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International Journal of Food Properties

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Antibacterial activity of proteins extracted from

the pulp of wild edible fruit of Bromelia pinguin L.

Jorge Carlos Ruiz-Ruiz, Jess Ramn-Sierra, Carolina Arias-Argaez, Denis

Magaa-Ortiz & Elizabeth Ortiz-Vzquez

To cite this article: Jorge Carlos Ruiz-Ruiz, Jess Ramn-Sierra, Carolina Arias-Argaez, Denis
Magaa-Ortiz & Elizabeth Ortiz-Vzquez (2017) Antibacterial activity of proteins extracted from
the pulp of wild edible fruit of Bromelia pinguin L., International Journal of Food Properties, 20:1,
220-230, DOI: 10.1080/10942912.2016.1154572

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Published online: 01 Mar 2016.

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Download by: [UNAM Ciudad Universitaria] Date: 22 February 2017, At: 16:19
2017, VOL. 20, NO. 1, 220230

Antibacterial activity of proteins extracted from the pulp of wild

edible fruit of Bromelia pinguin L.
Jorge Carlos Ruiz-Ruiz, Jess Ramn-Sierra, Carolina Arias-Argaez, Denis Magaa-Ortiz,
and Elizabeth Ortiz-Vzquez
Divisin de Estudios de Posgrado e Investigacin, Instituto Tecnolgico de Mrida, Mrida, Yucatn, Mxico


Bromelia pinguin L. is a natural source of bioactive compounds. The main Received 4 September 2015
purpose of this research was to isolate and characterize bioactive proteins Accepted 11 February 2016
from its fruit. B. pinguin proteins were fractionated by gel filtration chromato-
graphy, and analyzed using sodium dodecyl sulfate-polyacrylamide gel elec- Bromelia pinguin L.; Fruit;
trophoresis. The antibacterial activity of the proteins was analyzed against Antibacterial activity;
Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, and the Proteins
enzymatic activity was evaluated by protease activity and trypsin inhibitions
assays. Protein fraction obtained by gel filtration chromatography exhibited
antibacterial activity against E. coli (minimum inhibitory concentration [MIC]
0.3492 mg/mL) and S. aureus (MIC 0.6845 mg/mL). The proteolytic activity of
the fraction was 0.985 Ucas/mL. The substrate-sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis assay detected protease inhibitors with mole-
cular weights of 43 and 74 kDa. Antibacterial studies of E.coli and S. aureus were
determined by comparing the protein fraction with different antibiotics. The
antibacterial activity of proteins extracted from the pulp of the fruit of Bromelia
pinguin L. could be related to the presence of enzymes, protease inhibitors and

The continuous use of antibiotics has resulted in multi-resistant bacterial strains all over the world
and, as expected, hospitals have become breeding grounds for human associated microorganisms.
What is more, the same time-bomb effect is slowly developing with animal-associated pathogens in
commercially driven activities, such as aquaculture and confined poultry breeding, where the
indiscriminate use of antibiotics is perceived as essential for the survival of these industries.
Consequently, there is an urgent need to search for alternatives to synthetic antibiotics. The
discovery of two classes of antimicrobial peptides, non-ribosomally synthesized[1] present in bacteria,
lower eukaryotes, and plants, as well as ribosomally synthesized peptides of wider distribution,
provided a new therapeutic strategy to fight microorganisms. Thus, priorities over the next decade
should focus on the development of alternative drugs and/or the recovery of natural molecules that
would allow the consistent and proper control of pathogen-caused diseases. Ideally, these molecules
should be as natural as possible, with a wide range of action over several pathogens, easy to produce,
and less prone to resistance induction.[2]
Plants produce antimicrobial peptides (AMPs) in all organs either constitutively or in response to
microbial infection; however, information about the expression of AMPs in epidermal cells is
limited. As plants also biosynthesize protective secondary metabolites, AMPs may not be as crucial
for the first line of defense as in animals.[3] Plant AMPs were assigned to different classes according

CONTACT Dr. Elizabeth Ortiz-Vzquez Divisin de Estudios de Posgrado e Investigacin, Instituto
Tecnolgico de Mrida, Av. Tecnolgico km. 4.5 S/N, C.P. 97118, Mrida, Yucatn, Mxico.
Color versions of one or more of the figures in the article can be found online at
2017 Taylor & Francis Group, LLC

to their tertiary structures. The most common classes are thionins, defensins, and lipid transfer
proteins. Plant AMPs share the following important features: They are small cationic peptides with
molecular masses of 210 kDa. The structures of these small peptides are stabilized through
formation of 26 disulfide bridges. The activities of plant AMPs are primarily directed against
fungal, oomycete, and bacterial microorganisms, but certain members of a class can be directed
against other targets, including herbivorous insects.[4]
Fruit juices exhibit significant antibacterial effect, the activity being associated with mineral
content and biologically active constituents. Hence, these fruit juices with the properties of
bioavailability and retention of certain minerals, proteins, and polyphenolic compounds can be
recommended for their use as an alternative anti-infective agent in natural medicine for the
treatment of infectious diseases.[5] The use of herbal medicines as dietary supplements to fight
or prevent common diseases is increasing. These herbal products have fewer side effects and are
easily available at affordable cost.[6] Bromelia enguin L. fruit is an ovoid pointed berry of up to
5 cm length, the unripe fruit is green, changing to yellow after ripening. Before consumption,
the fruits are peeled and commonly heated or roasted to inactivate pinguinain, which can
damage palate tissue; this protease has been proposed as a meat tenderizer and for use in the
preparation of biologic detergents.[7] The juice of the fruit has been used as a refreshing
beverage as well as an anthelmintic agent.[8] The proteic fraction obtained from the juice of
Bromelia pinguin L fruits has shown activity against Lumbricus terrestris and Trichomonas
vaginalis.[9] In addition, extracts of the pulp of fruit have shown activity against Candida
albicans.[10] The research was carried out to isolate and characterize bioactive proteins from
the pulp of Bromelia enguin L. fruit, which have antibacterial activity, proteolytic activity, and
proteases inhibitory activity.

Materials and Methods

Fruit Collection and Storage
Ripen fruits of Bromelia pinguin L. were collected in the municipality of Tizimin, Yucatn, Mxico,
during the month of January 2013. Plants were identified on the field and their samples were
collected and brought to the laboratory for final identification with the help of literature.[11] After
collection, the fruits were selected, washed, and sanitized in chlorinated water and kept at 80C. An
extractor was used to obtain the pulp which was stored at 20C until protein extraction.

Protein Extraction
Protein extraction was performed according to Brito-Argez et al.[12] Briefly, the pulp from B. pinguin L.
fruits was mixed with Tris-HCl buffer (100 mM, pH 7.5) with cysteine (10 mM) and leupeptin (1.5 g/mL)
1:1 (w/v). The pulp was manually homogenized for 5 min at 4C and filtered through a cotton mesh. The
filtrate was centrifuged at 16,000 g for 30 min at 4C. The recovered supernatant is the crude protein
extract (CPE), which was then ultracentrifugated at 100,000 g or 45 min. The supernatant recovered in
this stage is the soluble protein extract (SPE); finally, the pellet re-suspended in Tris-HCl buffer is the
membrane protein extract (MPE). The extracts were stored at 20C until analysis. Protein content in CPE,
SPE, and MPE was estimated using a dye-binding colorimetric assay for measuring total protein
concentration.[13] In this assay, Coomassie Brilliant blue binds to the proteins in the fractions, as well as
to standards produced from bovine serum albumin stock (1 mg/mL) to generate a curve from 01 mg/mL.

Determination of Protein Profile and Molecular Weight

Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed
according to Bizani et al.[14] For the preparation of the samples, 5 g of each extract were taken and

diluted in 6 l of loading buffer (5); distilled water was added to achieve a final volume of 30 L.
The samples were incubated for 5 min at 95C in order to favor the denaturation of proteins. Gels
composed of two phases were used. The first was prepared with 4% of acrylamide in Tris-HCl buffer
(1.0 M, pH 6.8), the second was prepared with 15% of acrylamide in Tris-HCl buffer (1.5 M, pH 8.8).
Gels were run at 100 V for 2 h at room temperature. In order to obtain a good definition of bands
with high molecular weight, the method of silver nitrate staining was employed, and for bands with
low molecular weight, the method of Coomassie blue staining was used.[15] The relative molecular
weight of the polypeptides of samples was determined by plotting a standard curve of log MW versus
Rf for known samples (ladder), and reading off the log MW of the sample after measuring distance
migrated on the same gel.

Gel Filtration Chromatography (GFC) and Reverse-Phase High-Performance Liquid

Chromatography (RP-HPLC)
The protein extracts were fractionated by GFC. Briefly, 10 mL of extract was placed into a Sephadex
G-25 column (0.5 cm diameter 70 cm length) which had been previously equilibrated with 100
mM Tris-HCl buffer (pH 7.5). The volume of sample injected was 1.0 mL at a protein concentration
of 1.1683 mg/mL. The flow rate employed was 4 mL/min at 4C. Fractions of 1.0 mL were collected
and read at 280 nm. Samples were dissolved in deionized water and injected in a preparative HPLC
(Agilent, Model 1110) reverse-phase column (C18 Hi-Pore RP-318, 250 10 mm BIO-RAD
column).[16] The injection volume was 100 L, and the sample concentration was 1 mg/mL.
Elution was achieved by a linear gradient of acetonitrile in water (030% in 50 min) containing
0.1% trifluoroacetic acid at a flow rate of 4 mL/min at 30C. Elution was monitored at 280 nm.

Culture Preparation
Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 stock cultures were grown in
Luria Broth (LB) and sub-cultured every 4 weeks to maintain viability. To obtain working cultures, a
loopful of culture was inoculated into LB and incubated for 24 h at 37C. After incubation, the
cultures were diluted to 1 106 CFU/mL.

Disk Diffusion Test for Antibacterial Activity

Disk diffusion test[17] was conducted using sterile Petri dishes containing Muller Hinton-Agar. The
bacterium was spread over the plates. A sterile blank paper disc (0.625 cm in diameter) with Tris-
HCl buffer was placed on the agar. 59 L of sample at a concentration of (70 mg/mL) was added to
one of the disks. Ampicillin (5 mg/mL) was added to the control disc. The plate was incubated at 37
C for 24 h. A transparent ring around the paper disc signified antibacterial activity.

Micro-Broth Dilution Assay

Sterile 96-well microtiter plates with a well capacity of 300 L were used for micro-broth dilution assay.
A total volume of 250 L, consisting of 125 L of double strength LB, 100 L of sample and 25 L of
inoculum (1 106 CFU/mL), was used to determine minimum inhibitory concentrations of samples.
Microtiter plates were covered with a sterile lid and incubated for 24 h at 37C and absorbance (630
nm) of each well was read at 0, 3, 6, 12, and 24 h with a microtiter plate spectrophotometer Thermo
Scientific Multiskan FC. Waltham, Massachusetts, USA. Finally 100 L of triphenyltetrazolium
chloride (2% w/v) were added to each plate and read at 485 nm. The micro-broth dilution assays[17]
were performed in triplicate.

In Vitro Antibacterial Studies

Kinetic studies of the strains exposed to different antimicrobial agents were performed as reported
by Schneider et al.[18] with a slight modification. Briefly, bacterial strains were grown overnight in LB
medium and diluted in NaCl (0.85%) to an optical density at 600 nm of approximately 1. Total
extracts and different antimicrobial agents were added in concentrations corresponding to 2 of its
MIC to obtain a final bacterial OD 600 of approximately 0.1. The final mixtures were incubated at
37C and the OD 600 of the samples was determined every hour for a total time of 6 h.

Proteolytic Activity Assay

This assay was performed according to Abreu-Payrol et al.[19] In a test tube 100 L of sample was mixed
with 1100 L of substrate (casein 1%, w/v) in Tris-HCl buffer (0.05 mM, pH 8.0) with cysteine (5.0 mM).
The mixture was incubated at 37C for 20 min, and then quenched by adding 1800 L of trichloroacetic
acid (5%, w/v). Blanks were prepared by mixing 100 L of the sample, 1800 L of trichloroacetic acid (5%,
w/v) and 1100 L of substrate. The test tubes were then centrifuged at 7000 g for 20 min. Finally the
absorbance of the supernatant was measured at 280 nm. Each assay was performed in triplicate. The
proteolytic activity was expressed in caseinolytic units (Ucas), defined as the change in absorbance units
at 280 nm which produces 1 mL of enzyme solution, due to the digestion products of casein, soluble in
trichloroacetic acid to 5% (w/v) per minute in Tris-HCl buffer (0.05 mM, pH 8.0).

Characterization of Proteinases or Proteinaceous Proteinase Inhibitors by Substrate-Gel

Electrophoretic separation of proteinases or proteinaceous proteinase inhibitors in the samples was
performed using SDS-PAGEs followed by immersion of the gel in (1) a protein substrate solution for
detection of proteinases or (2) an appropriate proteinase solution, and then in a protein substrate
solution for detection of proteinase inhibitors.[20]

Results and Discussion

Determination of Protein Profile and Molecular Weight
Three types of protein extracts were obtained from the pulp of Bromelia pinguin L. fruit which were
denominated: CPE, SPE, and MPE. The highest concentration of protein was found in CPE (1.22 mg/mL),
followed by SPE (1.17 mg/mL), and MPE (0.79 mg/mL). CPE and SPE showed a similar electrophoretic
profile, indicating that most of the proteins present in CPE are soluble, in contrast to those associated with
membrane (Fig. 1). For CPE and SPE the pattern of the gel showed two groups of distinctive bands, the first
one with high molecular weights ranging from 17 to 142 kDa and the second one with low molecular
weights ranging from 3 to 8 kDa. One distinctive band was detected in the three protein extracts with a
molecular weight of 23 kDa.
The fruits of Bromelia pinguin contain a protease of low molecular weight called pinguinain
(crude extract [CE] which is immunologically related to fruit.[21] Abreu-Payrol et al.[7]
reported the presence of four cysteine endopeptidases with weights ranging from 23.23 to 23.68 kDa
in fruits of Bromelia pinguin from Cuba. There are three potential roles for cysteine endopeptidases
in plant defense: perception of the pathogen, activation of downstream signaling pathways, and
execution of a defense response.[22] This protease family has drawn attention as a potential pharma-
ceutical drug target in diseases characterized by excessive extracellular matrix degradation such as in
osteoporosis, arthritis, vascular diseases, and cancer. They have also been identified as critical
components of growth, cell differentiation, signaling, and host invasion of various human and
livestock pathogens (pathogenic parasites causing malaria, Chagas disease, and schistosomiasis), as
well as major allergens.[23,24]

Figure 1. SDS-PAGE patterns of proteins extracted from Bromelia pinguin L. fruit pulp. Lane 1: molecular marker; Lane 2: crude
protein extract; Lane 3: soluble protein extract; Lane 4: membrane protein extract. A: Silver staining; B: Coomassie staining.


The chromatography elution profile (Fig. 2) showed the fractionation of SPE into two peaks. A total
of 35 fractions (1 mL) were collected at a flow of 0.25 mL/min. The first peak (P1) consisted of
fractions 610 and the second one (P2) consisted of fractions 1420. Only P1 exhibited protein
content with 0.0776 mg/mL. Therefore, it refers to the presence of other components in the SPE,
such as phenolic compounds which absorb at the same wavelength (280 nm).[25] For P1 the SDS-
PAGE gel pattern showed two groups of distinctive bands, the first one with high molecular weights
ranging from 17 to 142 kDa and the second one with low molecular weights ranging from 3 to 8
kDa. For P2, in the SDS-PAGE gel pattern no bands were observed.
Additionally, SPE and P1 were injected into a RP-HPLC column (Fig. 3) in order to evaluate
whether an adequate purification of the extract was performed, given that the electrophoretic profiles
of the two samples were similar. In the chromatogram corresponding to SPE, peaks were observed
distributed between the retention times of 222 min, while in the chromatogram corresponding to
P1, peaks were observed between the retention times of 2 to 12 min. This shows that CFG effectively

Figure 2. Chromatography elution profile of soluble protein extract of Bromelia pinguin fruit pulp, on fractionation using gel
filtration chromatography.

Figure 3. Chromatograms obtained by reverse-phase high-performance liquid chromatography. A: Profile of soluble protein
extract (SPE); B: Profile of peak one (PI).

removes the non-proteic components from SPE. This is the first report of purification of proteins
present in the fruit of Bromelia pinguin.

Disk Diffusion Test for Antibacterial Activity

Antimicrobial activity was assessed using the following rating system: (1) <10.0 mm zone of
inhibition may be expressed as inactive; (2) 10.013.0 mm zone of inhibition is partially active; (3)
14.019.0 mm zone of inhibition is active; (4) >19.0 mm zone of inhibition is very active.[26] The
diameter of the inhibitory zone of PSE on E. coli ATCC 25922 and S. aureus ATCC 25923 is
presented in Table 1. The results presented in Table 1 are averages of three trials of disk diffusion
tests. PSE have significant antibacterial effect on both microorganisms with diameters of inhibitory
zone of 1.2 and 1.3 mm for E. coli ATCC 25922 and S. aureus ATCC 25923, respectively. Thus, PSE
was partially active against both microorganisms.
Subsequently, GFC antimicrobial activity was evaluated for P1 and P2. P1 was partially active
against both microorganisms and P2 was inactive. In order to confirm that the antimicrobial
activity was due to the presence of proteins, P1 was subjected to two treatments. In the first
treatment, the sample was heated at 95C for 30 min in order to denature the proteins. In the
second, proteinase K (20 mg/mL) was added in order to break peptide bonds. After the thermic
treatment, P1 remained partially active against both microorganisms (Table 1). These results
suggest that proteins are exceptionally heat-stable. The stability of proteins at high temperatures
is due to many factors[27] which include the co-occurrence of metabolites and sugars and the
presence of polar amino acids. Specific polar residues (Asp, Glu, Lys, Arg, Tyr) can enhance the
occurrence of intra-subunit ion-pair formation which confers thermal stability to the proteins.
This kind of proteases has great potential in the food, biotechnology, and pharmaceutical

Table 1. Extension of zone of bacteria growth inhibition around filter papers with samples in mm.
Sample E. coli ATCC 25922 S. aureus ATTC 25923
SPE 12.0 0.30 13.0 0.40
P1 13.0 0.30 12.0 0.70
P2 8.0 0.50 0.0
P1 + thermic treatment 12.0 0.70 12.0 0.90
Ampicillin (2 mg/mL) 10.0 0.10 25.0 0.10
SPE: soluble protein extract; P1: peak one; P2: peak two.

industries given their activity over a wide range of temperatures and pH. In contrast, after the
hydrolytic treatment with proteinase K, P1 was found to be inactive against both microorganisms
(Table 1). These results suggest that the antibacterial activity of P1 is caused by the proteins
extracted from the B. pinguin fruit pulp.

Micro-Broth Dilution Assay

The results of the micro-broth dilution assay showed that P1 inhibited the growth of both strains; E.
coli ATCC 25922, with 0.279 mg/mL and S. aureus ATCC 25923 with 0.594 mg/mL. The MIC values
for the ATCC strains exposed to ampicillin were 0.008 and 0.014 mg/mL for E. coli ATCC 25922 and
S. aureus ATCC, respectively. Po-Len et al.[28] evaluated the antibacterial activity against E. coli
ATCC 29213 and S. aureus ATCC 29212 of methanolic extracts (ME), and fractions obtained with
ethyl acetate (EaF) and water (AqF) from B. pinguin fruit pulp.[28] The MICPlease spell out MIC.
values were 16 mg/mL (CE); 4 mg/mL (EaF) and 16 mg/mL (AqF) against E. coli, and 16 mg/mL
(CE); 8 mg/mL (EaF) and 16 mg/mL (AqF) against S. aureus. According to these authors the
observed antibacterial activity of extract/fractions of B. pinguin fruit could be associated with the
content of phenolics. Fruits are rich in phenolics, tannins, and flavonoids[29] In the present study, the
MIC values were significantly lower indicating that proteins of B. pinguin fruit pulp are more active
against microorganisms than phytochemicals.

In Vitro Antibacterial Studies

In this study, growth kinetic studies of E.coli and S. aureus were determined by analyzing and
comparing protease inhibitors (PI) with four antibiotics (ampicillin, tetracycline, rifampicin, and
vancomycin). This study, unlike an MIC assay, allows the determination of the bactericidal activity
of P1.[30] The killing kinetics of P1 against tested strains is shown in Fig. 4. P1 exhibited bactericidal
effect at 0.558 mg/mL concentration against E. coli ATCC 25922 and at 1.188 mg/mL concentration
against S. aureus ATCC 25923.
Growth inhibition of P1 against E. coli ATCC 25922 could be compared with the mechanism of
inhibition of rifampicin. Rifampicin is active against both gram-positive and gram-negative bacteria
and its mechanism of action is based on its ability to bind to and inactivate the bacterial DNA-
dependent RNA polymerase, a mode of action not shared by any other antibiotic in use.[31] Growth
inhibition of S. aureus ATCC 25923 showed that P1 is similar to that reported for tetracycline. The
tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of
aminoacyl-tRNA to the ribosomal acceptor (A) site. Tetracyclines are broad-spectrum agents,
exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical
organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites.[32]

Figure 4. Time kill studies of E. coli ATCC 25922. A: and S. aureus ATCC 25923; B: exposed to ampicillin (AMP); tetracycline (TET);
rifampicin (RIF); vancomycin (VAN); and Peak one (P1).

Characterization of Proteases by Substrate-Gel Electrophoresis

Zymography technique facilitated the detection of proteolytic bands that contrasted very well in the
blue background of undegraded protein substrate in stained polyacrylamide gel. Fig. 5a showed
discrete lytic zones that represent the presence of proteolytic enzymes. Comparatively, discrete
proteolytic bands were noticed in the gel copolymerized with casein as substrate, where lytic zones
(white bands) were observed in the gel. The SPE and peak one obtained by GFC had similar profiles.
A zone of proteolytic activity observed within a range of 30250 kDa. Proteases may be contributing
to the antibacterial activity of the SPE and P1. The proteases can be found in the vacuole,[33]
lysosome,[34] and chloroplasts,[35] and have also been found extracellular, in the apoplast.[36] The
proteases catalyze the cleavage of peptide bonds of proteins or polypeptide chains. This feature plays
an important role in many biological processes such as protein degradation, and protein processing,
as well as plant defense against pathogens.[37]

Characterization of Proteinaceous PI by Substrate-Gel Electrophoresis

Other compounds related to plant defense against herbivores and pathogens are PI, which are
constitutively expressed in seeds, tubers, and in other storage organs, and are induced in injured
plants.[38] Because PI can affect bacterial growth, the presence of such compounds was determined in

Figure 5. SDS-zymograph of proteases extracted from Bromelia pinguin L. fruit pulp. Lane 1: molecular marker; Lane 2: soluble
protein extract; Lane 3: Peak 1; Lane 4: Peak 2.

this study. In Fig. 5, a substrate-SDS-PAGE gel showed PI. SPE has a greater content of this type of
proteins with molecular weights ranging from 57200 kDa and from 2950 kDa. Meanwhile, in P1
only two bands were observed with weights of 43 and 75 kDa; these proteins could be inhibitors.
Proteases are essential in the development and growth of microorganisms; some pathogens are
known to produce extracellular proteases, which may be involved in disease development. Plants
synthesize PI to respond to the attack of microorganisms and suppress proteolytic enzyme activity,
which results in a potent inhibition of the growth of a variety of bacterial and fungal strains.[39]

Antibacterial protein fraction specific to Escherichia coli ATCC 25922 and Staphylococcus aureus
ATCC 25923 was obtained by GFC from fresh fruit of Bromelia pinguin L. Protein fraction inhibited
the growth of E. coli with 0.279 mg/mL and S. aureus with 0.594 mg/mL. In vitro antibacterial studies
indicated that the protein fraction acted as rifampicin against E. coli and as tetracycline against S.
aureus. SDS-PAGE analysis indicated the presence of enzymes, PI and peptides in the protein
fraction. This kind of molecules plays an important role in plant defense process against pathogens.
The results indicate that proteins from the pulp of B pinguin could be antimicrobial agents, naturally
occurring and non-toxic, which may be used for the treatment of human diseases caused by
pathogenic bacteria due to the fact that their mechanisms of action are similar to that exhibited
by antibiotics which are chemically synthesized and pharmaceutically employed.

This research forms part of the project 4577.12-P Anlisis del extracto proteico de Bromelia pinguin con posible
aplicacin teraputica y/o biotecnolgica, financed by Tecnolgico Nacional de Mxico.

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