Saudi Journal of Biological Sciences 28 (2021) 1001–1009

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Original article

Applications of bromelain from pineapple waste towards acne

Sukaina Abbas ⇑, Tejashree Shanbhag, Amruta Kothare
Department of Life Sciences, Kishinchand Chellaram College, Churchgate, Mumbai, Maharasthra 400-020, India

a r t i c l e i n f o a b s t r a c t

Article history: Bromelain is a proteolytic mixture obtained from pineapple (Ananas comosus (L. Merr)). It has diversified
Received 23 July 2020 clinical properties and is used in alleviation of cancer, inflammation and oxidative stress. The current
Revised 26 October 2020 study focuses on extraction of bromelain from different parts of pineapple such as core, crown, fruit, peel
Accepted 4 November 2020
and stem. The extracted enzyme was precipitated using ammonium sulphate at 40% saturation followed
Available online 11 November 2020
by dialysis. The fold of purification obtained for peel, crown, core, fruit and stem were found to be 1.948,
1.536, 1,027, 1.989, and 1.232 respectively. Bromelain activity was estimated using Azocasein assay, the
Keywords:
highest activity was seen in peel at 3.417 U/lg. Antimicrobial activity and MIC of the bromelain purified
Bromelain
Acne
and crude fractions was studied against the test organisms. Peel crude and purified extract exhibited
Antioxidant highest inhibitory effect towards S. aureus followed by P. acne. The antioxidant activity was evaluated
Antimicrobial using DPPH antioxidant assay. IC50 values peel, fruit, stem and crown are found to be 13.158 lg/ml,
Formulation 24.13 lg/ml and 23.33 lg/ml and 113.79 lg/ml respectively. The purified bromelain from peel, stem
and crown was used to create a facewash formulation towards pathogens frequently associated with skin
infections. Common skin pathogens like S. aureus and P. acne were found highly sensitive to its action. The
aim of this study was to evaluate the potential of bromelain isolated from waste parts of pineapple in
alleviation of acne due to its diverse antimicrobial properties.
Ó 2020 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction peel and leaves in reduced quantities compared to the fruit

(Sriwatanapongse et al., 2000). Various purification strategies have
Pineapple (Ananas comosus) has been used as a traditional med- been discussed and developed for extraction of bromelain. It pre-
icine by several cultures throughout time and bromelain has been cipitates at 20% to 60% with ammonium sulphate showing highest
established since 1876. Bromelain gets its properties mainly due to recovery at 20%. Dialysis to be followed after ammonium precipita-
the presence of its sulfhydryl proteolytic enzymes. Bromelain is tion to concentrate and to purify bromelain further by a cost-
classified as stem bromelain or fruit bromelain depending on the effective manner (Pardhi et al., 2016).
origin of the protease. Bromelain is present throughout the pineap- Skin problems like wrinkles, acne and dry skin seemed to be
ple plant however the concentration and composition may vary solved using proteases such as papain and bromelain (Ozlen and
depending on the part of the fruit and its variety (Gautam et al., Chatsworth, 1995). Loon et al., (2018) observed sensitivity of S.
2010). Extraction of bromelain makes it feasible as its bioavailabil- aureus against pineapple extract. Partially purified bromelain from
ity rises during maturity of fruit rather than during fruit develop- the pineapple core also inhibited the growth of S. epidermidis and P.
ment (Maurer, 2001). acne (Hidayat et al., 2018). Bromelain has also been extensively
Extensive studies have been done with bromelain to explore its used as a line of treatment for periodontal gingivitis and found
clinical properties. Thus, finding effective extraction methods is to be effective against periodontal pathogens namely, S. mutans,
necessary. Bromelain is found in pineapple wastes such as core, A. actinomycetemcomitans, P. gingivalis (Praveen et al., 2014).
Edema caused by post-surgical trauma also has been effectively
⇑ Corresponding author. treated by bromelain (Mackay and Miller, 2003).
E-mail address: suk24me@gmail.com (S. Abbas). Acne is a skin condition affecting the skin’s oil glands is a very
Peer review under responsibility of King Saud University. prominent condition. It can create a lot of psychological distur-
bances and stress on the individual that it affects. P. acne is an
opportunistic pathogen that plays an important role in the growth
and cause of acne. S. aureus is a part of the normal skin microbiota
Production and hosting by Elsevier and is associated with skin conditions such as folliculitis and also

https://doi.org/10.1016/j.sjbs.2020.11.032
1319-562X/Ó 2020 The Authors. Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

reported to enhance the effect of other microbes in acne lesions 3.1.3. Dialysis
(Kumar et al., 2016). 3.1.3.1 Activation of dialysis membrane. 7 cm of dialysis membrane
Hence this study aims to evaluate the potential of bromelain in was added into boiling distilled water with 2% sodium carbonate
alleviation of acne owing to its diverse antimicrobial properties. for 45 min. The membrane was boiled again for 45 min in distilled
Active bromelain was isolated from waste parts of pineapple and water, the membrane was left overnight in Acetate buffer (pH-7.0).
its effects on bacterial pathogens like acne was studied. Bromelain
will aid as a potent anti-inflammatory agent that will act as bene- 3.1.3.2 Dialysis. The fractions from ammonium precipitation were
ficial additive to the formulation. Using bromelain from the waste loaded into the activated dialysis membranes and tagged. They
parts of pineapple will help in waste recycling as well as make the were equilibrated into a beaker with acetate buffer. The process
whole process cost-effective. was carried out for 24 h in an ice box with the replacement of buf-
fer every 6 h. The samples from membranes were then unloaded
and labelled as purified bromelain samples. The setup is observed
2. Materials
in Fig. 2.
Biological material: Pineapple, Turmeric, Aloe vera gel
Chemicals: Acetic acid (Chemdyes corp.), Agar Agar Type 1 3.2. Estimation of protein content by Folin-Lowry assay
(Hi-Media), Ammonium Sulphate (Loba Chemie), Ampicillin antibi-
otic discs (Hi-Media), Ascorbic Acid (Loba Chemi), Azocasein The protease mixtures were estimated for the protein content
(Sigma-Aldrich), Beef Extract (Hi-Media), Bovine Serum Albumin by using Folin-Lowry assay. Bovine serum albumin (BSA) was used
(Sigma-Aldrich), Conc. HCl (Chemdyes), Copper Sulphate, DPPH as a standard at 1 mg/ml. Freshly prepared alkaline copper sul-
(Hi-Media), Folin- Ciocalteu reagent (Thermofisher scientific), phate solution ((50:1) 2% Na2CO3 in 0.1 N NaOH: 0.5% CuSO4 in
Peptone (Hi-Media), Sodium Acetate Trihydrate, Sodium 1% NaK), Folin-Ciocalteu Phenol reagent (FC) 1 N was used. 1 ml
Carbonate, Sodium Chloride (SDFCL chemicals), Sodium Hydroxide, of protein was added to 5.5 ml of alkaline copper solution and
Sodium Potassium Tartrate, Tris Base (Hi-Media), Tris HCl incubated for 10 min. 0.5 ml of FC reagent was added to the mix-
(Hi-Media), Yeast Extract (Hi-Media) ture and incubated in dark for 30 min and absorbance was
Microbiological Strains: S. aureus (MTCC 96), C. diphtheriae, recorded (Km = 660 nm). (Plummer and Plummer, 1988)
E. coli (ATCC: 25922), Pseudomonas aeruginosa (ATCC: 227853),
P. acne (MTCC: 1951). 3.3. Estimation of proteolytic activity

3.3.1. Calibration curve

3. Methodology
Azocasein was used as the substrate in the concentration range
of 0.1–2%. Azocasein was digested using enzyme sample for
3.1. Sample preparation
300 min and using regression analysis, an equation was derived
correlating the absorbance/color intensity to the substrate
3.1.1. Crude extract preparation
concentration.
The pineapple parts were categorized as fruit, stem, peel, core
and crown. They were individually homogenized with 0.1 M of
sodium acetate buffer, (pH – 7.0). The extracts obtained was fil- 3.3.2. Proteolytic activity of samples
tered and centrifuged at 6000 rpm for 20 min at 4 °C. These Proteolytic activity of samples was tested varying the azocasein
extracts were labelled as fruit crude extract, stem crude extract, concentration from 0.1 to 2%. Equal quantities of substrate and
peel crude extract, core crude extract and crown crude extract of enzymatic samples (i.e. 100 ll) were incubated for 10 min. The
bromelain. The parts are observed in Fig. 1. reaction was terminated using 5% Trichloroacetic acid (TCA) and
the vials are centrifuged at 4 °C for 10 min. The supernatant is
taken by 1:1 dilution of 0.5 N NaOH and its absorbance is read
3.1.2. Ammonium sulphate precipitation (Km = 440 nm). The blank was obtained by mixing the TCA to
The crude extract was subjected to ammonium sulphate precip- the substrate prior to enzyme addition. Calculations were done
itation at 40% saturation (226 g/l). The extraction was carried out as specified by Coêlho et al. (2016)
in an ice box over a magnetic stirrer. Ammonium sulphate was
gradually added pinch by pinch in a period of 30 min. It was
3.4. Antimicrobial activity testing
allowed to settle for 24 h at 4 °C. The solution was then centrifuged
at 6000 rpm for 10 min at 4 °C. The pellet was later reconstituted in
3.4.1. Bacterial maintenance
minimum volume of 10 mM Tris, (pH – 7.0) and then subjected to
S. aureus (MTCC 96), C. diphtheria, E. coli (ATCC: 25922), P. aerug-
dialysis. The supernatant obtained was further precipitation at 60%
inosa (ATCC: 227853), P. acne (MTCC: 1951) were maintained on
saturation (120g/l). The solution was allowed to stand for 24 h at
Nutrient Agar slants and incubated at 37 °C. These cultures are
4 °C and then centrifuged at 6000 rpm for 10 min at 4 °C. The pellet
going to be further mentioned as test cultures.
obtained was reconstituted in minimum volume of 10 mM Tris,
(pH – 7.0).

Fig. 1. Parts of the pineapple, Crown, stem, peel (left to right). Fig. 2. Dialysis setup, Sample loaded in dialysis membrane (Left to right).

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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

3.4.2. Ditch plate technique 3.6.1. Physical characteristics of the facewash

Antimicrobial susceptibility of the organisms towards the crude Physical characteristics were studied such as the color, pH,
extract was observed by using the ditch plate technique. Bromelain washability, foamability and consistency (Ingle and Meshram,
extract was mixed with molten agar and poured into the ditch. The 2018).
test organism is plated across the ditch. The plates incubated at
37 °C for 24 h. The Zone of Inhibition was measured (Benson 3.6.2. Antibacterial testing of formulation
et al., 2015). The antimicrobial activity of formulation the crude extract was
determined using the disc diffusion method. Nutrient agar plates
3.4.3. Minimum inhibitory concentration by turbidity test were swabbed with the cultures. Sterile Whatmann discs impreg-
The MIC of the purified extracts of peel, fruit and stem against nated with the test samples were placed. The samples were tested
the test cultures in a 10–100% (v/v) dilution. Saline was used as a in four concentrations of (v/v) percentage at 25%, 50%, 75% and
negative control in place of test samples. Absence of turbidity sig- 100%. They were incubated at 37 °C for 24 h. The Zone of Inhibition
nifies no growth of culture whereas presence of turbidity signifies was measured. (Benson et al., 2015)
growth of culture (Wiegand et al., 2008).
3.7. Comparison of commercial products
3.5. Antioxidant activity testing by DPPH method
Four commercially available herbal facewashes were selected at
random and their antimicrobial action against bacterial cultures
Antioxidant activity (DPPH free radical scavenging activity):
were tested at four (v/v) % concentration levels (i.e. 25%, 50%,
The antioxidant activity of the extracts and standards was based
75%, 100%). These were named as Brand 1, Brand 2, Brand 3 and
on the radical scavenging effects of 2, 2 diphenyl-1-
Brand 4)
picrylhydrazyl (DPPH). The diluted working solutions of the sam-
ples were made in water. Ascorbic was used 10-100ug/ml.
0.002% DPPH was prepared in ethanol. The test sample/standard 4. Results and conclusion
was mixed with DPPH in 1:1 ratio and incubated in the dark for
30 min. The solution mixtures were read at 517 nm. The OD was 4.1. Estimation of protein content
recorded and percentage inhibition was calculated by the formula
below. Ethanol with DPPH (1:1) is used as a blank (Khalaf et al., The protein content was estimated by folin-lowry method. The
2008; Yuris and Siow, 2014). values obtained in crude extracts were fruit (170 lg/ml), crown
  (167 lg/ml), peel (128 lg/ml), stem (127 lg/ml) and core
AB (126 lg/ml). The crude extracts were purified by ammonium sul-
Percent inhibition ¼ x100
A phate precipitation and dialysis. The highest protein content after
where A is OD of blank and B is OD of sample: purification was observered in peel (103 lg/ml), followed by stem
(100 lg/ml), crown (93 lg/ml) and core (92 lg/ml). The results are
shown in Graph 1.
3.6. Formulation
4.2. Estimation of proteolytic activity
Three formulations were synthesized using the stem bromelain,
peel bromelain and crown bromelain as the primary base See Proteolytic activity was estimated using the Azocasein method.
Table 1 and Fig. 3. Azocasein content was calibrated for maximum digestion using
bromelain as seen in Graph 2. This was used for calculation of
Table 1 proteolytic activities of crude and purified extracts. An increase
Formulation of Bromelain Facewash. was seen in proteolytic activities after purification. Peel and fruit
Material Function
demonstrated highest proteolytic activity at 3.417 U/lg and
2.556 U/lg. Proteolytic activities have been depicted in Graph 3
Bromelain Antimicrobial/Antioxidant
Turmeric Antioxidant
and Table 2.
Aloe Vera Gel Moisturizer
Xanthum gum Thickener 4.3. Estimation of antimicrobial susceptibility testing of bromelain
Tea tree oil Essential oil/Preservative
extracts
NaOH, SLES, Sodium stearate, Propylene glycol Soap Base

Antimicrobial susceptibility testing of crude and purified

bromelain extracts was performed by ditch plate method. Peel
crude and purified extract exhibited highest inhibitory effect
towards S. aureus followed by P. acne, E. coli, C. diphtheria and P.
aeruginosa. P. aeruginosa was most susceptible to the action of
bromelain. According to inhibitory action, the potent extracts were
peel, fruit, stem, crown and core. The results are shown in Graphs
4, 5 and Fig. 4.

4.4. Estimation of minimum inhibitory concentration (MIC) by

turbidity test

Minimum Inhibitory concentration of the purified extracts was

studied against the test cultures. P. acne was found susceptible
against the purified fractionates at concentrations as low as
Fig. 3. Bromelain Facewash Formulations. 19 lg/ml of the fruit extract. While P. aeruginosa and C. diphtheria
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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

Graph 1. Estimation of protein content in bromelain crude and purified extracts.

Graph 2. Calibration Graph of Azocasein.

Graph 3. Specific activity exhibited by Bromelain fractions by Azocasein assay.

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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

Table 2
Azocasein assay summation.

Specific Activity of Crude (U/ug) Specific Activity of Purified fraction (U/ug) Fold purification Percentage Yield (%)
Peel 1.755 3.417 1.948 112.519
Crown 1.258 1.933 1.536 105.453
Core 1.615 1.658 1.027 102.435
Fruit 1.285 2.556 1.989 114.032
Stem 1.798 2.216 1.232 108.627

Graph 4. Antimicrobial activity exhibited by Crude extracts of Bromelain.

Graph 5. Antimicrobial activity exhibited by purified Bromelain.

are susceptible, they require higher concentration of the enzyme to of peel, fruit, stem and crown are 13.158 lg/ml, 24.13 lg/ml and
inhibit its growth. Turbidity test obsevations are shown in Table 4. 23.33 lg/ml and 113.79 lg/ml. The antioxidant activity of brome-
MIC values are shown in Graph 6 and Table 34. lain is compared with ascorbic acid (used as a standard). IC50 of
ascorbic acid being 9.92 lg/ml. The percentage inhibition of
bromelain samples are shown in Graph 7 and Table 5.
4.5. Antioxidant activity

Natural antioxidants are present in bromelain that help in com- 4.6. Physical parameter testing of test formulations
bating oxidative stress. The antioxidant activities of various
bromelain samples are found to significantly different. (One-Way The physical parameters of the test formulations have been
ANOVA; a-0.05; P value – 0.703). IC50 values bromelain samples depicted in Table 6.
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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

susceptible organism by the three test formulations followed by P.

acne. The zone of inhibition is graphically represented. Results are
depicted in Graph 8 and Fig. 5.

4.8. Antimicrobial susceptibility results of commercial products

All the facewashes were found to be effective against P. acne

and S. aureus. However, Brand 2 and brand 3 showed no inhibition
against E. coli. Results are shown in Graph 9 and Fig. 6.

5. Discussion

Pineapple is an age-old fruit, consumed and used traditionally

for many of its medicinal properties. It has been used for debride-
Fig. 4. A petriplate showing antimicrobial sensitivity by Bromelain by Ditch plate ment of skin (Houck et al., 1983). Bromelain is also used as a
technique. replacement during deficiency of pepsin and trypsin due to its
activity and stability over a wide pH range (Balakrishnan et al.,
1981). Generally, only the fruit is considered as the only edible
4.7. Antimicrobial susceptibility results of Bromelain test formulation member and the rest is treated as waste i.e. the peel, the core,
crown and the stem. Pineapple wastes are usually discarded and
The antimicrobial susceptibility of the formulation was tested then allowed to biodegrade which leads to environmental degrada-
by disc diffusion against the test organisms. S. aureus was the most tion mainly due to carbohydrate rich contents. According to our

Graph 6. MIC results of extracts against cultures.

Table 3
MIC results of extracts v/s culture.

Part Organism MIC (ug/ml)

Peel C.diphtheriae 45
E.coli 22.5
P.acne 30
P.aerurginosa 52.5
S. aureus 15
Fruit C. diphtheriae 67.998
E. coli 19.428
P. acne 19.428
P. aerurginosa 87.426
S. aureus 38.856
Stem C. diphtheriae 96.48
E. coli 48.24
P. acne 36.18
P. aerurginosa 96.48
S. aureus 48.24

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Table 4
MIC turbidity test observations.

Peel Purified Extract

Organism 100% 90% 80% 70% 60% 50% 40% 30% 20% 10%
C. diphtheria – – – – – + + + + +
E. coli – – – – – – – – + +
P. acne – – – – – – – + + +
P. aeroginosa – – – – + + + + + +
S. aureus – – – – – – – – + +
Fruit Purified Extract
Organism 100% 90% 80% 70% 60% 50% 40% 30% 20% 10%
C. diphtheria – – – – + + + + + +
E. coli – – – – – – – – – +
P. acne – – – – – – – – – +
P. aeroginosa – – + + + + + + + +
S. aureus – – – – – – – + + +
Stem Purified Extract
Organism 100% 90% 80% 70% 60% 50% 40% 30% 20% 10%
C. diphtheria – – – + + + + + + +
E. coli – – – – – – – + + +
P. acne – – – – – – – – + +
P. aeruginosa – – – + + + + + + +
S. aureus – – – – – – – + + +

study, active bromelain was obtained from the waste parts as well.
Peel demonstrated highest proteolytic activity at 3.417 U/lg fol-
lowed by fruit at 2.556 U/lg. Ammonium sulphate precipitation
and dialysis were applied for purification for the ease of usage, fea-
sibility and easy recovery of the enzyme with highest activity. This
step is in accordance with (Pardhi et al., 2016) which validate the
use of these methods for bromelain purification. Bromelain activity
was studied using azocasein assay. The highest activity was seen in
peel and fruit at 3.417 U/lg and 2.556 U/lg respectively. This
method is in accordance with (Coêlho et al., 2016) for estimation
of proteolytic activities using azocasein. Yuris and Siow (2014)
associated pineapple for its antioxidant activities to the presence
of phenolics, which motivated us to study if bromelain also exhib-
ited antioxidant activities. IC50 values of bromelain samples of
peel, fruit, stem and crown are 13.158 lg/ml, 24.13 lg/ml and
23.33 lg/ml and 113.79 lg/ml. Acne is a skin condition affecting
the skin’s oil glands is a very prominent condition. It can create a
lot of psychological disturbances and stress on the individual that
it affects. The microbial flora often associated with acne infection
includes P. acnes, S. epidermidis, and S. aureus. The antimicrobial
activity of bromelain was tested against P. acne, S. aureus, C. diph-
theria, E. coli and P. aeruginosa. S. aureus was the most susceptible
organism to the action of crude and purified bromelain extracts
followed by P. acne. Peel crude and purified extract exhibited high-
est inhibitory effect towards S. aureus. The highest resistance to
Graph 7. Antioxidant activity of Ascorbic acid and Bromelain extracts.
Bromelain activity was shown by P. aeruginosa. A facewash was
prepared using peel, stem and crown bromelain extracts. To the
Table 5
best of our knowledge, this is the first formulation that was pre-
IC50 values exhibited by the samples tested.
pared using bromelain extracted from the wastes of pineapple.
Sample IC50 value The antimicrobial susceptibility of formulation was tested by disc
Ascorbic Acid 9.92 lg/ml diffusion against the test organisms. S. aureus was the most suscep-
Peel 13.158 lg/ml tible organism by the three test formulations followed by P. acne.
Fruit 24.13 lg/ml
We chose four popular herbal facewashes acclaimed to be used
Stem 23.33 lg/ml
Crown 113.79 lg/ml
for anti-acne and compared the antimicrobial action of the formu-
lations. Brand 2 and Brand 3 showed no activity against E.coli.. The

Table 6
Physical observations of the formulations.

Factor Peel Formulation Stem Formulation Crown Formulation

pH 5.5 5.9 5.3
Color Golden Yellow Brownish Yellow Pale Green
Consistency Semi-solid Semi-solid Semi-solid
Washability Good Good Good
Foambility Low Foam appears Low Foam appears Low Foam appears

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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

Graph 8. Antimicrobial testing of Bromelain facewash formulations.

Fig. 5. AST of Bromelain Formulation: stem, peel and crown (Top to bottom) against E. coli, S. aureus, P. acne, and C. diphtheria (left to right).

Graph 9. Antimicrobial testing of commercial facewashes.

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S. Abbas, T. Shanbhag and A. Kothare Saudi Journal of Biological Sciences 28 (2021) 1001–1009

Acknowledgments

This project was funded by University of Mumbai minor

research grants scheme, URG project – 388. We would like to
extend our gratitude to National Facility for Biopharmaceuticals
at Guru Nanak Khalsa College for their sophisticated instrumenta-
tion and analysis support.

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Bromelain interactions with other skin proteases and antimicrobial Three Pineapple (Ananas comosus L.) Varieties. J. Food Stud. https://doi.org/
peptides such as psoriasin can also be explored. 10.5296/jfs.v3i1.4995.

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