Industrial Crops and Products 87 (2016) 150–160

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

The anti-biofilm potential of commonly discarded agro-industrial

residues against opportunistic pathogens
Sergio Luiz de Almeida Rochelle a , Janaina de Cássia Orlandi Sardi a , Irlan Almeida Freires a
, Lívia Câmara de Carvalho Galvão a , Josy Goldoni Lazarini a , Severino Matias de Alencar b ,
Pedro Luiz Rosalen a,∗
a
Department of Physiological Sciences, Piracicaba Dental School, University of Campinas, Avenue Limeira 901, 13414 903 Piracicaba, SP, Brazil
b
Department of Agro-Food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Avenue Padua Dias 11,
13418-900 Piracicaba, SP, Brazil

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

Article history: Brazil is one of the largest producers of agro-industrial residues in the world. The aim of the study was
Received 8 December 2015 to evaluate the anti-biofilm potential of hydroalcoholic extracts from agro-industrial residues. Organic
Received in revised form 11 March 2016 residues from different parts were obtained from companies and subjected to hydroalcoholic extraction.
Accepted 19 March 2016
A total of 14 extracts from the species Theobroma cacao (cocoa), Coffea arabica (coffee), Psidium guajava
(guava), Citrus sinensis (orange), Malus domestica (apple), geopropolis, Punica granatum (pomegranate)
Keywords:
and Solanum lycopersicum (tomato) were obtained and screened for their MIC/MBC against reference
Agro-industrial residues
microorganisms (eleven bacterial strains and one yeast strain). The most bioactive extracts were chem-
Adhesion
Biofilm
ically characterized by GC/MS and tested for their ability to inhibit adhesion, kill mature biofilm cells,
Opportunistic pathogens and disrupt biofilm morphology. Eight extracts showed antimicrobial activity with MIC values ranging
from 31.25 to 2000 ␮g/ml and MBC/MFC ranging from 62.50 to 4000 ␮g/ml. The extracts of geopropolis,
pomegranate batchs #1 and #2 and coffee were able to inhibit adhesion at their MICs by 23% to 80%
depending on the strain. At 10×MIC, three extracts also inhibited mature biofilm of seven pathogens.
Toxicity tests were performed in vivo with the extracts of geopropolis, pomegranate 1 and pomegranate
2 in G. mellonella larvae. At the dose of 50 mg/kg, the extracts did not exert considerable acute toxic effects
in the larvae over a period of 72 h. The extracts from agro-industrial residues have promising anti-biofilm
activity against opportunistic pathogens of clinical relevance in the medical and dental settings.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction pounds with antioxidant and antimicrobial activity (Dailey and

Throughout the history of mankind, naturally-occurring prod-
ucts have been considered as a valuable source of bioactive
molecules for drug development in several areas of medicine and
dentistry (Cragg and Newman, 2013). With more than 43,000 native
plant species cataloged, Brazil holds more than 20% of the world’s
biodiversity, generating a large number of research opportunities
for the discovery of new molecules (Brazil, 2015). As a country
of agriculture, Brazil is also a major producer of agro-industrial
residues which are commonly discarded by the industry. The search
for alternatives to make use of the organic (waste) material gen-
erated is growing within several research centers, especially by
center producers and industrialists, particularly interested on com-
∗ Corresponding author at: Department of Physiological Sciences, Piracicaba Den-
tal School, University of Campinas, 901 Limeira Ave., 13414 903, Piracicaba, SP,
Brazil.
E-mail address: rosalen@fop.unicamp.br (P.L. Rosalen).
Vuong, 2015; Leouifoudi et al., 2015).
Agro-industrial residues and agricultural byproducts are con-
stantly being despised and discarded as waste in water fountains
causing environmental degradation. The study and use of the com-
pounds existing in these materials are important not only to reduce
the amount of pollutants input, but also to aggregate value and find
substances that have biological activity and therapeutic potential,
thus contributing to sustainability of residue-producing compa-
nies. Agro-industrial residues can be potentially used as energy
sources (Moreira et al., 2008), alternative animal feed (Pandeya
et al., 2000), fertilizers for plants (Alburquerque et al., 2012) and
sources of chemical compounds with medicinal purposes (Liu et al.,
2010). A number of phytochemical classes have been identified
in residue-derived extracts, including saponins, flavonoids, thio-
sulfates, tannins, quinones, alkaloids, organic acids, in addition to
phenolic compounds (Tajkarimi et al., 2010).
The seeds (beans) of the tree Theobroma cacao L., commonly
found in tropical areas, are used by the industry to produce cocoa

http://dx.doi.org/10.1016/j.indcrop.2016.03.044
0926-6690/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160
Fig. 1. Inhibitory effects on biofilm adhesion. Biofilms were allowed to grow and treated with the extracts at MIC. All extracts showed inhibitory effects ranging from 23%
by coffee dreg on S. mutans to 80% by geopropolis on S. epidermidis. The values of the graph are compared with the measure of untreated biofilms (100% adherence) for each
strain (* P < 0.05, one-way ANOVA, with Tukey’s post-test. # P < 0.05, Student’s t test).
products, including chocolate. In the meantime, the part of the
cocoa bean that enfolds the nibs, named shells or husks, are com-
monly discarded during the production of cocoa powder and butter
(Bertoldi et al., 2016). As the cocoa shell is estimated to account for
52–76% of the cocoa fruit, its disposal generates large amounts of
waste (Chan and Choo, 2013).
Coffea Arabica L. produces one of the crops mostly used my
mankind throughout the history. Coffee is part of the daily routine
of millions of people worldwide. As a result, the generation of sol-
uble coffee processing waste is very high. The residues are mainly
produced during the industrial process of washing and removal of
the pulp from the fruit. The residues include parts of the pulp, bark,
mucilage and residual water. In the process of obtaining the coffee
beverage, a second residue is generated, the coffee dregs (Mussatto
et al., 2011; Murthy and Naidu, 2012).
Psidium guajava L. is widely known worldwide due to the use
of its fruits, guava, in natura or in food products (juices, jams, ice
creams, syrups etc.). The pomace residues resulting from industrial
processing of guava—mainly the seeds—consist of 10–15% of the
whole fruit (Schieber et al., 2001).
Citrus sinensis (L.) Osbeck is the source of orange, a well-known
citrus fruit, which can be consumed fresh or through a wide range
of food products (juices, syrups, jams, candy, etc.). One of the main
issues faced by orange juice processing industries is the large vol-
ume of solid and liquid residues generated every day. The solid
waste consists of the pressed fruit peel, seeds and pulp, and is
mostly transformed into pellets—commonly used as animal feed
additive. Among the liquid residues, the processing water has
aroused more attention due to its high levels of organic matter and
therefore high polluter potential.
Malus domestica Borkh. is a medium-sized tree growing in tem-
perate regions better known for its fruits, apples. It is estimated that
more than 60% of the apple production worldwide is commercial-
ized and consumed in natura (Alberti et al., 2016). However, there
is an important industrial use of apple in juices, syrups, jams, baby
food, vinegar etc., and, in turn, production of waste. For instance,
151
apple pomace (containing the fruit peel, pulp, seeds, and stems)
generated upon juice pressing can consist of up to 25% of the weight
of the fresh fruit (Rha et al., 2010).
Geopropolis is a different type of propolis whose resin is col-
lected by native stingless bees (Melipona spp.), which also contains
clay in its composition (da Cunha et al., 2013). To date, geopropolis
has not been used by the food and pharmaceutical industry, but
recent studies have shown that the extract of geopropolis and its
isolated compounds have biological activity (da Cunha et al., 2013,
2016). After production of the geopropolis extract, the remaining
material exposed to the organic solvents is considered the residue
of geopropolis and is commonly discarded.
P. granatum L. (pomegranate) is a plant species commonly
used in folk medicine and to a lesser extent, by the food indus-
try for production of juices and jams. The industrial processing of
pomegranate generates pomace residues with promising biological
properties (Tehranifar et al., 2011). The byproduct of pomegranate
is named pomace and contains about 78% of peel and 22% of seeds
in relation to wet weight (Qu et al., 2009).
Solanum lycopersicum L., popularly known as tomato, is widely
used by the industry for development of numerous food products,
particularly sauces and ketchup. The processing rates of tomato in
Brazil are estimated to 1.28 million tons, which generates approx-
imately 256,000 tons of waste, causing 20% of loss in production
during processing. Tomatoes residues are mainly formed by grind-
ing of seeds, peel and stem (Silva et al., 2009).
All these agro-industrial residues were studied herein due to
their potential has a new source of bioactive molecules with
biological properties. Some authors have demonstrated the use
of residues with antimicrobial potential against several Gram-
positive and Gram-negative microorganisms, as well as yeasts and
other fungi (Moody et al., 2004; Tarfa et al., 2004; Ponnusamy et al.,
2010; Zottich et al., 2011; Shad et al., 2014). Nevertheless, there
is no data in the literature on the potential of residues against
biofilms of opportunistic pathogens, such as streptococci, staphy-
lococci, Pseudomonas spp., Escherichia spp., among others. It is well
152 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160

Fig. 2. Effects of selected bioactive extracts on mature biofilm survival. Mature biofilms were developed, treated with the most bioactive extracts at 10x their MICs and
plated for CFU/ml. All data were compared to the group of untreated biofilm (One-way ANOVA with Tukey’s post-test, P < 0.05).

known that the ability to form biofilm confers advantages to the
microorganism when compared to its planktonic form. Hence, the
use of substances able to disrupt biofilm integrity and viability and
thus prevent infection is highly desirable, particularly in the current
scenario of microbial resistance and adverse effects of synthetic
drugs.
The present study reports on the bioprospection of fourteen
agro-industrial organic residues for their antimicrobial activity
against pathogenic biofilms, including effects on biofilm forma-
tion, adhesion and morphology. In the perspective of future clinical
use, the most bioactive residues were also tested for their systemic
toxicity in vivo using Galleria mellonella model.
2. Material and methods
2.1. Standards and reagents
Organic solvents were purchased from Merck (Germany);
Sabouraud Dextrose Agar, Yeast Nitrogen Base broth, and
Brain Heart Infusion were acquired from Difco® (Detroit,
MI, USA); resazurin, XTT (2,3-Bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt), TTC
(2,3,5-triphenyltetrazolium chloride) (Sigma-Aldrich, St. Louis,
MO, USA), Chlorhexidine digluconate, Gentamicin, Vancomycin
hydrochloride and Nystatin were obtained from Sigma-Aldrich
(St. Louis, MO, USA); chambered glass slides were purchased from
Corning® (BD Falcon, Bedford, MA, USA) and polystyrene plates
were obtained from TPP® (Trasadingen, Switzerland).
2.2. Plant material
Organic residues from different parts of eight plant species:
T. cacao L. (cocoa), C. Arabica L. (coffee), P. guajava L. (guava), C.
sinensis (L.) Osbeck (orange), M. domestica Borkh. (apple), geo-
propolis, Punica granatum L. (pomegranate) and S. lycopersicum L.
(tomato) were obtained from companies and suppliers through an
agreement between Embrapa (Brazilian Agricultural Research Cor-
poration), Luiz de Queiroz College of Agriculture (ESALQ) at the
University of São Paulo (Piracicaba, SP, Brazil), and Piracicaba Den-
tal School at the University of Campinas (Piracicaba, SP, Brazil).
The plant material—which is commonly considered as waste—was
Table 1
Results of MIC-MBC/MFC of fourteen crude hydroalcoholic extracts from agro-industrial residues against twelve opportunistic pathogens with clinical relevance. The results are expressed in ␮g/ml.

S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160

Id Extract Residue C. albicans E. coli F. nucleatum P. gingivalis P. intermedia P. aeruginosa S. aureus MRSA S. epidermidis S. mitis S. mutans S. parasanguinis

MIC/MFC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC MIC/MBC

1 Cocoa Shell – – 1000/# – – – – – – – 1000/2000 –

2 Coffee Straw – – – – – – – – – – – –
3 Coffee Straw – – – – – – – – – – – –
4 Coffe Bark – – – – – – – – – – – –
5 Coffe Dreg – – – – 1000/# – 1000/2000 – – – 250/500 –
6 Guava Pomace – – – – – – – – – – – –
6* Guava Pomace – – 500/# 1000/# – – – – – – – –
7 Orange Pomace – – – – – – – – – – – –
7* Orange Pomace – – – – – – – – – – – –
8 Orange Clear Water – – – – – – – – – – – –
8* Orange Clear Water – – – – – – – – – – – –
9 Orange Dark Water – – – – – – – – – – – –
9* Orange Dark Water – – – – – – – – – – – –
10 Apple Pomace – – – – – – – – – – – –
10* Apple Pomace – – – – – – – – – – – –
11 Geopropolis Residue – – 1000/# – 500/# – 125/# 125/# 62.5/# 2000/# 250/# 31.25/62.5
12 Pomegranate 1 Pomace 250/# 2000/# – – – – – 2000/# – 2000/# – –
12* Pomegranate 1 Pomace 62.5/# 2000/# – – – – – 1000/# – 2000/# – 125/#
13 Pomegranate 2 Pomace 500/# 2000/# – – – – – 2000/4000 – 2000/# – 125/#
13* Pomegranate 2 Pomace 250/# 2000/# 1000/# – 500/# 125/# 500/# 500 – 500/# – 125/#
14 Tomato Pomace – – – – – – – – – – – –
14* Tomato Pomace – – – – – – – – – – – –

Note: (*) sample without sugar content; (−) absence of antimicrobial activity; (#) CBM/CFM value higher than 4000 ␮g/ml.

153
154 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160
grounded, lyophilized, homogenized, weighed and then stored at
−20 ◦ C.
2.3. Preparation of extracts
The residues were subjected to hydroalcoholic extraction (80%
EtOH/H2 O and 1:10, v/v) for 48 min using an ultrasound appa-
ratus. The obtained extracts were filtered, rotaevaporated and
freeze dried to give the crude dried extracts (Ribeiro et al., 2007;
Tiveron et al., 2012). A total of fourteen extract were obtained, dried
and stored at −20 ◦ C until use. For some extracts, such as guava,
orange, apple, pomegranate and tomato, a solid-phase extraction
of sugars was performed in order to concentrate active compounds
(with less interferences) leading to increased biological activity.
The hydroalcoholic extracts of sugar-containing residues were sub-
jected to solid-phase extraction, as previously described (Tiveron
et al., 2012).
2.4. Phytochemical analysis by gas chromatography coupled to
mass spectrometry (GC–MS)
Gas Chromatography analyses were performed in a gas chro-
matograph (Shimadzu model GC 2010) coupled to a mass
spectrophotometer (Shimadzu QP 2010 Plus) using a capillary col-
umn (RTX5MS 30 m × 025 mm × 025 ␮m). GC–MS was carried out
at an initial column temperature of 80 ◦ C for 1 min, then 200 ◦ C at a
heating ramp of 5 ◦ C/min for 1 min, 250 ◦ C at a rate of 5 ◦ C/min for
8 min, 300 ◦ C at a rate of 10 ◦ C/min for 5 min, and final temperature
of 320 ◦ C at a rate of 10 ◦ C/min for 10 min, totalizing 66 min of anal-
ysis. Helium was used as a carrier gas. The injector temperature was
280 ◦ C and the split injection volume was 0.1 ␮l at a ratio of 1:30.
For geopropolis residues, GC–MS was carried out at an initial col-
umn temperature of 80 ◦ C for 1 min, then 320 ◦ C at a heating ramp
of 5 ◦ C/min for 1 min, for a total period of 69 min of analysis. The
injector temperature was 280 ◦ C and the splitless injection volume
was 0.4 ␮l. The data integration was completed on the software
LabSolutions (GCMS solutions) and compounds were detected by
comparison with the mass spectra data in Wiley 8 libraries based
on their mass-to-charge ratio (m/z) (Tiveron et al., 2012).
2.5. Microorganisms and growth conditions
A total of twelve reference strains were used in this study:
Staphylococcus aureus (ATCC 25923), S. aureus MRSA (ATCC 33591),
S. epidermidis (ATCC 12228), Escherichia coli EHEC (ATCC 43895),
Pseudomonas aeruginosa (ATCC 27853), Streptococcus mutans
(UA159), Staphylococcus. parasanguinis (ATCC 903), Staphylococcus
mitis (ATCC 7073), Porphyromonas gingivalis (ATCC 25586), Pre-
votella intermedia (ATCC 25611), Fusobacterium nucleatum (ATCC
25586) and Candida albicans (CBS 562). All strains were stored at
−80 ◦ C until use and grown in appropriate culture media and under
specific atmosphere and temperature conditions.
2.6. Determination of the minimum inhibitory concentration
(MIC) and minimum bactericidal/fungicidal concentration
(MBC/MFC)
The MIC microdilution tests were carried out in accordance
with the CLSI protocols for bacteria (CLSI, 2012) or yeasts (CLSI,
2002), with modifications. Ethanol (17.5%, v/v) and DMSO (1%, v/v)
were used as a vehicle for geopropolis and for the other extracts.
Chlorhexidine digluconate, Gentamicin, Vancomycin hydrochlo-
ride and Nystatin were used as positive controls. Bacterial and
fungal inocula were prepared (625 nm and 530 nm, respectively,
with absorbance 0.08–0.1) and diluted to reach final concentra-
tions of 1.0 × 105 CFU/ml and 2.5 × 103 CFU/ml, respectively. Plates
were incubated under different conditions based on strain require-
ments (35 ◦ C/37 ◦ C, 5% CO2 /1 atm) for 24 h. The MIC100 was defined
as the lowest concentration of the agent that inhibited visible
microbial growth indicated by 0.1% resazurin. The MBC/MFC was
determined by subculturing an aliquot from each well with a con-
centration equal to or higher than the MIC on solid culture media.
The MBC/MFC was defined as the lowest concentration of the agent
causing no visible growth on the solid media.
2.7. Inhibition of biofilm adhesion
This assay was carried out with the most bioactive extracts
showing MIC values lower than 500 ␮g/ml, which has been con-
sidered as the cut off point for a strong antimicrobial activity
(Freires et al., 2015). Initially, 100 ␮l of a 1 × 108 cells/ml culture
in saline were added to the wells of a 96-well plate. The plates
were incubated at 35 ◦ C or 37 ◦ C at 70 rpm for 3 h for adhesion.
Then the supernatant was removed from each well of the plate.
Subsequently, 200 ␮l of the extracts at their MICs were diluted in
culture media and added to the wells. After 24 h incubation, the
supernatant was removed and the wells rinsed with 200 ␮l of ster-
ile phosphate buffered saline (PBS). The readings were performed
by spectrophotometry at 490 nm and confirmed through metabolic
analysis using XTT (for yeast) and TTC (for bacteria) (Djordjevic
et al., 2002; with modifications, Galvão et al., 2012).
2.8. Effects on mature biofilm formation
C. albicans was cultured on Sabouraud Dextrose Agar at 35 ◦ C
for 24 h. A loopful of the growth was inoculated into Yeast Nitro-
gen Base broth supplemented with 50 mM glucose and incubated
for 18 h at 35 ◦ C. Then cells were harvested and washed twice
with PBS (pH 7.2), suspended in YNB supplemented with 100 mM
glucose and adjusted to a density of approximately 107 CFU/ml
(Samaranayake et al., 2015). Subsequently, 200 ␮l of the suspension
were added to the wells of 96-well plates, which were incubated
at 35 ◦ C for 48 h. Biofilms were washed three times with PBS to
remove non-adhered cells.
Bacterial cultures were grown in appropriate media for 24 h and
adjusted to a density of approximately 108 CFU/ml (Lázaro-Díez
et al., 2016). Then 200 ␮l of the suspension were added into the
wells of 96-well microplates, which were incubated at 37 ◦ C for
24/48 h to allow for biofilm formation. All following procedures
were carried out as described above for yeasts.
Semi-quantitative analysis of treated biofilms. Mature fungal
and bacterial biofilms were developed in 96-well microplates as
described above. Then the wells were washed three times with PBS
to remove planktonic (non-adhered) cells. The test extracts were
added to the culture media (YNB for yeasts and BHI for bacteria) to
be at a concentration of 10xMIC. Subsequently, 200 ␮l of this mix
solution (culture media plus the extracts) were added to the wells
of the plate containing the developed biofilms and the plates were
incubated at 37 ◦ C for 24 h. The metabolic activity of the biofilms
was quantified by reduction assay with XTT (yeast) and TTC (bac-
teria) after 4 h at 37 ◦ C. A control corresponding to the extracts
alone with XTT or TTC was performed to exclude the probability
of salt reduction by compounds from the extract. Color changes
were measured using a microplate reader at 490 nm.
2.9. Killing assay (quantitative analysis)
The ability of the extracts to kill bacterial or fungal biofilm cells
was investigated as previously reported (Martinez and Casadevall,
2006), with modifications. After development and treatment with
the extracts for 24 h, biofilms were scraped from the bottom of
the wells with a micro-pipette for dissociation of the cells. Then
 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160 155

Fig. 3. Effects of geopropolis residues on the morphology/integrity of mature pathogenic biofilms. SEM photomicrographs showing S. aureus (A), S. aureus (MRSA) (B), S.
epidermidis (C), S. mutans (D) and S. parasanguinis (E) biofilms untreated (1) and treated with geopropolis residue extract at 2500 ␮g/ml (2).

serial dilutions were carried out and aliquots of each suspension
were plated onto BHI (bacteria) or Sabouraud (yeast) agar plates.
The percentage of CFU/ml (colony forming units/ml) survival was
determined by comparing the survival of treated biofilms with that
of untreated biofilms. Chlorhexidine (0.12%) was used as a positive
control in this assay.
2.10. Effects on biofilm morphology
Biofilms were first developed on tissue culture treated cham-
bered glass slides, washed with PBS buffer to remove planktonic
cells and treated with the crude extracts at a concentration 10 times
their MIC. A negative control group with untreated biofilm was also
included. After 24 h, the samples were washed and kept in 2.5%
glutaraldehyde/PBS for 2 h at room temperature. The slides were
156 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160
Fig. 4. Effects of pomegranate residues on the morphology/integrity of mature pathogenic biofilms. SEM photomicrographs showing C. albicans (A), S. parasanguinis (B) and
P. aeruginosa (C) biofilms untreated (1) and treated with pomegranate extract #1 (2) or pomegranate extract #2 (3) at 2500 ␮g/ml.
dehydrated with ethanol (from 50% to 100%) and dried. The samples
were coated with gold in a metallizer machine at 40 mA (BAL-TEC
SCD 050) and observed using a scanning electron microscope (Jeon
JSM 5600LV, Tokyo, Japan) (Freires et al., 2014).
2.11. Toxicity in G. mellonella model
This assay was carried out to evaluate the acute toxic effects of
the most bioactive extracts in G. mellonella model, as previously
described with modifications (Megaw et al., 2015). This is a rele-
vant alternative model to the use of vertebrates which allows the
preliminary assessment of systemic toxicity upon treatment with a
given drug (Megaw et al., 2015). A total of 15 larvae were randomly
selected for each group weighing between 0.2 and 0.3 g with no or
few signs of melanization. Five microliters of the extracts (10xMIC)
or controls (17.5% ethanol, 1% DMSO and saline) were injected into
the hemocoel of each larva via the last left proleg using a Hamil-
ton syringe (Hamilton, Reno, NV). The larvae were incubated in the
dark at 37 ◦ C and their survival was recorded at selected intervals
for 72 h. The larvae displaying no movements upon touch and with
high levels of melanization were counted as dead.
2.12. Statistical analysis
All assays were performed in triplicate of three independent
experiments. The data were analyzed on GraphPad Prism 5.0 using
one-way analysis of variance (ANOVA) with Tukey’s multiple com-
parison test. For the G. mellonella toxicity model, Kaplan-Meier
killing curves were plotted and estimations of differences in sur-
vival were compared using the log rank test. In all tests, type one
error (␣) was set at 0.05.
3. Results
3.1. Antifungal and antibacterial activity of agro-industrial
residue extracts
As seen in Table 1, a screening of fourteen hydroalcoholic
extracts of agro-industrial residues was carried out, including:
cocoa (shell), coffee (straw, bark and dreg), guava (pomace), orange
(pomace, clear processing water and dark processing water), apple
(pomace), geopropolis (residue), pomegranate batch #1 and #2
(pomace) and tomato (pomace). All samples were tested against
twelve different opportunistic pathogens. The results demon-
strated that eight extracts showed antimicrobial activity against the
tested strains (Gram-positive and Gram-negative), with MIC val-
ues ranging from 31.25 to 2000 ␮g/ml and MBC/MFC ranging from
62.50 to 4000 ␮g/ml (Table 1), as follows: cocoa shell, coffee dreg,
guava pomace, geopropolis, pomegranate #1 pomace (either with
or without sugar), pomegranate #2 pomace (either with or without
sugar). Most extracts were found to be active against the Gram-
positive bacteria tested (streptococci and staphylococci) while only
the extract of pomegranate #2 was effective against the Gram-
negative bacterium P. aeruginosa. The extracts of pomegranate #1
and #2 were also active against C. albicans yeasts, with lower MIC
values for sugar-free extracts.
3.2. Inhibition of biofilm adhesion in vitro
This assay was carried out with the most bioactive extracts
showing MIC values lower than 500 ␮g/ml (geopropolis residue,
pomegranate #1 and #2 pomace, and coffee dreg). At MIC value,
geopropolis residue inhibited S. epidermidis, S. aureus (MRSA), S.
aureus and S. mutans biofilm adhesion by 80%, 64%, 52% and 50%
respectively (P < 0.05); pomegranate 1 inhibited the adhesion of C.
 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160
albicans and S. parasanguinis biofilms by 55% and 62% respectively
(P < 0.05); pomegranate 2 inhibited the adhesion of P. aeruginosa
and C. albicans by 45% and 50%, respectively (P < 0.05); whereas
coffee dreg extract inhibited S. mutans biofilm by approximately
23% (Fig. 1). The extracts with inhibition values lower than 50%
were discarded and subsequent analyses were carried out only with
geopropolis and pomegranate #1 and #2 pomace.
3.3. Phytochemical analysis by gas chromatography coupled to
mass spectrometry (GC–MS)
Table 2 shows the compounds found in the hydroalcoholic
extracts of geopropolis and pomegranate #1 and #2 by GC–MS
analysis. Compound 1,2-benzenedicarboxylic acid, diethyl ester
was the most abundant compound found in geopropolis extract
(10.73%) and pomegranate #1 (31.23%), while M = 217 (not identi-
fied, 8.89%) was predominant in pomegranate #2. The compound
1,2-benzenedicarboxylic acid, diethyl ester was also found in
pomegranate #2 extract in lower proportions (0.33%). Overall,
pomegranate #1 and #2 showed similar peaks, with small differ-
ences in their percentual areas. Several compounds could not be
identified based on the device library.
3.4. Effects on mature biofilm survival
Mature biofilms were developed and treated with the most
bioactive extracts at 10x their MICs (Fig. 2). The treatment with geo-
propolis extracted led to a significant reduction of CFU/ml (between
2 and 10 Log10 ) (P < 0.05) in the biofilms of S. aureus, S. aureus
(MRSA), S. epidermidis and S. mutans; pomegranate #1 was effec-
tive against S. parasanguinis and C. albicans, with CFU/ml reduction
between 1 and 2.5 Log10 , respectively (P < 0.05); and pomegranate
#2 against P. aeruginosa and C. albicans, with CFU/ml reduction of
about 2.5 Log10 (P < 0.05). The geopropolis extract proved to be the
most active agent against the biofilms, in most cases leading to com-
plete cell elimination. Chlorhexidine was used as a positive control
and markedly led to total killing of biofilms.
3.5. Effects on biofilm morphology
SEM analysis was performed to evaluate the effects of the
most bioactive extracts on the morphology and integrity of mature
biofilms (Figs. 3 and 4). Overall, both bacterial and fungal biofilms
treated with geopropolis or pomegranate extracts (2500 ␮g/ml)
showed damaged architecture and amorphous cell cluster areas,
accompanied by reduced numbers of microbial population. All
biofilms were found to be susceptible to the action of the residue
extracts at the tested concentration. The vehicles did not affect
biofilm growth and morphology.
3.6. In vivo toxicity in G. mellonella model
Toxicity tests were performed in vivo with the extracts of geo-
propolis, pomegranate #1 and #2 in G. mellonella larvae. This
model is advantageous for its low-cost, feasibility, rapid results and,
particularly, for being an alternative to the use of mammals (ver-
tebrates) for preliminary assessment of toxicity, thus reducing the
number of sacrificed animals and refining subsequent toxicology
studies. The tested concentrations of geopropolis, pomegranate #1
and #2 were the same as those used in the biofilm assays, that is,
2500 ␮g/ml (corresponding to a dose of 50 mg/kg in the larvae).
As seen in Fig. 5, at the dose of 50 mg/kg (effective dose against
the biofilms in vitro), the extracts did not exert considerable acute
toxic effects in the larvae over a period of 72 h. Geopropolis and
pomegranate #2 caused a non-significant 10% decrease in percent
157
Fig. 5. Percentage of survival of G. mellonella over 72 h after treatment with different
extracts. The concentrations used were those found to be effective in the biofilm
assay (10xMIC of the extracts) (P > 0.05, log rank test).
survival after 24 h, whereas pomegranate #1 did not affect sur-
vival at all. The vehicles (1% DMSO for pomegranate and 17.5%
ethanol for geopropolis) did not cause considerable toxic effects
either (P > 0.05).
4. Discussion
The use of agro-industrial residues for therapeutic purposes
may constitute a sustainable resource to prevent environmental
impact while broadening the antimicrobial arsenal in medicine and
dentistry. This is a pioneer bio-guided study reporting on the anti-
biofilm potential of fourteen extracts of agro-industrial residues
(which are commonly discarded by the industry) against medically
relevant pathogens.
It was demonstrated that eight out of the fourteen extracts
screened have antimicrobial activity with low MIC values and thus
were selected for further analyses using biofilm models. There have
been reports in the literature on the antimicrobial activity of agro-
industrial residues against planktonic microorganisms (Ćilerdžić
et al., 2015; Rodrigues et al., 2015). Gerhardt et al. (2012) found that
the hydroalcoholic extracts of residues from several types of cit-
rus (Citrus reticulata Blanco, Citrus maxima (Burm.) Merr. and Citrus
limonia Osbeck) inhibited bacterial growth of pathogens, suggest-
ing their use as an alternative for disinfection and food preservation.
Martin et al. (2012) studied residues of beet stalks, peanut pellicles,
Pinot Noir grape pomace, pulp and seeds of Petit Verdot grapes,
fermentation dregs of grapes, and guava pomace against S. aureus
and L. monocytogenes with MICs ranging from 0.78 to 25 mg/ml,
which are much higher than those reported in our study. Nev-
ertheless, as these residues are different from the ones studied
herein, variations in their antimicrobial properties are expected.
The authors of the aforementioned studies pointed out that the
residues with the best antimicrobial activity were those presenting
high levels of total phenolic compounds in their chemical com-
position based on phytochemical analysis. In the present study,
GC/MS analysis revealed the major presence of the compound 1,2-
benzenedicarboxylic acid, diethyl ester (diethyl phthalate) in all
three extracts (geopropolis, pomegranate #1 and #2), although
at different proportions in each sample. Diethyl phthalate is also
present in other naturally-occurring agents and has been reported
to have antimicrobial properties (Velanganni et al., 2011). In gen-
eral, phthalates are known to be active against pathogens and to
have other pharmacological activities, including anticancer effects
(Mohansrinivasan et al., 2015). Hence, the presence of this com-
pound may contribute to the antimicrobial activity of the extracts.
Microbial adhesion to a given substrate (hydroxyapatite, resin
composite, acrylic resin, etc.) is an important step for surface col-
onization and mature biofilm formation thereon. It is mediated by
a large number of adhesins present on the cell wall of bacteria
and fungi (Sardi et al., 2014). Thus, inhibition of biofilm adhesion
constitutes a strategic target for the development of antimicrobial
158 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160

Table 2
Retention Times, % Area of Each Component, and important Ions Presents in the Mass Spectra of Silylated Compounds in Pomegranate 01, Pomegranate 02 and Geopropolis
Samples by CG-MS.

Peak Compounds Rt(min) Geopropolis(%Area*) Ion (m/z, abundance in parenthesis)

2 N.I. 5.20 0.11 84(78) 69(100) 55(65) 41(65) 27(55)

4 N.I. 15.40 0.15 217(24) 147(28) 143(14) 75(28) 73(100)
21 2-Propenoic acid, 3-phenyl-trimethylsilyl ester 18.20 3.03 205(100) 161(90) 131(87) 103(60) 75(48)
35 1,2-Benzenedicarboxylic acid, diethyl ester 19.35 10.73 177(23) 150(15) 149(100) 105(12) 76(12)
38 Benzoic acid, 3,4,5-tris trimethylsiloxy trimethylsilyl ester 27.16 0.10 459(32) 458(78) 443(33) 281(100) 73(70)
39 N.I. 28.47 0.12 313(72) 132(40) 117(74) 75(71) 73(100)
40 Oleic acid, trimethylsilyl ester 31.52 0.10 129(45) 117(63) 75(88) 73(100) 55(54)
41 N.I. 31.96 0.06 132(64) 117(78) 75(74) 73(100) 43(42)
42 N.I. 35.66 0.20 97(77) 83(88) 69(78) 57(100) 55(68)
43 N.I. 36.02 0.09 99(26) 85(72) 71(80) 57(100) 43(62)
45 N.I. 39.61 0.13 432(25) 418(24) 417(63) 73(100) 45(15
48 N.I. 41.44 0.17 111(56) 97(95) 83(100) 55(69) 43(53)
50 N.I. 41.61 0.11 529(15) 528(42) 527(100) 147(31) 73(69)
62 Resveratrol 43.33 1.40 446(13) 445(37) 444(100) 443(15) 73(68)
66 N.I. 44.05 1.04 97(52) 83(58) 73(100) 69(61) 55(69)
72 N.I. 44.69 0.46 428(81) 75(46) 73(100) 43(40) 41(16)
73 N.I. 44.84 0.67 73(100) 69(43) 57(48) 55(44) 43(46)
76 N.I. 45.25 1.44 281(25) 207(46) 147(16) 133(12) 73(100)
79 N.I. 45.68 0.84 609(22) 608(50) 607(100) 517(33) 73(85)
86 N.I. 46.60 1.17 531(25) 464(18) 463(52) 147(20) 73(100)
90 N.I. 47.18 1.33 425(41) 89(38) 73(100) 57(58) 43(62)
93 N.I. 47.58 1.73 218(100) 203(49) 133(42) 119(45) 107(42)
95 N.I. 47.89 3.32 624(33) 356(29) 147(32) 75(22) 73(100)
99 N.I. 48.67 1.94 535(16) 233(24) 147(44) 75(22) 73(100)
109 N.I. 49.85 1.17 281(25) 207(46) 147(16) 133(12) 73(100)
112 N.I. 50.21 1.28 144(37) 132(34) 131(100) 75(50) 73(62)

Peak Compounds Rt (min) Pomegranate #1 Pomegranate #2 Ion (m/z, abundance in parenthesis)

(%Area*) (%Area*)

2 N.I. 5.26 0.28 0.26 71(44) 59(100) 56(36) 43(72) 41(56)

4 1,2-Benzenedicarboxylic acid, diethyl ester 19.35 31.23 0.33 177(23) 150(15) 149(100) 105(12) 76(12)
14 N.I. 25.12 8.89 8.12 217(100) 204(44) 132(32) 129(70) 73(99)
15 N.I. 25.92 0.27 0.26 217(10) 147(14) 103(26) 74(8) 73(100)
16 N.I. 26.21 1.33 1.07 205(20) 204(100) 191(46) 147(23) 73(99)
19 N.I. 26.63 0.92 0.45 217(62) 205(34) 147(38) 103(46) 73(100)
20 N.I. 27.01 2.71 1.78 217(38) 147(36) 117(18) 103(39) 73(100)
21 N.I. 27.09 1.05 12.54 319(61) 217(25) 205(32) 147(35) 73(100)
22 1,2-Benzenedicarboxylic acid, dibutyl ester 27.27 0.68 4.63 150(9) 149(100) 104(6) 76(7) 41(15)
24 N.I. 28.21 0.59 1.46 205(19) 204(94) 191(44) 147(22) 73(100)
25 Epicatechin (silanizada) 47.07 0.66 0.24 650(23) 369(34) 368(100) 355(46) 73(56)
27 Benzeneacetic acid, alpha 3,4-tris(trimethylsilyl)oxy trimethyl silyl ester 47.80 0.27 2.07 356(21) 355(33) 147(12) 74(10) 73(100)

therapies. In this study, the residues of geopropolis, pomegranate
#1 and #2 pomace (without sugar) and coffee dreg presented the
best antimicrobial activity (based on their MICs) and thus were
tested for the ability to inhibit biofilm adhesion in vitro. At con-
centrations corresponding to their MICs, geopropolis inhibited S.
epidermidis, S. aureus (MRSA), S. aureus and S. mutans; pomegranate
#1 inhibited C. albicans and S. parasanguinis; pomegranate #2
inhibited P. aeruginosa and C. albicans; and finally, coffe dreg
inhibited S. mutans biofilm adhesion. Percent inhibition ranged
from 23% to 80% depending on the extract and strain used. Other
authors in the literature have also reported anti-adhesion activ-
ity of herbal extracts. Limsuwan et al. (2014) studied the ethanolic
extract of Rhodomyrtus tomentosa (Aiton) Hassk. against S. aureus, S.
mutans and C. albicans biofilms, and observed decreased adhesion
of biofilms treated with the sub-inhibitory concentrations of the
extracts. Gomaa and Gaweesh (2013) investigated the hydroalco-
holic extract of Egyptian propolis against oral Candida spp. adhesion
and found inhibitory concentrations ranging from 25 to 125 mg/ml.
Other studies with natural products against biofilms of opportunis-
tic pathogens have also been reported (Bakkiyaraj et al., 2013;
Wojtyczka et al., 2013; Capoci et al., 2015; Süntar et al., 2015; Veloz
et al., 2015).
It is estimated that 40% of the biofilm proteins in the cell
wall are different from planktonic cell proteins which may conse-
quently lead to non-specific binding of the antimicrobial agent (Ren
et al., 2005). Given this, the effects of the most bioactive extracts
(10xMIC) against mature biofilms were also investigated. It has
been well documented that microorganisms growing in biofilms
are much less susceptible to the action of antimicrobial agents
than are those growing in planktonic form. Studies have shown
that the effective antibiofilm concentration of antimicrobials can
be up to 10–1000 times higher than that for planktonic cultures
(MIC) grown in conventional liquid media (Costerton et al., 1999;
Sardi et al., 2014; Brilhante et al., 2015) Thus, 10xMIC was the
minimal selected concentration used in our study for the treat-
ment of mature biofilms. The results showed that geopropolis led
to a significant reduction in the numbers of CFU/ml of S. aureus,
S. aureus (MRSA), S. epidermidis and S. mutans biofilms. In addi-
tion, pomegranate extracts (#1 and #2) were also effective against
C. albicans and P. aeruginosa killing biofilm cells. To the best of our
knowledge, this is the first study in the literature on the use of agro-
industrial residues against biofilms of opportunistic pathogens.
In the perspective of future clinical use, the most bioactive
residues were also tested for their systemic toxicity using G. mel-
lonella model. This is an in vivo model for the assessment of toxicity
of drugs as well as of microbial virulence, validated in the inter-
national literature (Fuchs and Mylonakis, 2006; Desalermos et al.,
2012; Megaw et al., 2015). In the present study, the extracts of geo-
propolis and pomegranate #1 and #2 at their effective anti-biofilm
concentration in vitro did not affect the larvae survival over a period
of 72 h, thus indicating low acute toxicity according to this model.
 S.L.d.A. Rochelle et al. / Industrial Crops and Products 87 (2016) 150–160
Further research should focus on the in vivo effects of these extracts
using other methods to subsidize future clinical use.
The findings of the present study may help in the national
development of novel bioactive compounds, reduce the national
dependence on imported pharmaceutical ingredients and assist in
therapy of infectious diseases of medical and dental interest.
5. Conclusions
The extracts from agro-industrial residues of geopropolis and
pomegranate seem to have promising anti-biofilm activity against
opportunistic pathogens of clinical relevance in the medical and
dental settings, and showed low acute toxicity in vivo. These find-
ings support the use of these residues as potential candidates for
drug development under a sustainable perspective.
Conflict of interests
The authors declare no conflict of interest.
Acknowledgements
The authors thank the National Council for Scientific and Tech-
nological Development (CNPq, Brazil, grants no. 140029/2014-1
and no.308644/2011-5) and the Coordination for the Improvement
of Higher Education Personnel (CAPES-Proex, 2013) for financial
support, as well as the companies that provided the residues:
Esalq/Embrapa collaboration (cocoa shell), Café Iguaçú Paraná (cof-
fee dreg), Citrovita (orange pomace and processing water), Yakult
Santa Catarina (apple pomace), Cepera São Paulo (guava and tomato
pomace), Marcos Cunha (geopropolis residues), Obatã Café São
Paulo (coffee straw and dreg). The authors also thank the Depart-
ment of Morphology (Microscopy Core), Piracicaba Dental School,
University of Campinas, for the technical support in the scanning
electron microscopy analysis.
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