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
Article views: 94
Download by: [UNAM Ciudad Universitaria] Date: 22 February 2017, At: 16:19
INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. 1, 220230
http://dx.doi.org/10.1080/10942912.2016.1154572
Introduction
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 eortiz@itmerida.mx 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 www.tandfonline.com/ljfp
2017 Taylor & Francis Group, LLC
INTERNATIONAL JOURNAL OF FOOD PROPERTIES 221
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.
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.
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.
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.
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.
Figure 2. Chromatography elution profile of soluble protein extract of Bromelia pinguin fruit pulp, on fractionation using gel
filtration chromatography.
INTERNATIONAL JOURNAL OF FOOD PROPERTIES 225
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.
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.
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).
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]
Conclusion
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.
Funding
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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