was then placed in a water bath
for 20 min at 20 °C. Yellow to orange colour developed was
read at 480 nm using a spectrophotometer (Libra S12,
Biocrom, Holliston, Massachusetts, USA) and concentration
of glucose was calculated using a standard curve of D-glucose
with a concentration range from 20 to 100 mg/L.
7105
Determination of protein content
Total nitrogen content in the crude pineapple extracts was
determined using a Total Organic Carbon (TOC) anaylzer
with Total Nitrogen (TN) detector (TOC-V, Shimadzu, Kyoto,
KYT, Japan). Sample preparation involved dilution to below
50 mg/L of nitrogen. Standard solution of 50 mg/L potassium
nitrate (KNO3) was used to confirm the original calibration.
The result was displayed as total nitrogen and conversion
factor of 6.25 was used to calculate the protein content from
the obtained value. This method is comparable to the Kjeldahl
method for protein determination (Hausmann et al. 2013).
Determination of proteolytic activity and its specific
activity
The proteolytic activity of the crude pineapple extracts was
determined by the casein digestion unit (CDU) assay, using
casein and L-tyrosine as substrate and standard, respectively
(Murachi 1970). The extract was initially diluted to an appro-
priate concentration (5–16 dilution factors) with an enzyme
diluent consisting of 0.03 M L-Cysteine HCL and 0.006 M
EDTA. An aliquot of this diluted extract (1.0 ml) was then
mixed with 5.0 ml of a casein substrate containing 0.6 % (w/v)
of casein in a phosphate buffer at pH 7.0. The reaction was
carried out at 37 °C for 10 min and was stopped by the addi-
tion of 5 ml of TCA (5 % (w/v). The reaction mixture was then
centrifuged at 8000×g for 10 min (Beckman Coulter, USA).
The absorbance of the supernatant was read at 275 nm using a
spectrophotometer (Biocrom, USA). One unit of protease ac-
tivity was defined as the amount of enzyme required to release
a product equivalent to 1 μg of tyrosine in 1 min per ml of
sample under the standard assay conditions and expressed as
CDU/ml. The specific activity of the enzyme was calculated
using Eq. 1 and expressed as CDU per mg of protein.


CDU Proteolytic activity
Specific activity ¼ ð1Þ
mg Protein content
Determination of particle size distribution
Particle size of the samples was determined by using a particle
size analyzer (Zetasizer 2000, Malvern Instruments, Malvern,
Worcestershire, U.K.). The refractive index for all crude pine-
apple extracts (ranging from 1.337 to 1341) was pre-
determined using a refractometer (Abbe no.302, Atago,
Minato-ku, Tokyo, Japan) and a value of 1.33 was used for
the water dispersant. Viscosity of the samples was following
the water viscosity of 1.0031 cP. The measurement was car-
ried out at 20 °C and at scattering angle of 173°. All samples
were measured using square polystyrene cuvette (DTS0012).
7106
Preparation of pectin powder derived from CWM extract
Preparation of pectin powder from the CWM extract was ob-
tained following the method of Guo et al. (2012) with minor
modifications. A volume (3 L) of the CWM extract was pre-
pared from 6 fruits and the pH was adjusted to 1.5 using 0.5 M
HCl. The mixture was then heated and kept at 80(±2) °C with
continuous stirring for 1 h using a water bath oscillator. The
hot mixture was then filtered through a Whatman no. 4 filter
paper by vacuum filtration; and the filtrate was collected and
cooled to a room temperature. The filtrate was mixed with an
equal volumes of 95 % (v/v) ethanol and kept undisturbed
overnight at 4 °C to precipitate pectin. The precipitated pectin
was separated by centrifugation at 8000×g for 10 min
(Beckman Coulter, USA) and then washed three times using
95 % ethanol to remove the monosaccharides and disaccha-
rides. After purification, the wet pectin was dried at 40 °C in a
drying oven until constant weight was observed, followed by
grinding to powder. The pectin yield was calculated as fol-
lows.
 .  Final dried pectin weight
Pectin yield %; w v ¼
Extract volume
 100 ð2Þ
Protein sizing
The proteins present in the CWM and other crude pineapple
extracts were separated according to their molecular weight
using a lab-on-a-chip technology (2100 Bioanalyzer, Agilent
Technologies, California, US) with a Protein 250 LabChip kit.
All chips were prepared according to the protocol provided by
the manufacturer and a specific protein ladder was used as a
standard. Initially, samples and ladder were labelled with a dye
solution and incubated in ice for 30 min. They were then
diluted with water in a ratio of 1:200, mixed with a denatur-
ation buffer and heated up at 100 °C for 5 min to denature
them. Subsequently, the samples and ladder were pipetted into
chip wells. The chip was placed in the bioanalyzer and the
assay was started within 5 min. Each sample was sequentially
separated in the separation channel and detected by a laser-
induced fluorescence detector (670–700 nm). All the process
was controlled using Agilent 2100 Expert Software and the
results of the analysis were presented as quantitative profiles
and also as simulated gel-electrophoresis patterns.
Measurement of rheological properties
The rheological properties of the samples were established
using a rheometer (MCR 301, Anton Paar, Graz, Austria) with
a double gap cylinder geometry (DG.26.7/Q1, Anton Paar).
The temperature was controlled by a Peltier system (Anton
J Food Sci Technol (November 2015) 52(11):7103–7112
Paar GmbH). The data of the rheological measurements were
analysed with a supporting Rheoplus rheometer software
(Version 2.3, Anton Paar). The rheological behaviour of the
pineapple extracts was determined with the increment of shear
rate from 0 to 500 s−1 which is mostly applied in juice industry
(Shamsudin et al. 2009). The temperature during all determi-
nations was maintained at 20±0.1 °C apart from the evalua-
tion of rheological properties at different temperatures. Data
from the experiments were fitted to existing rheological
models including Newtonian, Bingham and Herschel-
Bulkley. These models are presented as:
Newtonian : σ ¼ μ⋅γ̇ ð3Þ
Bingham : σ ¼ σ0 þ μ0 ⋅γ̇ ð4Þ
Herschel−Bulkley : σ ¼ σ0 þ k⋅γ̇ ð5Þ
Where σ is the shear stress (Pa), γ̇ is the shear rate (s−1), η is
the viscosity (Pa.s), η′ is the plastic viscosity, σo is the yield
stress (Pa), k is the consistency index (Pa1/2sn) and n is the
flow behaviour index.
All samples examined for the rheological properties were
adjusted to a total solid content of 2.8 % to eliminate concen-
tration influence; and on the CWM extract under different
temperature and pH conditions. Evaluation of the effects of
the temperature on the rheological properties of the CWM
extract was achieved by adjusting the temperature controller
of the rheometer to 5, 10, 15, 20 or 25 °C. Effects of different
pH level on the rheological properties of the CWM extract
was assessed at 1, 4, 7 or 10. The pH of 30 ml CWM extract
was adjusted with either 0.5 M HCl or 1.0 N NaOH and final
volume of the samples was then made up to 35 ml with MilliQ
water.
Statistical analysis
All tests were carried out in triplicate with at least one sub-
sampling. The results were analysed by either one-way or
two-way analysis of variance (ANOVA) using Statistical
Analysis System (SAS) software (Version 9.2, SAS Institute,
USA). Significant differences between mean were compared
by t-test at 5 % level of significance.
Results and discussion
Proportion and FTIR spectra of pineapple parts
Table 1 shows the results of the different proportion of the
pineapple parts. Non-edible parts of the commercial grade
Smooth Cayenne pineapple consisting of a crown, peel and
core, which are considered waste, represent almost half of the
total fruit proportion. The waste percentage of the pineapple in
J Food Sci Technol (November 2015) 52(11):7103–7112
Table 1 Proportion percentage of commercial Smooth Cayenne
pineapple parts
Pineapple parts Fruit proportion (%) Waste proportion (%)
Flesh 51.2±4.6 a
−
Crown 13.9±3.7b 28.3±5.9a
Peel 27.8±3.4c 57.0±4.8b
Core 7.0±1.6d 14.7±4.6c
Different letters in the same column indicate the significant differences
(p<0.05)
our study was close to the waste percentage of 50 % (w/w) and
58 % (w/w) in Nang Lae and Phu Lae cultivars, respectively,
reported by Ketnawa et al. (2012). The current progress in
pineapple industry has led to a waste generation due to selec-
tion and removal of the components unsuitable for human
consumption including the skins, peels, crowns, cores, pom-
ace of the fruit, leaves and other non-edible parts. These parts
are prone to microbial spoilage, which leads to an environ-
mental issue, thus their utilization for production of certain by-
products such as bromelain enzyme and dietary fibre appears a
plausible approach that would generate additional profit for
the industry and at least partially alleviate a problem of the
waste disposal.
The peel constituted the largest portion of total waste
(57 %, w/w) following by the crown and core (28 and 15 %,
w/w, respectively). These proportions were used in the prepa-
ration of crude waste mixture (CWM) extract for subsequent
characterization analyses. The CWM was prepared to repre-
sent a real form of raw materials obtained in the pineapple
industry, which would normally be all mixed together without
sorting process. Hence, the CWM extract was analyzed in
order to provide a general understanding of this complex sys-
tem and generate suggestions for utilization of all the waste in
the pineapple industry for bromelain production.
FTIR has been used to assess the composition of the pine-
apple parts due to its non-invasive nature. FTIR spectra of
pineapple powders and commercial bromelain are shown in
Fig. 1. Bromelain can be characterised by the presence of
peaks corresponding to C-N group (1149–1179 and 1255–
1290 cm−1), amide I group (1600–1690 cm−1), C=O group
(1640–1700 cm−1) and NH- group (3338–3380 cm−1) as re-
ported by Soares et al. (2012) which were observed in our
spectra as well. All the prepared pineapple powders showed
similar spectrum as the commercial bromelain powder indi-
cating the presence of bromelain in all the prepared samples
tested.
Additionally, the spectra in Fig. 1 also show the existence
of peaks characteristics of polysaccharide that are identified
by an intense C-O stretching bands between 1000 and
1040 cm −1 , COO- group (1400 cm −1 ) and C-H group
(2800–3000 cm−1) (Gnanasambandam and Proctor 2000;
7107
2
1.5 (v)
Absorbance
(iv)
1
(iii)
(ii)
0.5
(i)
0
3600 2600 1600 600
Wavenumber (cm-1)
Fig. 1 FTIR of commercial bromelain and freeze-dried pineapple pow-
ders; (i) commercial bromelain derived from pineapple stem; (ii) flesh;
(iii) crown; (iv) peel; (v) core
Saha et al. 2007). This confirmed that the prepared sampled
contained both bromelain and polysaccharides.
Physicochemical properties of crude pineapple extracts
Feed properties of the crude pineapple extracts would drive
direction in terms of appropriate membranes configurations,
membrane materials, pore sizes, operating parameters and
cleaning methods for the bromelain purification. Various
chemical properties of the crude pineapple extracts are pre-
sented in Table 2. In general, the extracts obtained from dif-
ferent parts of the pineapple varied in their composition. The
overall properties of the CWM extract were clearly similar to
the peel extract, which was likely related to its proportion, as
more than a half of the mixture was made up of the peel.
The pH of CWM was 4.08, and was close to the average
pH of individual crude pineapple extracts which had pH rang-
ing from 3.49 to 4.25. The low pH in the pineapple is due to a
high acidity caused by citric and malic acid content
(Bartolomé et al. 1995).
Proteins and bromelain in particular, are present in the pine-
apple parts in different proportions in various concentrations
and activities as shown in Table 2. Among the extracts, the
flesh and crown parts showed the highest specific enzyme
activity of 561 and 520 CDU/mg protein, respectively, while
the core extract had the lowest specific enzyme activity (81
CDU/mg protein). Such a wide variation in specific enzyme
activity among these extracts is likely influenced by different
types of proteolytic enzymes present in the pineapple
(Ketnawa et al. 2012). At least eight distinct proteolysis active
compounds of similar molecular mass, including fruit brome-
lain, stem bromelain, ananain and comosain have been iden-
tified in the pineapple plant extract (Bhattacharyya 2008).
In our study, the CWM extract showed considerable spe-
cific enzyme activity of 395 CDU/mg protein, which demon-
strated its potential for further utilization for bromelain
7108 J Food Sci Technol (November 2015) 52(11):7103–7112

Table 2 Characteristics of the crude pineapple extracts

Crude pineapple pH Protein Enzyme Specific activity Total sugar Total solid
extract (mg/ml extract) (CDU/ml extract) (CDU/mg protein) (mg/ml extract) (%, w/w)

CWM* 4.08±0.02a 1.75±0.02a 689.8±7.7a 394.9±1.4a 26.06±0.86a 4.03±0.23a

Flesh 3.49±0.01b 1.02±0.04b 573.6±12.2b 561.3±33.9b 52.74±1.61b 6.16±0.37b
Crown 4.25±0.00c 2.13±0.03c 1106.1±57.8c 519.5±34.3b 17.08±1.44c 2.82±0.24c
Peel 4.00±0.02d 1.37±0.04d 429.7±35.8d 313.9±17.9c 24.70±1.41a 4.48±0.24a
Core 3.71±0.03e 0.94±0.02e 76.6±2.9e 81.8±4.9 51.03±2.66b 6.14±0.12b

Different letters in the same column indicate the significant differences (p<0.05). *CWM extract was prepared from the combination of 57 % peel, 28 %
crown and 15 % core parts respectively

extraction. Most of the commercial applications of bromelain
do not require a high purity of the enzyme and therefore, the
purification process can be designed depending on the desti-
nation of the bromelain (Costa et al. 2014). For food industry
applications, the bromelain powder is commercially available
in a specific enzyme activity ranging from 750 to 1600 CDU/
mg protein (Biozym 2014) and even in a higher range for
pharmaceutical and research applications. Thus, the CWM
extract requires at least 2 to 4 fold purification for commer-
cialization purpose, which may be achieved by using mem-
brane filtration processes (Doko et al. 1991; Hebbar et al.
2012; Lopes et al. 2009).
However membrane filtration must also consider the ef-
fects of other components that may compromise performance,
for example polysaccharides, which may interfere with mem-
brane operation. Significant membrane fouling was reported
in sugarcane juice clarification using UF due to interactions
between polysaccharide and membrane (Saha et al. 2006). In
the current study, the existence of polysaccharides and other
carbohydrate components in the CWM and other crude pine-
apple extracts was established by determining the amount of
total sugars and total solids (TS) in different pineapple parts
(Table 2). As shown in Fig. 1, their presence was already
observed by FTIR. The CWM extract contained considerable
amounts of high molecular weight sugars. The mixture had
26 mg/ml of total sugar and 4 % (w/w) TS, most of which
originated from the core portion. The core extract exhibited
1000 nm). This particle distribution in the flesh and core ex-
tracts is reflective of the presence of larger molecules such as
polysaccharides which is in agreement with the total sugar and
TS results in Table 2, which may also exist in the CWM
extract. The presence of polysaccharides in the extract may
result in formation of high molecular weight aggregates re-
sponsible for membrane fouling, which in turn may hinder the
membrane performance.
Since the presence of high molecular weight polysaccha-
rides in the extract was indicated by findings in Fig. 1 and
Table 2, isolation of crude pectin from the CWM extract was
performed for further characterization. The yield of crude pec-
tin extracted from the CWM extract was fairly low at 0.06 %
(w/v). The FTIR characterization of the CWM pectin is pre-
sented in Fig. 3 and was compared to commercial pectin de-
rived from the citrus fruit. There are five characteristic peaks
in this spectrum that can be used to identify pectin which
includes R-O-R bond (1100 cm−1), C-C bond (1200 cm−1),
COO- stretching (1650 cm−1), COO-R bond (1750 cm−1) and
C-H bond (2800–3000 cm−1) (Monsoor et al. (2001). The
presence of the typical peaks for pectin (Fig. 3) confirmed
the presence of pectin in the CWM extract.
Various approaches such as feed pre-treatment, optimiza-
tion of operating conditions and selection of different
40
Volume (%)

35 15 (i) (ii)

the highest amount of total sugar and total solid content 30 10

(iii)

51 mg/ml and 6.14 %, respectively, compared to the other (iv)

Volume (%)

25 5
waste extracts, which was in agreement with a previous report
(Ketnawa et al. (2012). 0
20 5 15 25 35 45 55
Membrane processes involve separation of principal com- Size (d.nm)
15
ponents based on size exclusion. These particles would pre-
10 (iv)
sumable composed of the proteins and polysaccharides pres-
ent in the crude pineapple extracts. Particle size distributions 5
observed in the crude pineapple extracts is shown in Fig. 2.
0
Particle size distribution of the CWM extract varied between 3 1 10 100 1000 10000
and 1000 nm, with irregular peaks between 10 and 100 nm. In Size (d.nm)
comparison, the flesh extract had a larger size distribution Fig. 2 Particle size distribution of the crude pineapple extracts; (i) crown;
from 20 to 1500 nm followed by the core extract (20– (ii) peel; (iii) CWM; (iv) core; (v) flesh
J Food Sci Technol (November 2015) 52(11):7103–7112
2.0
1.5
Absorbance
(ii)
1.0
0.5 (i)
0.0
3600 2600 1600 600
Wavenumber (cm-1)
Fig. 3 FTIR of pectin powder (i) commercial pectin derived from citrus
fruit; (ii) pectin derived from CWM extract
membrane materials can be chosen in order to reduce the
fouling caused by polysaccharide component in the mem-
brane process (Saha et al. 2006). The application of emerging
ceramic membrane in the filtration process is believed to re-
duce the fouling caused by polysaccharides compared to con-
ventional polymeric membranes (Finley 2005). Besides that,
some studies have reported on success with using pectinase
enzyme to hydrolyze both high and low esterified pectin in
fruit juice that would reduce the juice viscosity and improve
membrane filtration process including in the guava juice pro-
duction (Akesowan and Choonhahirun 2013). If this option is
chosen as a pre-treatment, selection of pectinase enzyme with
higher molecular weight than the bromelain is important so
that it can be separated from the extract during UF process and
might be recycled back in the system.
Electrophoretic analysis of proteins present in the CWM
and individual crude pineapple extracts was performed for
determination of a molecular weight cut-off (MWCO) selec-
tion of the membrane filters in the bromelain purification pro-
cess. The results in Fig. 4 show the protein patterns of all the
crude pineapple extracts in comparison with a commercial
stem bromelain as a reference (lane 6). In general, most of
the proteins in all the crude pineapple extracts had different
molecular weights (MW) of either 11.7, 19.6 and 26.9 kDa
with the CWM extract was having all the bands (lane 5).
Different parts of the pineapple contained different MW of
the protein similarly to the report by Ketnawa et al. (2012)
who found proteins with MW ranging of 18 to 39 kDa from
crude extracts of different pineapple parts. The commercial
stem bromelain showed MW of 27.1 kDa (lane 6) indicating
the differences between the crude and the purified bromelain
extract. Several authors characterized the crude and purified
bromelain as having molecular weights ranging from 14.9 to
39.2 kDa (Chaurasiya and Hebbar 2013; Costa et al. 2014;
Hebbar et al. 2012; Ketnawa et al. 2012; Soares et al. 2012).
The variation in the molecular weight of the enzyme might be
due to differences in the bromelain source e.g. peel, fruit, stem
7109
Fig. 4 Protein patterns of the crude pineapple extracts; lane 1: flesh; lane
2: crown; lane 3: peel; lane 4: core; lane 5: CWM and lane 6: commercial
bromelain from stem
and core; and other parameters such as pineapple species, age,
harvesting and size (Bresolin et al. 2013; Chaurasiya and
Hebbar 2013).
The CWM extracts would be filtered by microfiltration
(MF), where the permeate would then be fed to ultrafiltration
(UF) to concentrate bromelain. This strategy is known as 2-
stage membrane filtration and can be used for protein separa-
tion (Datta et al. 2009). The selection of MWCO should be
above and below a specific protein size range. For bromelain
purifcation, the 2-stage membrane filtration were performed at
different size range of 0.1–8 μm and 5–10 kDa of the MF and
UF, respectively (Doko et al. 1991; Hebbar et al. 2012; Lopes
et al. 2009).
Based on the current result, the membrane process should
have MWCO above 30 kDa to first pass the bromelain and
filter remaining larger molecules. The permeate of the MF is
then fed to a membrane with pore size below 10 kDa for
protein capture and concentration in the retentate. Commercial
membranes with pore sizes as small as 10 kDa are available
from either polymeric or ceramic materials for this purpose.
For instance, a range of pore sizes is offered by Pall Corpora-
tion for its Schumasiv ceramic membrane series which have
high mechanical strength, good thermal resistance and high
separation efficiency (Heidenreich 2011).
Rheological characteristics of crude pineapple extracts
Table 3 shows the fitted parameters of Newtonian, Bingham
and Herschel-Bulkley rheological models for all the crude
pineapple extracts at 20 °C with standardized total solid (TS)
at 2.82 % (w/w). Viscosity of the feed is an important property
7110 J Food Sci Technol (November 2015) 52(11):7103–7112

Table 3 Data fitted to parameters of rheological models of the crude pineapple extracts with standardized 2.82 % (w/w) total solid

Crude pineapple extract Newtonian Bingham Herschel-Bulkley

η R2 σo η′ R2 σo k n R2

CWM* 0.0011a 0.9971 0.0014a 0.0012a 0.9984 0.0014a 0.0013a 0.9696a 0.9998
a
Flesh 0.0011 0.9965 0.0014a 0.0011a 0.9984 0.0010a 0.0015a 0.9518a 0.9996
Crown 0.0011a 0.9980 0.0011a 0.0011a 0.9987 0.0011a 0.0013a 0.9736a 0.9999
Peel 0.0011a 0.9966 0.0013a 0.0011a 0.9979 0.0012a 0.0013a 0.9660a 0.9998
Core 0.0011a 0.9977 0.0010a 0.0011a 0.9986 0.0010a 0.0013a 0.9662a 0.9997

Different letters in the same column indicate the significant differences (p<0.05). *CWM extract was prepared from the combination of 57 % peel, 28 %
crown and 15 % core parts respectively

affecting the flow behaviour in the membrane processing
(Cheryan 1986). Thus, it is important to characterize the rhe-
ological properties of the CWM and other crude extracts since
the feed viscosity in process stream effects mass transfer and
pressure drop of the membrane filtration. The viscosity (η)
values of the CWM extract and other crude extracts were
similar (p>0.05) at low values among each other and were
maintained at satisfactory high R2 for Newtonian model.
Thus, the result described the behaviour of the CWM and
other pineapple extracts were comparable to Newtonian fluid.
The Newtonian behaviour of the extracts may be attributed to
the low molar mass of the extracts since the rheological be-
haviour of fluids depends on their molecular structures
(Belibağli and Dalgic 2007). Low feed viscosity will directly
influence better flux performance in membrane process espe-
cially for the UF and MF systems. The results were also sim-
ilar (p>0.05) in terms of yield stress (σo), plastic viscosity (η′),
consistency index (k) and flow behaviour index (n) among all
extracts. The best rheological model for describing the flow
behaviour of the extracts was selected by comparing the value
of coefficient of determination, R2. The experimental results
showed the Herschel-Bulkley model fitted the best R2 value in
the range of 0.9996–0.9999.
To further understand the rheological behaviour of the
CWM extracts, evaluation of the rheological properties was
performed at different temperatures and pH levels. Table 4
shows the parameters of the Newton, Bingham and Her-
schel–Bulkley models along with the correlation coefficient
(R2) for each flow model. The rheological models show that
CWM extract had a significantly (p<0.05) lower viscosity (η)
at a higher temperature. Viscosity decline associated with a
rise in temperature has been contributed to thermal expansion
of the molecules, which increases the intermolecular distances
and consequently a reduction in viscosity (Kumoro et al.
2009). Viscosity variations with temperature has been report-
ed for pineapple juice extracted from Josapine variety and was
described by the Arrhenius relationship especially at a higher
concentration (Shamsudin et al. 2009). Available literature
reports focused on investigating the effects of temperature
on rheological properties of pineapple juice; however there
are no reports on CWM extract. Nevertheless, these studies
can be used as a base to analyse our findings.

Table 4 Data fitted to parameters of rheological models of the CWM extract at different temperature and pH conditions

Conditions Newtonian Bingham Herschel-Bulkley

η R2 σo η′ R2 σo k n R2

Different temperatures
5 °C 0.0018a 0.9992 0.0017a 0.0018a 0.9997 0.0017a 0.0018a 0.9919a 0.9999
10 °C 0.0015b 0.9998 0.0015a 0.0015b 0.9999 0.0015a 0.0014b 1.0032ab 0.9999
15 °C 0.0013c 0.9998 0.0016a 0.0013c 0.9999 0.0016a 0.0012c 1.0066ab 0.9999
20 °C 0.0012d 0.9995 0.0017a 0.0012d 0.9998 0.0017a 0.0012c 1.0108b 0.9998
25 °C 0.0010e 0.9993 0.0012a 0.0010e 0.9998 0.0012a 0.0010d 1.0142b 0.9997
Different pH
pH1 0.0013a 0.9995 0.0026a 0.0012a 0.9999 0.0025a 0.0013a 0.9964a 0.9999
pH4 0.0013a 0.9951 0.0031a 0.0012a 0.9981 0.0026a 0.0015a 0.9580a 0.9999
pH7 0.0011b 0.9997 0.0015b 0.0011b 0.9997 0.0015b 0.0010c 1.0198b 0.9997
pH10 0.0011b 0.9999 0.0013b 0.0011b 0.9997 0.0013b 0.0010c 1.0202b 0.9997

Different letters in the same column indicate the significant differences (p<0.05)
J Food Sci Technol (November 2015) 52(11):7103–7112
In membrane processing, higher temperature would gener-
ally result in higher flux due to lower viscosity of the stream in
pressure-controlled and mass transfer-controlled regions
(Cheryan 1986). It also important to consider in the case of
pineapple extracts, high temperature may lead to protein de-
naturation which would not only reduce the bromelain activity
but also potentially cause fouling phenomena by particle-
particle interactions. Although the optimum temperature for
the highest bromelain activity appeared to be in the range
between 50 and 70 °C (Ketnawa et al. 2012; Silvestre et al.
2012), a 17 % loss of the enzyme activity has been reported
when it was exposed to 50 °C for 60 min (Jutamongkon and
Charoenrein 2010). In contrast, the proteolytic activity of con-
centrated bromelain solutions remains relatively stable for at
least 1 week at room temperature (Hale et al. 2005). The
current study suggested in applying processing temperature
of 20–25 °C in the membrane purification process in regards
to the low feed viscosity for better flux performance, and to
reduce the possibility of protein denaturation as well as the
processing utility cost.
Solute-membrane and solute-solute interactions are also
influenced by environmental pH. Table 4 indicates the
CWM extract had significantly (p<0.05) higher viscosity (η)
at its natural pH (pH4) and pH 1 in comparison to that obtain-
ed at a more alkaline pH (7 and 10). Such rheological charac-
teristics of the extract were attributed to its components re-
sponsible for the microstructure, related to the zeta potential
of the extract. Stem and fruit bromelain are reported to have an
isoelectric point (IEP) of pH 9.5 and 4.6, respectively
(Chaurasiya and Hebbar 2013). In the current study, the pI
of the CWM extract was determined at pH 2.37 indicating a
mixture of bromelain with other components. At pH near to its
pI, particles in the extract tend to agglomerate to each other
due to neutral surface charge among them. Thus, changing the
pH of the extracts away from its isoelectric point will lead to
repulsion between the particles and hence, reducing the
viscosity.
The feed pH is an important factor in protein separations
through membrane process. Beside influencing the viscosity,
the pH of the feed can also affect electrical charge on both the
protein and the membrane due to the ionization or deioniza-
tion of various acidic/basic groups on the protein and mem-
brane surface (Burns and Zydney 1999). This can cause either
attractive or repulsive response in the protein-membrane in-
teraction based on their isoelectric point. The isoelectric point
of a ceramic membrane coated with zirconium dioxide (ZrO2)
is at pH 6.3–7.1 while for a polymeric membrane is in a range
of pH 4–5 (Hofs et al. 2011). Better feed-membrane electro-
static interaction during the UF process is expected if the pH
of the CWM extract is adjusted above its pI and below the
membrane’s pI resulting in desired flux.
Lopes et al. (2009) reported the best pH condition for bro-
melain concentration using membrane process was at pH 7–
7111
7.5 with approximately 85–100 % enzyme activity recovery.
The pH range between 6 and 7 was also reported to be the
optimal values of the bromelain specific activity (Silvestre
et al. 2012). Thus, altering the CWM extract to pH 7 might
be a good alternative in order to optimize the membrane pu-
rification process and to obtain the best end product.
Conclusions
The CWM extract has been prepared from 57 % peel, 28 %
crown and 15 % core according to the pineapple proportion.
With a specific enzyme activity of 394.9 CDU/mg protein, the
CWM extract should be purified at least 2–4 folds for com-
mercialization purpose. Elimination of polysaccharide, in par-
ticular pectin, in the CWM extract by physical or chemical
means was proposed prior to the membrane purification pro-
cess to minimize the fouling issue. The membrane used in the
purification process of the extract should have a molecular
cut-off (MWCO) above 30 kDa or below 10 kDa for protein
selectivity as permeate or as retentate, respectively. Based on
the rheological properties evaluation, the current study sug-
gests that the membrane-based bromelain purification process
should be performed at temperature of 20–25 °C and at pH 7
of the extract.
Acknowledgments The authors gratefully acknowledge the Ministry
of Education Malaysia and Universiti Putra Malaysia (UPM) for provid-
ing the PhD scholarship for M.Z.M. Nor.
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