MINI-REVIEW
Bromelain: an overview of industrial application
and purification strategies
Zatul Iffah Mohd Arshad & Azura Amid & Faridah Yusof &
Irwandi Jaswir & Kausar Ahmad & Show Pau Loke
Received: 19 March 2014 /Revised: 8 June 2014 /Accepted: 10 June 2014 /Published online: 26 June 2014
#
Springer-Verlag Berlin Heidelberg 2014
Abstract This review highlights the use of bromelain in
various applications with up-to-date literature on the purifica-
tion of bromelain from pineapple fruit and waste such as peel,
core, crown, and leaves. Bromelain, a cysteine protease, has
been exploited commercially in many applications in the food,
beverage, tenderization, cosmetic, pharmaceutical, and textile
industries. Researchers worldwide have been directing their
interest to purification strategies by applying conventional and
modern approaches, such as manipulating the pH, affinity,
hydrophobicity, and temperature conditions in accord with
the unique properties of bromelain. The amount of down-
stream processing will depend on its intended application in
industries. The breakthrough of recombinant DNA technolo-
gy has facilitated the large-scale production and purification of
recombinant bromelain for novel applications in the future.
Keywords Bromelain
.
Cysteine protease
.
Purification
.
Recombinant bromelain
Introduction
Modern technological advancements have raised the protease
production industrially worldwide. Today, proteases dominate
with approximately 60 % market share of the total enzyme
market worldwide where the major producers are Novo
Industries, Gist-Brocades, Genencor International, and Miles
Laboratories (Feijoo-Siota and Villa 2011). This growth is
entrenched with the rising general awareness of the need to
protect the environment from the impact of chemical indus-
trialization. Recently, bromelain has drawn attention in di-
verse industrial applications owing to its properties and higher
commercial values (Heinicke and Gortner 1957). Bromelain is
a protease derived fr om the stem and fruit of pineapples
(Ananas comosus). Stem bromelain (EC 3.4.22.32), ananain
(EC 3.4.22.31), and comosain are extracted from pineapple
stems, while fruit bromelain (EC 3.4.22.33) is mainly from
fruit juice (Rowan et al. 1990). Similar proteases are also
present in pineapple peel, core, crown, and leaves, with the
highest proteolytic activity and protein contents detected in
the extract of pineapple crown (Ketnawa et al. 2012).
Stem bromelain can be isolated from stem juices by means
of precipitation and centrifugation (Devakate et al. 2009;
Heinicke and Gortner 1957). Moreover, using chromato-
graphic methods, other basic proteolytic (ananain, comosain,
F4, F5, and F9) and acidic components of stem bromelain
fraction A (SBA/a) and fraction B (SBA/b) have been partially
and fully purified (Feijoo-Siota and Villa 2011; Harrach et al.
1995, 1998; Murachi et al. 1964;Napperetal.1994;Rowan
et al. 1990;Wharton1974). The molecular weight of purified
stem bromelain is 23.40–35.73 kDa, fruit bromelain
31.00 kDa, ananain 23.43–23.42 kDa, and comosain 23.56–
24.51 kDa. The isoelectric point of stem and fruit bromelain is
at pH 9.55 and 4.6, respectively (Murachi et al. 1964; Yamada
et al. 1976). There is minimal difference in the amino acid
composition between stem bromelain, ananain, and comosain
Z. I. M. Arshad
:
A. Amid (*)
:
F. Yusof
:
I. Jaswir
Bioprocess and Molecular Engineering Research Unit, Department
of Biotechnology Engineering, Faculty of Engineering, International
Islamic University Malaysia, P.O. Box 10, 50728,
Kuala Lumpur, Malaysia
e-mail: azuraamid@iium.edu.my
Z. I. M. Arshad
Faculty of Chemical and Natural Resources Engineering, Universiti
Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan,
Pahang, Malaysia
K. Ahmad
Kuliyyah of Pharmacy, International Islamic University Malaysia,
P.O. Box 10, 50728 Kuala Lumpur, Malaysia
S. P. Loke
Manufacturing and Industrial Processes Division, Faculty of
Engineering, Centre for Food and Bioproduct Processing,
University of Nottingham Malaysia Campus, Jalan Broga,
Semenyih, 43500, Selangor Darul Ehsan, Malaysia
Appl Microbiol Biotechnol (2014) 98:7283–7297
DOI 10.1007/s00253-014-5889-y
as shown in Table 1. Stem bromelain contains 285 amino
acids where the most abundant amino acids are alanine and
glycine, while histidine and methionine are present in the
lowest amounts (Murachi 1964; Ota et al. 1964). It appeared
to differ significantly in the number of lysine, arginine, and
isoleucine compared to ananain and comosain (Napper et al.
1994). Ananain possesses more hydrophobicity in the region
near histidine-157 and sequence insert between 170 and 174
residues which cannot be found in stem bromelain (Lee et al.
1997). Stem bromelain also contains four hexosamines, and
2.1 % carbohydrate, meaning it is a glycoprotein (Murachi
1964; Murachi et al. 1964). This observation was corroborated
by the findings of Ota et al. (1964), who detected 1.5 %
carbohydrate, including six glucosamines, in purified stem
bromelain. Later, Murachi et al. (1967) isolated 30 mg of
glycopeptides from 1 g of stem bromelain using gel filtration
in an attempt to confirm the presence of a glycoprotein.
Murachi et al. (1967) and Yasuda et al. (1970)reportedthat
the carbohydrate composition in stem bromelain consists of
mannose, fucose, xylose, and glucosamine in the ratio of
3:1:1:4 using gas chromatography analysis, whereas Scocca
and Lee (1969) obtained the same oligosaccharide group with
a different ratio of 2:1:1:2 using automated borate
chromatography.
Stem bromelain is an endoprotease that breaks peptide
bonds within the protein molecule. Stem bromelain is under
the papain family which resembles papain unequivocally in
the alignment of amino acid sequence. A prominent similarity
is observed in their mechanism of action as shown in Fig. 1.
Initially, noncovalent bonding involves the free enzyme
(structure a) and substrate to form the complex (structure b).
Then, the next step is the acylation of the enzyme (structure c)
which produces amine, with R′-NH
2
as the first product.
Finally, the deacylation reaction step takes place between the
acyl-enzyme and water to r elease the carboxylic acid,
RCOOH, and free enzyme (Rao et al. 1998). The presence
of asparagine-175 hydrogen-bonded with histidine-159 favors
the catalytic function in papain. Stem bromelain is distin-
guished from papain by its lower value of second-order rate
constant of inactivation by iodoacetate and iodoacetamide due
Table 1 Amino acid composition of stem bromelain, ananain, and
comosain (Napper et al. 1994)
Amino acid Stem bromelain Ananain Comosain
Asp 18 19 18
Thr 9 8 7
Ser 17 18 17
Glu 16 13 13
Gly 22 24 25
Ala 25 20 20
Va l 1 4 1 4 1 3
Met 3 2 3
IIe 17 14 12
Leu 6 9 9
Tyr 14 12 12
Phe 6 5 7
His 1 2 2
Lys 15 11 10
Arg 6 10 11
Cys 7 7 7
Fig. 1 Catalytic mechanism of cysteine proteinase action. Im and
+
HIm
referred to imidazole and protonated imidazole (Rao et al. 1998)
7284 Appl Microbiol Biotechnol (2014) 98:7283–7297
to the absence of asparagine-175 and two adjacent residues as
well as the mutation of serine-176 to lysine in its structure
(Ritonja et al. 1989). It is noteworthy that stem bromelain
exhibits a weak inhibition by
L-3-carbox y-2,3-trans-
epoxypropiony l-leucylamid o (4-guanidino) butane (E64)
about 678 M
−1
s
−1
in contrast to pa pain which is about
638,000 M
−1
s
−1
(Barrett et al. 1982; Rowan et al. 1990).
Specificity of stem bromelain was markedly examined
using different substrates, such as gelatin (gelatin digestion
unit), casein (casein digestion unit), azocasein and
azoalbumin, hemoglobin, sodium caseinate, Z-Arg-Arg-NH-
Mec, and Z-Arg-Arg-ρNa (Corzo et al. 2012;Gautametal.
2010; Iversen and Jørgensen 1995;Napperetal.1994;Rowan
et al. 1990). Its proteolytic ability efficiently had hydrolyzed
glucagon at arginine-alanine and alanine-glutamic acid bonds
where it shows preference for glutamic acid, aspartic acid,
lysine, or arginine in the P1 site yet leaves intact the arginine-
arginine and lysine-tyrosine bonds. The release of DNP (2,4-
dinitrophenyl)-alanine and DNP-glutamic acid after FDB
(fluorodinitrobenzene) treatment becomes an evidence of the
successful cleavage at its preference bonds (Murachi and
Neurath 1960; Napper et al. 1994). Profiling of substrate
specificity for stem bromelain using positional scanning syn-
thetic combi natorial libraries (PS-SCLs) had displayed a
cleavage site at arginine (Choe et al. 2006). Generally, stem
bromelain is in full activity under the presence of ethylenedi-
aminetetraacetic acid (EDTA) and cysteine, and the addition
of mercury (II) chloride (HgCI
2
) causes inhibition of its activ-
ity (Murachi and Neurath 1960). The optimum pH and tem-
perature for stem bromelain act ivity are in the range of
pH 6.5–8.0 and 55–60 °C, respectively, for most of the sub-
strates which are presented in Table 2. Studies of the stability
of pineapple fruit in storage without preservative at −4°C
indicate that bromelain retains 75±5 % of its activity, and no
microbial growth is detected after 180 days (Bhattacharya and
Bhattacharyya 2009). The bromelain degradation profile in
aqueous solution at various temperatures follows first-order
kinetics, and its activation energy is 41.7 kcal/mol (Yoshioka
et al. 1991).
While the biochemical propertie s such as molecular
weight, optimum pH and temperature as well as carbohydrate
composition in this enzyme display varying degrees of ho-
mology from various researchers, judgment on the variation of
bromelain resource and purification process has to be scruti-
nized extensively before awaiting for further industrial devel-
opment. Thanks to the major breakthrough of modern bio-
technology, new analytical technologies to characterize and
ensure the safety of the source and end product application
have been developed (Staub et al. 2011). Today, escalating
growth of commercial manufacturers of stem bromelain (re-
trieved from http://www.chemicalbook.com) worldwide has
swiftly found usage in a wide array of industrial applications
such as food, textile, brewing, cosmetic, and dairy products
(Polaina and MacCabe 2007). Thus, this review will start with
an overview on various applications of bromelain in tenderi-
zation, baking industry, protein hydrolysate, enzymatic brow-
ning inhibition agent, animal feed, textile industry, tooth whit-
ening as well as cosmetic industry. Next, an in-depth summary
on recent purification strategies of bromelain in the last
10 years which include ultrafiltration, precipitation, aqueous
two-phase system (ATPS), adsorption, reverse micelle extrac-
tion (RME), and chromatography will be provided.
Industrial applications of bromelain
Bromelain is a plant protease isolated from pineapple. Its
strong proteolytic activity has created a wide interest in nu-
merous applications, mainly in tenderization, foods, deter-
gents, and the textile industry. Bromelain has also been envis-
aged to have extensive applications as an active ingredient in
tooth-whitening dentifrices and skin products. In this part of
the review, the major applications of bromelain are summa-
rized (see Table 3).
Table 2 Optimum pH and temperature for bromelain with different substrates
Type of bromelain Optimum pH Optimum temperature (°C) Substrates References
Fruit 6.5 55 Azocasein Corzo et al. (2012), Bhattacharya
and Bhattacharyya (2009)7.5 55 Azoalbumin
7.7 59 Casein
6.5 59 Sodium caseinate
2.9 37 Hemoglobin
Peel 8 60 Casein Ketnawa and Rawdkuen (2011)
Recombinant 4.6 45 N
α
-CBZ-L-lysine p-nitrophenyl ester (LNPE) Amid et al. (2011)
Stem 7 40 Casein Khan et al. (2003)
8 – Bz-Phe-Val-Arg-pNA Benucci et al. (2011)
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Tenderization
Tenderness is an important characteristic of meat. The over-
whelming demand for guaranteed tender meat has attracted
players in the meat industry to provide an acceptable quality of
product. In the USA, certified Angus beef was introduced in
the late 1970s. Consumers have found it consistently flavor-
ful, juicy, and tender (Koohmaraie and Geesink 2006). Many
approaches have been employed to improve postmortem ten-
derness, including blade tenderization (Pietrasik et al. 2010;
Pietrasik and Shand 2011), moisture enhancement technology
(Streiter et al. 2012), and enzymatic treatment (Gerelt et al.
2000; Pietrasik and Shand 2011). In a traditional tenderization
method, meat is kept cool for up to 10 days to allow postmor-
tem proteolysis by proteolytic enzymes: cathepsins and
calpains (Nowak 2011).
The potential of proteolytic enzymes such as bromelain,
papain, and ficin has been recognized, as shown by their long
history of use in meat tenderization. Compared with papain
and ficin, bromelain is commercially available in the market
under name brands such as McCormick and Knorr.
Commercial papain, bromelain, and zingibain efficiently hy-
drolyze a few meat myofibril proteins, such as actomysin,
titin, and nebulin, as revealed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) (Hage et al.
2012). Sullivan and Calkins (2010)reportedthatbromelain
can improve the sensory evaluation rating and tenderness of
meat comparable to other exogenous enzymes. The addition
of bromelain can also produce a tenderizing effect on myosin
and other myofibrillar proteins of coarse dry sausage. A
number of methods of tenderization using bromelain have
been reported. Lizuka and Aishima (1999) successfully
applied continuous monitoring using attenuated total reflec-
tance infrared spectroscopy (ATR-IR) to observe the structural
changes in meat protein after treatment with pineapple juice.
Melendo et al. (1997) studied the effect of pH, temperature,
cutting method, and cooking time during marinating with
bromelain. Ketnawa and Rawdkuen (2011)demonstratedthat
treatment of bromelain at up to 20 % (w/w) leads to extensive
proteolysis on beef, chicken, and squid. Besides, bromelain
can be a good substitute for hydrochloric acid as a hydrolysis
agent for oyster meat and, at the same time, receives higher
acceptability scores in sensory evaluation in oyster sauce
production (Chuapoehuk and Raksakulthai 1992).
Baking industry
Gluten is a functional component of wheat food products,
such as flour. It consists of two major proteins, gliadin and
glutenin. Gluten becomes insoluble and forms lattice-like
structures when it is hydrated. Therefore, gluten must be
degraded to avoid resistance to do ugh stretching (Walsh
2002). The use of proteolytic enzymes such as bromelain
can improve dough relaxation, enhance solubility, and prevent
dough shrinkage. This will allow the dough to rise evenly
during the baking process (Kong et al. 2007; Polaina and
MacCabe 2007). Bromelain also has been used to produce
hypoallergenic flour that is suitable for wheat-allergic patients.
The immunoglobulin E (IgE)-binding epitope, Gln-Gln-Gln-
Pro-Pro, is a major allergen in flour. Thus, the addition of
bromelain can help to hydrolyze peptide bonds near proline
(Pro) residues, which degrades the epitope structure (Tanabe
et al. 1996; Watanabe et al. 2000).
Table 3 Industrial applications of bromelain
Applications Reasons References
Baking industry – Improve dough relaxation and allow the dough to rise evenly Kong et al. (2007), Tanabe et al. (1996), Watanabe et al. (2000)
– Produce hypoallergenic flour
Tenderization – Hydrolyze meat myofibril proteins Hage et al. (2012), Sullivan and Calkins (2010), Ketnawa and
Rawdkuen (201 1), Chuapoehuk and Raksakulthai (1992)– Hydrolyzing agent for meat, oyster, chicken, and squid
Fish protein
hydrolysate
– Hydrolyze fish protein to generate fish protein hydrolysate Elavarasan et al. (2013), Ren et al. (2008), Tanuja et al. (2012)
Antibrowning agent – Inhibit browning of fruits and phenol oxidation Lozano-De-Gonzalez et al. (1993), Srinath et al. (2012)
Alcohol production – Enhance protein stability of beers Benucci et al. (2011)
– Prevent haze formation
Animal feed – Estimate protein degradation in ruminant feed Tománková and Kopečný (1995)
Textile industry – Minimize softening time in cocoon cooking Koh et al. (2006), Devi (2012), Singh et al. (2003)
– Remove scale and impurities of wool and silk fibers
– Enhance dyeing properties of protein fibers
Tooth whitening – Remove stains, plaque, and food debris on the outer
surface of teeth
Chakravarthy and Acharya (2012), Kalyana et al. (2011)
Cosmetic industry – Treat acne, wrinkles, and dry skin Ozlen et al. (1995), Levy and Emer (2012)
– Reduce post-injection bruising and swelling
7286 Appl Microbiol Biotechnol (2014) 98:7283–7297
Protein hydrolysate
Protein hydrolysate has a long history of use as a nitrogen
source for growth media for microbial, plant, and animal cell
culture at the laboratory and industrial scales. It is widely used
as a nutritional supplement, pharmaceutical ingredient, and
flavor enhancer and in cosmetics and beverages. Current
practice in the manufacturing of protein hydrolysate involves
acid, alkali, and enzyme h ydrolysis. However, enzymatic
hydrolysis is preferable due to the ease in controlling the
degree of hydrolysis as well as shortening the hydrolysis time.
The significant parameters for enzymatic hydrolysis are tem-
perature, hydrolysis time, pH, and degree of hydrolysis (DH)
(Pasupuleti and Braun 2010). The addition of proteolytic
enzymes can make the enzymatic hydrolysis process efficient
and reproducible (Chalamaiah et al. 2012). There has been
considerable research on the flavor profile (sweet, salty, sour,
bitter, bouillon, and umami taste) and functional properties
(hypoantigenic, antioxidant, and antimicrobial) of protein hy-
drolysate isolated from fish and plant sources using commer-
cial bromelain.
Protein hydrolysate derived from food processi ng by-
products can be used as precursors in the production of meat,
savory food, and chicken flavor substitutes. For example, the
hydrolysis of mungbean protein by bromelain has released 16
free amino acids which give a meaty flavor characteristic such
as bouillon, sweet smell, green, and fatty (Sonklin et al. 2011).
Besides, after bromelain hydrolysis, several amino acids such
arginine, lysine, and leucine were found from the protein by-
product hydrolysis of Gracilaria fisheri (red seaweeds) which
characterize se af ood flav oring (Lao haku njit et al. 2014).
Sangjindavong et al. (2009) reported on the production of fish
sauce from the fermentation of surimi waste accelerated with
pineapple core and peel.
In spite of the fact that protein hydrolysate can be used as a
flavor agent, the hydrolysis process is often accompanied by
bitterness effect due to the reaction of endoprotease with the
protein substrate during the hydrolysis. The bitter intensity
can be expressed as the taste dilution (TD) factors. Partial
hydrolyzation of soy protein isolate (SPI) by bromelain
showed lower (TD) factors of 4 and 16 in 10 and 15 % DH,
respectively, compared to alcalase and neutrase. This phenom-
enon could be due to the remaining nonpolar amino acid
residues at the C-terminus of the pe ptide after bromelain
hydrolyzes the hydrophobic amino acid residues (Seo et al.
2008). Cheung and Li-Chan (2014) found that after 8 h of
hydrolysis, shrimp hydrolysate by bromelain demonstrated
less bitter and high umami taste in the instrumental taste
sensing system rather than alcalase and protamex hydrolysate.
The bitterness characteristic results from enzymatic hydrolysis
and is due to the presence of N-terminal peptide residues
which are glycine, leucine, isoleucine, phenylalanine, and
valine (Hevia and Olcott 1977).
Recent studies have demonstrated that protein hydrolysates
from fish source possess antioxidant activities after enzymatic
hydrolysis using various proteolytic enzymes such as brome-
lain, papain, flavorzyme, protamex, neutrase, and alcalase
(Lamsal et al. 2007; Ren et al. 2008; Tanuja et al. 2012).
Among these enzymes, bromelain hydrolysate from freshwa-
ter carp (Catla catla) is appearing to give high disappearance
rates of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)
(Elavarasan et al. 2013). Antimicrobial peptides from natural
sources can be promising ca ndidate s to rep lace synthetic
antimicrobial agents in the treatment of a wide spectrum of
bacterial infections. Thus, Ghanbari et al. (2012)exploredthe
application of Actinopyga lecanora (sea cucumber) in the
production of antimicrobial peptide. They found that after
7 h of hydrolysis, hydrolysates prepared by bromelain exhib-
ited the highest antimicrobial activities against Pseudomonas
sp., Pseudomonas aeruginosa,andEscherichia coli compared
to other hydrolysates generated by papain, alcalase, pepsin,
trypsin, and flavorzyme. That study corroborates a previous
study performed by Salampessy et al. (2010), which showed
the antimicrobial capability of bromelain hydrolysate from
leatherjacket (Meuschenia sp.) against Staphylococcus aureus
and Bacillus cereus.
Enzymatic browning inhibition agent
The discoloration of fruits into brown color, especially apples
and pears, occurs due to the oxidation of phenols to quinones
in the presence of oxygen. Further reactions of quinones with
other substances, such as phenolic compounds and amino
acids, lead to the development of brown pigments.
Enzymatic browning may deteriorate the nutritional quality
and flavor of the fruits. To control the browning of fresh and
canned fruit, ethylene diaminetetraacetic acid (EDTA), ascor-
bic acid,
L-cysteine, citric acid, and phosphoric acid are com-
monly used (Ozoğlu and Bayındırlı 2002). However, due to
health concerns over the side effects caused by chemical-
based enzymatic browning inhibition agents, there has been
an increasing amount of research on agents derived from
plant-based sources. According to Lozano-De-Gonzalez
et al. (1993), eluate from cation exchange of the pineapple
juice is effective as a sulfite agent to inhibit browning of fresh
and dried apple rings. Later, Srinath et al. (2012)extendedthat
study by partitioning the fruit and crown leaf using cation-
exchange chromatography after ammonium sulfate precipita-
tion. They concluded that the protease activity and browning
inhibition effects were higher in the fruit extract compared to
the crown leaf extract. Similarly, Chaisakdanugull et al.
(2007) employed pineapple juice that was fractioned by a
solid-phase C
18
cartridge as an inhibitor of enzymatic brow-
ning in bananas. The study reported a 100 % inhibitory effect
of bromelain toward polyphenol oxidation (PPO). In contrast
to earlier findings, however, stem bromelain had the weakest
Appl Microbiol Biotechnol (2014) 98:7283–7297 7287
