processes

Article
Extraction and Characterization of Bromelain from Pineapple
Core: A Strategy for Pineapple Waste Valorization
Alex Fissore 1 , Mauro Marengo 1 , Valentina Santoro 2 , Giorgio Grillo 1 , Simonetta Oliaro-Bosso 1 ,
Giancarlo Cravotto 1 , Fabrizio Dal Piaz 3 and Salvatore Adinolfi 1, *

1 Department of Drug Science and Technology, University of Turin, 10125 Turin, Italy;
alex.fissore@unito.it (A.F.); mauro.marengo@unito.it (M.M.); giorgio.grillo@unito.it (G.G.);
simona.oliaro@unito.it (S.O.-B.); giancarlo.cravotto@unito.it (G.C.)
2 Department of Pharmacy, University of Salerno, 84084 Fisciano, Italy; vsantoro@unisa.it
3 Department of Medicine, Surgery and Dentistry, University of Salerno, 84081 Baronissi, Italy;
fdalpiaz@unisa.it
* Correspondence: salvatore.adinolfi@unito.it

Abstract: Bromelain is a mixture of cysteine endopeptidase usually extracted from pineapple juice
and is used for the treatment of various human diseases and in industrial applications. Bromelain
demand is quickly increasing, and its recovery from pineapple residues appears to be a sustainable
waste management strategy. Pineapple core is among the most significant waste materials in the
production of canned pineapple and is richer in bromelain than other pineapple residues. In this
project, we compared the enzymatic properties and composition of bromelain extracts from either
pineapple core or pulp to address the recovery of bioactive bromelain from pineapple core, thus
contributing to the valorization of this waste material. Although significant differences were detected
in the protein content of the two preparations, no differences could be detected for their proteolytic
activity and for the effect of pH on their enzymatic activity. Mass spectrometry (MS) approaches
identified the same peptidases in the fruit and in the core. This confirmed the possibility of using
pineapple core to obtain relevant amounts of bioactive bromelain by applying a relatively simple
procedure, thus paving the way to implementing a circular economy in this specific industrial sector.
Citation: Fissore, A.; Marengo, M.;
Santoro, V.; Grillo, G.; Oliaro-Bosso,
Keywords: bromelain; pineapple core; proteolytic activity; mass spectrometry; waste valorization
S.; Cravotto, G.; Dal Piaz, F.; Adinolfi,
S. Extraction and Characterization of
Bromelain from Pineapple Core: A
Strategy for Pineapple Waste 1. Introduction
Valorization. Processes 2023, 11, 2064. Bromelain is a well-known mixture of different cysteine endopeptidases from pineap-
https://doi.org/10.3390/pr11072064 ple and has been widely used for the treatment of various human diseases due to its
Academic Editor: Carla Silva demonstrated anti-inflammatory, anti-thrombotic, anti-edematous and fibrinolytic activ-
ities that have made pineapple quite popular in traditional medicine [1,2]. Bromelain
Received: 3 May 2023 anti-inflammatory and analgesic activity was first reported for the treatment of both
Revised: 3 July 2023
osteoarthritis and rheumatoid arthritis [3] and was confirmed by several studies after-
Accepted: 10 July 2023
ward [4,5]. Bromelain was effective in the treatment of acute sinusitis, rhinitis, and chronic
Published: 11 July 2023
rhinosinusitis [6] and has been demonstrated to decrease platelet aggregation, blood vis-
cosity, and the risk of both thrombus formation and angina pectoris in various in vitro
and in vivo studies [7,8]. Bromelain preparations have also been extensively used in the
Copyright: © 2023 by the authors.
nutraceutical and cosmeceutical sectors and in the food industry for beer manufacture, meat
Licensee MDPI, Basel, Switzerland. tenderization, gluten degradation in baking, or improvement of cheese properties [9,10].
This article is an open access article Bromelain is present in almost all parts of pineapple (peel, leaves, stem, and fruit)
distributed under the terms and but can be found in the highest amounts in fruit and stems. The pineapple stem primarily
conditions of the Creative Commons provides stem bromelain (EC 3.4.22.32) [1] but also contains other proteinases, including
Attribution (CC BY) license (https:// ananain, comosain [11], and acidic stem bromelain [2]. Fruit bromelain (EC 3.4.22.33) is the
creativecommons.org/licenses/by/ major proteolytic enzyme in the fruit flesh [12], and two active isoforms of this enzyme have
4.0/). been identified in pineapple fruit crude extracts [13]. Whereas bromelain from pineapple

Processes 2023, 11, 2064. https://doi.org/10.3390/pr11072064 https://www.mdpi.com/journal/processes

Processes 2023, 11, 2064 2 of 10

stems has an isoelectric point (pI) of 9.5 and an optimum pH range of 6–7, the bromelain
extracted from the fruit is characterized by a pI of 4.6 and an optimum pH range of 3–8 [2,14].
These differences are most likely due to the presence of different thiol-endopeptidases in
bromelain preparations from either the fruit or the stem and most likely also explain the
great heterogeneity of clinical and preclinical results for the therapeutic effects of bromelain
in the treatment of inflammatory diseases and immune dysfunctions [8].
Since the demand for bromelain has been quickly increasing in recent years due to its
broad application as a protease in various industrial fields [15], its recovery from pineapple
residues appears as a sustainable and effective waste management strategy. This approach
may indeed counterbalance the serious environmental problems raised by the dramatic
amounts of waste produced during pineapple fruit processing, transportation, and storage,
which are often degraded in landfills, resulting in a dramatic release of harmful greenhouse
gases and substances to the environment [16,17]. In this frame, pineapple core is one of the
most significant waste materials in the production of canned pineapple, jam, or juice, but it
usually goes to landfills or for animal feeding. Pineapple core contains relevant amounts
of glucose and fructose that make it suitable as a substrate for microbial fermentation
processes aimed at producing organic acids that find applications, among others, in the
food industry [18]. The core has been widely used as a source of fibers, phenolics and
antioxidant compounds, and vitamins [10] and is generally richer in bromelain than other
pineapple residues [19,20]. As for this latter issue, a number of researchers have focused
their attention on measuring the proteolytic activity of core-derived bromelain preparations,
but specific efforts are required for the molecular identification of the enzymes from the
pineapple core [18,20].
In this project, we focused on the preparation and the biochemical characterization of
bromelain extracts from either pineapple core or fruit. The application of enzymatic activity
tests and specific proteomics approaches allowed the comparison in terms of enzymatic
activity and composition of the bromelain preparations obtained from the core or the
pulp. In particular, this study will help identify the peptidase(s) extracted from either
the pineapple pulp or core and may contribute to addressing the possibility of recovering
biologically active bromelain from the core. Taking into account that fruit bromelain can be
easily extracted from pineapple pulp juice [9,21], our approach may pave the way to the
valorization of this specific waste material and to the application of a circular economy in
this specific industrial sector.

2. Materials and Methods

2.1. Preparation of Protein Extracts
Pineapples (Ananas comosus (L.) Merr., MD-2 variety) were purchased in a local super-
market, based on size uniformity, at a similar ripening stage, and free of visual defects. The
pineapples were washed with water and manually peeled, and the core was mechanically
separated from the pulp. The core and the pulp were cooled down at 4 ◦ C, mechanically
blended by a lab-scale blender (8010ES two-speed blender, Waring Commercial, Stamford,
CT, USA), and eventually centrifuged at 5000× g at 4 ◦ C for 20 min to separate the insoluble
particles from the juice by using a refrigerated J2-21 centrifuge (Beckman Instruments,
Palo Alto, CA, USA). Dry matter was determined on 20 g of blended material, dried to a
constant weight at 105 ◦ C overnight in a laboratory dry oven (Falc Instruments, Treviglio,
Italy) according to standard requirements (AOAC method 922.10) [22]. The residue was
weighed and reported as a percentage of the starting material. Results are expressed as
grams of solid matter/100 g of fresh pineapple.

2.2. Protein Quantification and SDS-PAGE

Soluble proteins in the juice from either the pulp or the core were quantified by using
the PierceTM bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Milan,
Italy) and were separated by SDS-PAGE [23]. For protein quantification, 0.01 mL of each
juice was used. In the case of SDS-PAGE, samples were diluted at a 1:1 ratio with denaturing
Processes 2023, 11, 2064 3 of 10

buffer (0.125 M Tris-HCl, pH 6.8; 50% glycerol (v/v); 17 g/L SDS; 0.1 g/L Bromophenol
Blue) in the presence of 2-mercaptoethanol and heated at 100 ◦ C for 5 min. SDS-PAGE
was carried out in a MiniProtean Tetra Cell apparatus (Bio-Rad, Milan, Italy) on a 12%
acrylamide-bisacrylamide (37.5:1 ratio) gel, using a Tris/glycine buffer system. Gels were
stained with GelCode™ Blue Safe Protein Stain (Thermo Fisher Scientific, Milan, Italy).

2.3. Enzymatic Assay

Bromelain activity was measured by slightly modifying the procedure used by Ket-
nawa and co-workers [17]. In particular, the juice obtained from either the core or the
pulp was centrifuged at 13,000× g for 15 min at 4 ◦ C, and 0.02 mL of the supernatant was
mixed with 1 mL of 1% (w/w) sodium caseinate in PBS buffer, pH 7.0, in the presence of
6 mM EDTA and 30 mM cysteine, and incubated at 37 ◦ C for 10 min. After the addition of
0.85 mL of 5% (w/v) trichloroacetic acid (TCA), the reaction mixture was centrifuged at
10,000× g for 10 min. The number of soluble peptides in the supernatant was determined by
measuring the absorbance at 280 nm, and the bromelain proteolytic activity was expressed
as the amount of enzyme that releases 1 µmol of tyrosine per minute under the assay
conditions, considering an extinction coefficient for tyrosine at 280 nm of 1490 M−1 cm−1 .
A blank solution was prepared by mixing the caseinate solution and the various samples
and by immediately stopping the reaction by TCA addition.

2.4. Mass Spectrometry

After protein separation by SDS-PAGE, the gel bands were excised and underwent
a trypsin in-gel digestion procedure. NanoUPLC-hrMS/MS analyses of the resulting
peptides mixtures were carried out on a Q-Exactive orbitrap mass spectrometer (Thermo
Fisher Scientific, Milan, Italy), coupled with a nanoUltimate300 UHPLC system (Thermo
Fisher Scientific, Milan, Italy). Peptide separation was performed on a capillary easy spray
(0.075 mm × 150 mm, 1.7 mm) using aqueous 0.1% formic acid (buffer A) and CH3 CN
containing 0.1% formic acid (buffer B) as mobile phases and a linear gradient from 5% to
35% of B in 60 min and a 300 nL/min flow rate. Mass spectra were acquired over a m/z
range from 350 to 1500. To achieve protein identification, MS and MS/MS data underwent
Mascot software (v2.5, Matrix Science, Boston, MA, USA) analysis using the non-redundant
Data Bank UniprotKB/Swiss-Prot (Release 2022_01). Parameter sets were trypsin cleavage;
carbamidomethylation of cysteine as a fixed modification and methionine oxidation as a
variable modification; a maximum of two missed cleavages; false discovery rate (FDR),
calculated by searching the decoy database, 0.05.

2.5. Statistical Analysis

Data presented in Tables 1 and 2 and in Figure 1 are the average of three replicates
from three independent measurements and are reported as average ± standard deviation.
Analysis of variance (one-way ANOVA) was carried out by using Statgraphic Plus v. 5.1
(StatPoint Inc., Warrenton, VA, USA). Data from MS analysis reported in Table 3 were
obtained by performing three replicates from three independent measurements.

Table 1. Dry weight and amount of soluble proteins in the centrifuged extracts of pineapple fruit
pulp and core. Amounts of soluble proteins were measured by using the PierceTM BCA Protein Assay
Kit (Thermo Fisher Scientific, Milan, Italy).

Dry Weight Soluble Proteins

Sample
(g/100 g Fresh Product) (mg/g Dry Material)
Pulp 12.0 ± 1.1 15.6 ± 1.0
Core 10.9 ± 0.8 6.8 ± 0.4
 Table 1. Dry weight and amount of soluble proteins in the centrifuged extracts of pineapple fruit
pulp and core. Amounts of soluble proteins were measured by using the PierceTM BCA Protein As-
say Kit (Thermo Fisher Scientific, Milan, Italy).
Processes 2023, 11, 2064 Dry Weight Soluble Proteins 4 of 10
Sample
(g/100 g Fresh Product) (mg/g Dry Material)
Pulp 12.0 ± 1.1 15.6 ± 1.0
Core 10.9 ± 0.8 6.8 ± 0.4
Table 2. Enzymatic activity on sodium caseinate of bromelain preparations from pineapple pulp and
core.
TableBromelain proteolytic
2. Enzymatic activity
activity on is expressed
sodium caseinate as the amountpreparations
of bromelain of enzymes from that release: (i) one
pineapple pulpµmol
of
andtyrosine per minute
core. Bromelain from casein
proteolytic under
activity the assay conditions;
is expressed (ii) of
as the amount one µg of tyrosine
enzymes equivalents
that release: i) one per
µmol from
hour of tyrosine
caseinper minute
under the from
assaycasein under (papain
conditions the assayunit,
conditions;
PU); andii) (iii)
one one
µg ofmg
tyrosine equiva-
of amino nitrogen
lents per
from hour from
a standard casein
gelatin under the
solution assay
after conditions
20 min (papain
of digestion unit, PU);
(gelatin and iii)
digesting oneGDU).
unit, mg of amino
nitrogen from a standard gelatin solution after 20 min of digestion (gelatin digesting unit, GDU).
Sample Bromelain Enzymatic Activity
Sample Bromelain Enzymatic Activity
µmol
µmol Tyrosine/mg
Tyrosine/mg 6
PU/g
PU/g Protein
Protein (×610
(×10 GDU/g
) ) GDU/g Protein
Protein
Protein × min
Protein × min
Pulp
Pulp 3.32± ±
3.32 0.06
0.06 36.1
36.1 ± 0.7
± 0.7 ± 45± 45
24072407
Core
Core 3.38 ± 0.09
3.38 ± 0.09 36.7 ± 1.0
36.7 ± 1.0 2446 ± 71± 71
2446

Figure 1.
Figure 1. pH
pHdependence
dependenceofofthe proteolytic
the activity
proteolytic on on
activity sodium caseinate
sodium of bromelain
caseinate extracts
of bromelain fromfrom
extracts
either pineapple pulp or core.
either pineapple pulp or core.
Table 3. Identification of fruit bromelain (BROM1_ANACO) and stem bromelain (BROM2_AN-
Table 3. Identification of fruit bromelain (BROM1_ANACO) and stem bromelain (BROM2_ANACO)
ACO) peptides in the three analyzed samples based on MS/MS data. * These peptides have the same
peptides in the
amino acid three analyzed
sequence samples
and cannot based on MS/MS
be discriminated baseddata. * Thesedata.
on MS/MS peptides have the same amino acid
sequence and cannot be discriminated based on MS/MS data.
Experimental BROM1 Experimental BROM2
Sample BROM1 BROM2
Sample MW
Experimental MW Peptide Experimental MW Peptide
MW
Peptide Peptide
1070.550 1–9 *
1070.550 1–9 *
938.471
938.471 10–1810–18
1070.551 121–129 * 1927.008
1927.008 43–5943–59
1070.551 121–129 * 750.345
Pulp
Pulp 2477.145
2477.145 164–185
164–185 750.345 65–7065–70
1067.565 71–79
1540.669
1540.669 300–313
300–313 1067.565
1124.587 71–7980–90
1583.758
1124.587 80–9098–112
950.461 180–187
1583.758 98–112
1070.5506 1–9 *
938.4709 10–18
2470.1471 19–40
1070.5506 121–129 * 1927.0154 43–59
Core 2477.1453 164–185 750.347 65–70
1540.6688 300–313 1067.5662 71–79
1124.5867 80–90
1583.7587 98–112
950.4613 180–187
Processes 2023, 11, 2064 5 of 10

Table 3. Cont.

BROM1 BROM2
Sample Experimental MW Experimental MW
Peptide Peptide
1070.5506 1–9 *
938.4709 10–18
1927.0083 43–59
1070.5506 121–129 * 750.3452 65–70
Commercial 2477.145 164–185 1067.5655 71–79
1540.6665 300–313 1124.5893 80–90
1583.7583 98–112
1940.9632 128–144
950.4615 180–187

3. Results
3.1. Sample Preparation
Crude protein extracts were prepared from either pineapple core or pulp after the
mechanical separation of the core from the rest of the fruit without adding additional water
or any other buffer or solvent for extraction. The core and the pulp were blended and cen-
trifuged to remove insoluble particles. This allowed the recovery of a clear supernatant that
contains the soluble bromelain proteinases. Total proteins in the extracts were quantified
by the BCA assay (Table 1) and normalized according to the total dry weight of the starting
material. Table 1 highlights a total protein content in the pulp more than double that in the
core, as already reported in the literature [17]. This difference could be attributed to the
specific biological composition of the pulp and the core, the latter being richer in fibers [24].

3.2. SDS-PAGE
Proteins in the extracts were separated by SDS-PAGE that provided very similar
tracings for the various samples, with a main protein band at around 23–25 kDa (Figure 2).
This indicates that proteins extracted from the fruit flesh and core were almost the same and
most likely corresponded to bromelain. In this frame, our results are comparable to those
obtained in previous studies, where bromelain from pineapple core and pulp were reported
to be 26 kDa and 23 kDa, respectively [2,25]. A commercial bromelain preparation (Merck,
Milan, Italy) was used as a reference and showed a slightly different electrophoretic pattern
than that of our extracts. In particular, the SDS-PAGE tracing of the commercial preparation
is characterized by a main protein band with a molecular weight of ∼25 kDa but also by a
number of bands at lower MW that might result from proteolytic degradation. Differences
in the electrophoretic pattern might be due to differences in terms of post-translational
modifications and/or proteolytic maturation between the proteins present in our extracts
and those in the commercial sample. As for these issues, the bromelain source (e.g., fruit,
core, stem) and the specific pineapple variety, harvesting, and ripening stage are to be
considered [26,27].
SDS-PAGE measurements also highlighted that bromelain is the most abundant pro-
tein that could be detected in the various preparations with very few contaminants, thus
indicating that its purification from the pineapple core does not require dramatic efforts.
Given that the various extracts considered in this study were obtained by an aqueous
extraction of the blended fruit pulp or core followed by a centrifugation step, this appears
quite relevant from a practical standpoint, with a special focus on the identification of a
procedure that would be effective and efficient as for providing bromelain with high yields
and purity level, while limiting the production costs.
Processes2023,
Processes 2023,11,
11,2064
x FOR PEER REVIEW 66 of 10
of 10

Figure 2. SDS-PAGE
Figure 2. SDS-PAGE of of the
the proteins
proteins from
from the
the centrifuged
centrifuged extracts
extracts of
of pineapple
pineapple fruit
fruit pulp
pulp and
and core.
core.
Proteins
Proteins were
were separated
separated ononaa12%
12%acrylamide-bisacrylamide
acrylamide-bisacrylamide (37.5:1
(37.5:1ratio)
ratio)gel
gelusing
usingaaTris/glycine
Tris/glycine
buffer
buffer system.
system. Gels
Gels were
were stained
stained with
with GelCode™
GelCode™ Blue Safe Protein Stain (Thermo Fisher Scientific,
Milan, Italy).
Milan, Italy). A
A commercial
commercial bromelain
bromelain preparation
preparation(Merck,
(Merck,Milan,
Milan,Italy)
Italy)was
wasused
used as
as aa reference
reference and
and
analyzed by
analyzed by SDS-PAGE.
SDS-PAGE.

3.3. Mass Spectrometry

SDS-PAGE Identification
measurements also highlighted that bromelain is the most abundant pro-
To better
tein that couldelucidate
be detectedthe protein patternpreparations
in the various in either the withpulp very
or thefew
core extracts, samples
contaminants, thus
underwent
indicating thatan SDS-gel separation
its purification fromof the
the proteins
pineapple followed
core doesby not
tryptic in situ
require digestion
dramatic and
efforts.
nanoLC-hrMS/MS analysis of the resulting peptide mixtures.
Given that the various extracts considered in this study were obtained by an aqueous ex-The same protocol was
also applied
traction of theto blended
a commercial bromelain
fruit pulp or corepreparation,
followed byused as a control. step,
a centrifugation For each
this sample,
appears
various bands with apparent molecular weights in the range of
quite relevant from a practical standpoint, with a special focus on the identification 17–25 kDa were excised
of a
and subjected to the MS-based identification procedure. Similar
procedure that would be effective and efficient as for providing bromelain with high results were obtained for
all the bands
yields and purity(Table 3), showing
level, that they
while limiting the contain
production twocosts.
different proteins: fruit bromelain
(BROM1_ANACO) and stem bromelain (BROM2_ANACO). Although the mature form of
these two Spectrometry
3.3. Mass proteins has aIdentification
70% sequence identity, there are several tryptic peptides unique to
each isoform, which allow the two proteins to be discriminated.
To better elucidate the protein pattern in either the pulp or the core extracts, samples
Both electrophoretic and LC-MS/MS analyses suggested that the fruit and the core
underwent an SDS-gel separation of the proteins followed by tryptic in situ digestion and
contain the same proteins. Indeed, the differences observed in the identified peptides
nanoLC-hrMS/MS analysis of the resulting peptide mixtures. The same protocol was also
may stem from slight variations in the abundances of each species in the gel bands rather
applied to a commercial bromelain preparation, used as a control. For each sample, vari-
than from modifications in the protein sequences. Furthermore, the peptides from the
ous bands with apparent molecular weights in the range of 17–25 kDa were excised and
two bromelains identified in the samples were substantially the same as observed for
subjected
the to thepreparation.
commercial MS-based identification
The differentprocedure. Similar
electrophoretic resultsobtained
profiles were obtained
for the for all
latter
the bands (Table 3), showing that they contain two different
sample found no correspondences in the MS data. This might be due to differences in the proteins: fruit bromelain
(BROM1_ANACO)
glycosylation of the and stemin
proteins bromelain (BROM2_ANACO).
the commercial sample compared Although
to thethe mature
other form
samples,
of these two proteins has a 70% sequence identity, there
although it was not possible to observe the glycopeptides in the LC-MS/MS analyses.are several tryptic peptides
unique
As to each
for this issue,isoform,
it shouldwhich allow the twothat
be emphasized proteins to be discriminated.
the purpose of this work was rather to
Both electrophoretic and LC-MS/MS analyses
identify and compare by a well-established proteomics protocol the suggested thatproteins
the fruit and thefrom
extracted core
contain the same proteins. Indeed, the differences observed in the
different parts of the same fruit, and not to demonstrate that the bromelain preparations identified peptides may
stem from slight
considered in thisvariations
study wereinidentical
the abundances of each species
to the marketed in the gel
one, of which, bands rather
however, than
the precise
from modifications
source is ignored. in the protein sequences. Furthermore, the peptides from the two bro-
melains identified in the samples were substantially the same as observed for the com-
3.4. Enzymatic
mercial Kinetic The
preparation. Measurements
different electrophoretic profiles obtained for the latter sample
found nodetection
The correspondences in the MS activity
of the enzymatic data. Thisof might be duebromelain
the various to differences in the
extracts glyco-
is essen-
tial to measuring their bromelain content, which in turn reveals the yield efficiencyalt-
sylation of the proteins in the commercial sample compared to the other samples, of
hough
the it was not
extraction possible
process, as to observe
well as thethe glycopeptides
stability in the LC-MS/MS
of the proteolytic activityanalyses. As for
under various
Processes 2023, 11, 2064 7 of 10

operational and storage conditions (pH, temperature). Different substrates can be used
to assess bromelain activity, ranging from casein and gelatin to the more sensitive casein
and albumin azo-derivatives and various artificial peptides that release easily detectable
molecules following proteolysis [28]. Casein (often in the form of sodium caseinate) is the
most frequently used substrate for proteolytic activity measurements [29].
In this work, the extracts from either the pulp or the core were tested for their prote-
olytic activity on sodium caseinate by spectrophotometrically measuring the amount of
released soluble peptides at 280 nm. In this frame, enzymatic kinetics for both preparations
appeared comparable, as highlighted in Table 2. Enzymatic activity was also calculated and
expressed as papain unit (PU) and gelatin digesting unit (GDU), which are conventionally
used to provide bromelain activity of commercial preparations [30]. In particular, one PU
is defined as the amount of enzyme that liberates one microgram of tyrosine equivalents
per hour from casein under the assay conditions [31], whereas one GDU is the amount
of enzyme which liberates 1 mg of amino nitrogen from a standard gelatin solution after
20 min of digestion and conventionally corresponds to ∼15,000 PU [31].
Our preparations showed an enzymatic activity comparable to most of the bromelain-
based products commercially available in the Italian market (data not shown) but also
to that reported in a number of studies that used different physical approaches to obtain
bromelain from various pineapple waste materials and by-products [19,32]. These results
also confirm that our extraction procedure does not impair bromelain proteolytic activity,
although further investigation is required to address potential effects on the enzyme when
shifting from a lab-scale to the industrial processing of pineapple.
The effect of pH on the enzymatic activity of the peptidases from the pulp or the core
was also addressed, and a similar trend was observed for both preparations that show the
highest activity at around pH 7.0, although no significant differences in peptide release
were observed in the pH range 7–10 (Figure 1). These results further confirm the presence
of very similar (if not the same) peptidases in the pulp and in the core.
As for the pH effect on the activity of bromelain from various pineapple parts, our
results are consistent with the observations of Gul and coworkers [33], although other
studies highlighted a relevant decrease of the proteolytic activity of fruit bromelain on
sodium caseinate at pH higher than 8 [19,29]. This behavior may be explained by differences
in terms of bromelain source and of variety, harvesting, and ripening stage of the pineapples
used in the various studies [17].

4. Discussion
Pineapple wastes are generated from the processing of juice, canned, and frozen
products, but also from the harvesting and processing practices, and can account for 50%
of pineapple weight, and their disposal represents a dramatic issue that may be related to
serious environmental problems [18]. In this frame, an effective waste management strategy
may involve (i) the use of waste as a substrate for bacterial growth and the biotechnological
production of ethanol, citric acid, and antioxidant compounds and ii) the recovery of added-
value products that include cellulose, hemicellulose, and other carbohydrates, and various
enzymes, among which bromelain is the most representative and commercially-valuable
(its cost can range up to 2400 USD/kg) [17,19].
In the present work, we addressed the extraction and enzymatic characterization of
bromelain from the pineapple core, which is one of the most significant waste materials in
the production of canned pineapple, but usually goes to landfills or is used for animal feed-
ing. Pineapple core is a relevant bromelain source since it is generally richer in bromelain
than other pineapple residues and represents around 15% of the total processing waste [19].
In this work, bromelain was extracted from either pineapple fruit pulp or core, and the
comparison of the two preparations showed significant differences in the protein content
due to their specific biological composition and the higher amount of fibers in the core
(Table 1). However, no differences could be detected for the specific proteolytic activity
on caseins by the two different preparations (Table 2) and for the effect of pH on their
Processes 2023, 11, 2064 8 of 10

enzymatic activity (Figure 1). Similar observations stem from the SDS-PAGE separation
of the proteins in the extracts (Figure 2) and from their MS identification (Table 3). This
latter result appears dramatically relevant since it confirms that the same peptidases are
present both in the core and in the pulp, where we were able to identify peptides from both
the fruit and the stem bromelain. This confirms the possibility of using pineapple core to
obtain relevant amounts of biologically active bromelain since it is fully comparable—if not
the same—to the enzyme obtained from the pulp, which is nowadays the primary source
of industrial fruit bromelain [21].
In our study, highly pure bromelain preparations were obtained by applying a rel-
atively simple procedure that only included homogenization and centrifugation of the
pineapple core. In this frame, it has to be highlighted that several limitations, including
temperature- or process-induced inactivation and/or aggregation, still impair the efficiency
and the effectiveness of bromelain recovery from pineapple extracts, although novel purifi-
cation strategies and biotechnological processes have been applied to mitigate production
costs [8].
Production costs strongly depend on the purification process applied to obtain com-
mercial bromelain at the desired purity level, which may involve ammonium sulphate
precipitation, an aqueous two-phase system, membrane filtration, and various chromato-
graphic approaches (ion exchange and/or size-exclusion) [18,34]. However, the direct
comparison of the various bromelain extraction and purification techniques reported in the
literature is not a straightforward task because of the wide heterogeneity in terms of brome-
lain source, extraction strategies, enzymatic activity test conditions, and ways of expressing
bromelain activity [8]. The selection of an effective purification procedure should take into
account the feasibility, ease of scale-up, and the cost of the specific approach, but also the
commercial fate of the bromelain, considering that most of the bromelain applications do
not require an enzyme completely free of contaminants.
The use of a simplified and mild procedure—as the one carried out in this project—
may allow a decrease in the overall production costs for bromelain production and further
contribute to its use in nutraceutical and pharmaceutical preparations or in various indus-
trial, technological processes [10,35]. Further efforts are surely required for the successful
shift from the laboratory to the commercial scale of the process considered in this study,
but our approach is paving the way to the valorization of pineapple core as a relevant
bromelain source, which provides the same enzyme as the one obtained from the pineapple
fruit juice. The implementation of a circular economy in this specific industrial sector
would allow the use as a valuable resource of the biomass associated with (and resulting as
a waste material from) pineapple production and processing and its conversion into high-
value-added products, thus contributing to improved societal economics. This approach
may lead to the development of more sustainable and innovative industrial technologies
and to a zero-waste scenario [36,37], both of which represent highly relevant issues for the
food industry that need immediate action [16].

Author Contributions: Conceptualization, S.A., F.D.P., and G.C.; methodology, M.M. and V.S.;
investigation, A.F., G.G., and V.S.; writing—original draft preparation, A.F., and V.S.; writing—
review and editing, M.M. and S.O.-B.; supervision, F.D.P., and G.C.; funding acquisition, M.M.,
and S.A.; project administration, S.A. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the University of Turin (Ricerca Locale MARM_RILO_20_04
and ADIS_RILO_21_01).
Data Availability Statement: Data are available upon request to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Processes 2023, 11, 2064 9 of 10

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