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Research on Chemical Intermediates ISSN 0922-6168 Res Chem IntermedDOI 10.1007/s11164-013-1267-0
Potential effluent oil mills and antibacterialactivity polyphenols against somepathogenic strains
Majdouline Larif, Mohammed Ouhssine,Abdelmajid Soulaymani & AzzedineElmidaoui
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Potential effluent oil mills and antibacterial activitypolyphenols against some pathogenic strains
Majdouline Larif • Mohammed Ouhssine •
Abdelmajid Soulaymani • Azzedine Elmidaoui
Received: 1 February 2013 / Accepted: 7 May 2013
� Springer Science+Business Media Dordrecht 2013
Abstract The recovery of polyphenols from olive mill wastewaters (OMWs)
provides the double opportunity to obtain high-added value biomolecules and to
reduce the phytotoxicity of the effluent. The purpose of this study is to gain
information about the hygienic quality of the raw material by performing a
microbiological analysis of olive mill wastewater, from an extraction system of
three phases. The determination of total coliforms,fecal coliforms,aecal strepto-cocci, and another group of microorganisms have been explored, namely yeasts,
molds and total aerobic mesophilic flora. We examined the results of the confron-
tation of olive mill wastewater polyphenols with certain strains known for their
pathogenicity, and the minimum concentrations inhibiting bacteria and the minimal
bactericidal concentration (MBC) were determined by the dilution method. Poly-
phenols are particularly the delipidated fraction of OMW extracts according to
liquid–liquid phase. The overall yield is 1.367 ± 0.108 %. The quantitative esti-
mation of total polyphenols was assessed by the colorimetric reagent Folin–Cio-
calteu, gallic acid and tyrosol, tannic acid and total flavonoids catechin (the method
using aluminum trichloride). The results of microbiological analysis confirmed that
there is no fecal contamination. For the in vitro bioassay, polyphenols showed a
variable antimicrobial activity against six bacteria: Escherichia coli, Proteus sp.,
Pseudomonas aeruginosa, Enterroccus feculis, Staphylococcus aureus, and
M. Larif (&) � A. Elmidaoui
Separation Processes Laboratory, Department of Chemistry, Faculty of Science, University Ibn
Tofail, Kenitra, Morocco
e-mail: majdoulinelarif@yahoo.com
M. Ouhssine
Microbial Biotechnology Laboratory, Improvement and Microbial and Plant Transformation,
Department of Biology, Faculty of Science, University Ibn Tofail, Kenitra, Morocco
A. Soulaymani
Genetics and Biometry Laboratory, Department of Biology, University Ibn Tofail, Kenitra, Morocco
123
Res Chem Intermed
DOI 10.1007/s11164-013-1267-0
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Klebsiella pneumoniae. This variability depends essentially on the microorganism
tested and the nature of the product tested. Indeed, we found complete inhibition of
the growth of Proteus sp. and E. fecalis by the sinks method. Some microorganisms
were sensitive to phenols with MBC values between 1/50 and 1/25.
Keywords Olive mill wastewater � Polyphenols � Hygienic quality � Antimicrobial
agents
Introduction
Pseudomonas aeruginosa and Klebsiella pneumoniae are common nosocomial
pathogens [1, 2]. Because these bacterial pathogens are multiply resistant to many
antibiotics, such as ceftazidime and gentamicin, the infections caused by them not
only require expensive antibiotic treatment but also increase the morbidity and
mortality in hospitalized patients [3, 4]. In order to control these infections, there is
a need for the development of agents with marked antibacterial activity, greater
sensitivity, and less toxicity [5].
Faced with this, the technologies dedicated to the recovery and purification of
bioactive compounds from natural effluents and biowastes are under implementa-
tion [6, 7]. The recovery of polyphenols from olive mill wastewaters (OMWs) has
been mostly undertaken by membrane processes [8–10] and liquid–liquid extraction
[11, 12].
Polyphenols of OMW extracts have shown their effectiveness in several areas,
with excellent biological properties as biopesticides against several species of fungi
and weeds [13] and against the pests, Aphis citricola and Euphylura olivina [11, 14].
In previous work, we also tested the effect of polyphenols extracted from olive
vegetation water as a corrosion inhibitor for carbon steel in 1 M HCl, using different
methods: weight loss, potentiodynamic polarization, and spectroscopy and electro-
chemical impedance measurements [15].
Thus, the selective recovery of phenolic compounds from OMWs can accomplish
both the reduction of the intrinsic wastewater environmental toxicity and the
obtainment of high added-value molecules [16]. Given that phenolic compounds are
known for their antibacterial activity [17, 18], we proposed to confirm this activity
using extracts of vegetable polyphenols. In addition, phenolic extracts could be
more beneficial than isolated constituents because the bioactivity of a single
component can change its properties in the presence of other compounds [19, 20].
The purpose of this study is to evaluate the sanitary quality of OMW proceeding
by microbiological analysis on the one hand, and to test the antimicrobial activity of
polyphenols by the methods of diffusion disks and agar dilution on the other hand.
Escherichia coli, Proteus, P. aeruginosa, Enteroccus feculis, Staphylococcusaureus, and Klebsiella pneumoniae were chosen for this study because they are the
source of many infections (urinary, intestinal, respiratory, etc.). The valorisation of
polyphenols as of bactericidal vegetable nature contributes firstly to serving as an
inexhaustible source and for their potential health benefits, and secondly, to avoid
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the use of antibiotics phytochemical synthesis of waste material and the time taken
for the synthesis of a molecule that may or may not be active [21].
Materials and methods
Origin of olive mill wastewater (OMW)
The olives were from Moroccan Picholine, Meknes-Tafilalt, Morocco. The OMW
were collected from a three-phase crushing unit (producing olive residue oil and
OMW) equipped with two centrifugations. The first separates the oil from the olive
cake and OMW, while the second separates the oil and OMW. The samples of
OMW collected were fresh from the second centrifugation of the industrial
apparatus and were placed in sterile plastic containers and brought back to the
laboratory for microbiological analysis. One sample was kept in the dark at 4 �C
until further use by the standards of Rodier [22].
Extraction of polyphenols
The amount of 100 g of vegetable filtered through a Whatman filter paper No.1
were delipidated by 29 100 mL of hexane. The delipidated OMW was then
extracted with 29 100 mL of ethyl acetate. The organic phase was dried over 3 g of
anhydrous sodium sulfate and then concentrated using a rotary evaporator set at
60 �C and a speed of 140 rpm [23].
Estimation of quantitative total polyphenols, total flavonoids and total tannins
The assay methods of total polyphenols equivalent/gallic acid and tannins/
equivalents/tannic acid were by the colorimetric reagent Folin–Ciocalteu [24].
The method of aluminum trichloride (AlCl3) was used to quantify flavonoids
equivalents/catechin [25].
Microbiological analysis of OMW
The analysis of microorganisms associated with OMW was performed by the
method of inoculation in liquid medium according to Ref. [26].
As the OMW are loaded in organic matter and are dark in color, we proceeded to
a series of dilutions from 10-1 to 10-7 to facilitate the counting of bacteria with the
naked eye. Thus, 1 mL of OMW crude was collected and poured into 9 mL of
sterile distilled water. A series of dilutions to 10-7 was then performed. The
appropriate dilution is set at 0.1 mL on the surface of the culture medium, with
Tergitol agar specific, total coliforms (TC), and fecal coliforms (FC). These bacteria
are recognizable by a yellow domed appearance. These seeds are incubated at 37 �C
for 24 h for TC and 44 �C for 48 h for FC. Fecal streptococci (FS) have a brick red
appearance after incubation at 37 �C in a culture medium (Slanetz yellow). To
determine the presence of total aerobic mesophilic flora (TAMF), 1 mL of the
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dilution was further placed in the culture medium, plate count agar (PCA).
Incubation was performed at 37 �C for 24 h. For molds and yeasts, we incubated
them by spreading 0.1 mL of the appropriate dilution on Sabouraud medium. The
boxes were then incubated at 30 �C for 2 days for yeasts and 3 days for molds.
Preparation of biological material
The biological material was isolated from the pathological products in the
bacteriology laboratory. Different bacterial species were subcultured by the
streaking method, and then incubated at 37 �C to obtain isolated colonies that
were used to prepare the inoculum.The six bacterial strains: E. coli, Proteus sp., P.aeruginosa, E. feculis, S. aureus and Klebsiella selected during this study are the
origins of several infections (urinary, intestinal, respiratory, etc.). These bacterial
strains were obtained from the American Type Culture Collection (ATCC). They
were maintained by subculture on nutrient agar supports growth for 24 h in the dark
at 37 �C.
Disk diffusion method
The qualitative antibacterial study was carried out by the disk diffusion method. It
was performed using a culture growth at 37 �C for 18 h and adjusted to *5 colony-
forming units per milliliter (CFU/mL). The culture medium used for the bacteria
was Mueller–Hinton Agar (MHA) [27, 28]. Five hundred microliters (lL) of the
inoculums were spread over plates containing MHA, and a Whatman paper disc
(6 mm) impregnated with 5 lL of the polyphenols was placed on the surface of the
media. Other discs impregnated with sterile distilled water served as a negative
control. The plates were left for 30 min at room temperature to allow the diffusion
of the polyphenols. They were then incubated for 24 h at 37 �C [29, 30]. After the
incubation period, the inhibition zone obtained around the disc was measured. The
experiments were run in triplicate, and the developing inhibition zones were
compared with those of reference discs.
Dilution method
Preparation and cell concentration of inocula
Preparations of inocula were made according to traditional methods. Bacterial
inoculum concentrations were evaluated by turbidity and expressed by measuring
the optical density (OD at 600 nm) on a spectrophotometer; OD of 0.08–0.1
corresponds to 108 CFU/mL.
Minimum inhibitory concentrations (MIC) of polyphenols were determined
according to the methods reported by Remmal et al. [31] and Satrani et al. [32].
Dilutions at 1/10, 1/25, 1/50, 1/100 1/200, 1/300, and 1/500 of polyphenols in
distilled water were prepared. Test tubes, each containing 13.5 mL of solid medium
Tryptic Soy Agar (TSA) for bacteria, were autoclaved for 20 min at 121 �C and
cooled to 45 �C and final concentrations at 1/100, 1/250, 1/500, 1/1,000, 1/2,000,
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1/3,000, and 1/5,000 (v/v) were added aseptically. The tubes were stirred to
thoroughly disperse the extract in the culture medium before pouring into Petri
dishes. Controls, containing the culture medium and agar solution at 0.2 % alone,
were also prepared. The inoculation was done by streaking using a calibrated
platinum loop in order to collect the same volume of inoculum. The latter was in the
form of a broth cultured for 24 h. Each test was repeated three times.
Data analysis
Conventional statistical methods were used to calculate averages and standard
deviations. All measurements were replicated three times, and the data are presented
as mean ± standard deviation.
Results and discussion
Yield and quantitative estimation of total polyphenols, total flavonoids and total
tannins
The overall yield was 1.367 ± 0.108 of dry extract of polyphenols per 100 g of
(OMW) according to five repetitions. The value found belongs to the domain found
by Amaral et al. [33] which is 1–1.5. By membrane processes, the potential of
polyphenols reached a beach of 0.5–19.3 g/L polyphenols [10], which is compa-
rable to a liquid–liquid extraction, which is 13.67 g/L. This difference in
performance can be attributed to several factors, essentially the origin, species,
period of harvesting, drying time, and extraction technique [34, 35].
The quantitative estimations of total polyphenols, total flavonoids, and tannins
were carried out on a dry weight basis as the equivalent of gallic acid, equivalent
catechin, and tannic acid equivalent, respectively, and shown in Table 1.
The content in total polyphenols in gallic acid equivalent was lower than that
found by Nefzaoui [36], which was 0.86 g/L. The value obtained was higher in
flavonoids than found by Debo et al. [16], which was 6.67 g/L. The value tyrosol
was higher than the one found by Jail et al. [37], which was 0.2 g/L. Total tannin
value was found by Leulmi et al. [38], which was still below 0.45 %. The difference
in results can be explained by several factors that have an influence on the content
was phenolic compounds: Extrinsic factors (such as geographic and climatic) and
Table 1 Results of the
determination of total
polyphenols, flavonoids of
condensed tannins and total
tannin in fresh OMW
Parameters Mean Standard
deviation (CV)
Total phenols/Ac. Gallic (g/L) 11.600 0.160
Flavonoids (catechin) (g/L) 7.335 0.930
Total tannins/tannic acid (g/L) 1.832 0.462
Tyrosol (g/L) 0.393 0.051
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genetic factors, but also the degree of maturation of the olives and the duration of
storage, have an effect on the polyphenol content [39, 40].
Bacteriological analysis
The results for the microbiological analysis are shown in Table 2.
Yeasts and molds are the most common flora which seem to adapt better to the
acidity of the OMW; they are, respectively, of about 4.6 9 105 and 7.33 9 106
CFU/100 mL, and these values are comparable to those obtained by Jouraiphy [41].
For total aerobic mesophilic flora (TAMF), it is around 6–7 9 105 compared with
domestic wastewater where the load is of the order of 6 9 106 [42], and can be
considered as revivable spoilage flora whose abundant presence indicates a process
of ongoing degradation and presents no health risk [43]. It was noted that there was
a complete absence of total coliforms and fecal coliforms in OMW, which
confirmed that there is no fecal contamination.
The antimicrobial capacity of effluent from olive oil mills is essentially linked to
the action of monomeric phenols and brown pigments or dark-colored catechol [44].
These effluents are acting on bacteria by denaturing proteins and by altering the
cellular membranes [45].
Antimicrobial activity of polyphenols
Method of wells
The diffusion method with disks allowed us to highlight the antibacterial
polyphenols, with a negative control with distilled water, against pathogenic
bacteria (E. coli, Proteus sp., P. aeruginosa, E. fecalis, S. aureus and K.pneumoniae). Results of the antibacterial activity of polyphenols in its fractions are
shown in Table 3.
The results show that the antibacterial activity is not the same, with the same
concentration of the product applied to the six bacteria giving diameters of
inhibition zones between 11 and 21 mm.
Table 2 Evaluation of the
hygienic quality of OMWMicrobial flora UFC
(mL)
UFC
(mL)
UFC
(mL)
Total coliforms in 37 �C/
100 mL
0 0 0
Fecal streptococci in 37 �C/
100 mL
0 0 0
Fecal coliforms in 44 �C/
100 mL
0 0 0
Yeast 4 9 105 5 9 105 5 9 105
Molds 8 9 106 7 9 106 7 9 106
Total aerobic mesophilic flora
in 30 �C
7 9 105 6 9 105 6 9 105
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We note that the polyphenols have an inhibitory effect which is greater in the
presence of Proteus sp. (Gram-negative) and E. fecalis (Gram-positive) as for these
strains the diameters are the highest compared to other bacteria and in contrast to the
control which does not have an antibacterial effect (Fig. 1).
Minimum inhibitory concentration of polyphenols (MIC)
The MIC is the lowest concentration inhibiting bacterial growth visible to the eye
after 18 or 24 h of contact at 37 �C. The results of the antibacterial activity of
polyphenols and its fractions are presented in Table 4.
Polyphenols present in vitro, a good inhibitory activity against the tested germs.
However, the microorganisms studied did not exhibit the same sensitivity by
polyphenols. For bacteria, Proteus sp., S. aureus and P. aeruginosa showed a
greater sensitivity to an inhibitory concentration of 1/25 compared with E. coli, K.pseudomonas, and E. fecalis, the growth of which was inhibited at 1/50 (Fig. 2).
Table 3 Antibacterial activity by the disk method
Germs Minimum Maximum Mean Standard deviation
(CV)
Gram (-) Pseudomonas aeroginosa 11.000 14.000 13.000 1.732
Proteus sp. 16.000 18.000 17.000 1.000
Echerichiacoli 13.000 16.000 14.667 1.528
Klebsilla pneumoniae 15.000 16.000 15.333 0.577
Gram (?) Enteroccus fecalis 12.000 21.000 17.000 4.583
Staphylococcus aureus 12.000 20.000 15.667 4.041
Fig. 1 Disk diffusion inhibitor activity of polyphenols is most important on Proteus sp. and E. fecalis
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Concentration minimal bactericidal of the polyphenols (MBC)
The minimal bactericidal concentration (MBC) is the lowest concentration of
polyphenols leaving only 0.01 % or less of the initial inoculum surviving after 18 h
of culture at 37 �C according to Table 5. This value characterizes the bactericidal
Table 4 Antibacterial activity of polyphenols on different germs
Germs 1/10 1/25 1/50 1/100 1/200 1/300 1/500 C.
Gram (-) Echerichia coli - - - ? ? ? ? ?
Proteus sp. - - ? ? ? ? ? ?
Klebsilla pseudonomas - - - ? ? ? ? ?
Pseudomonas aeroginosa - - ? ? ? ? ? ?
Gram (?) Enteroccus fecalis - - - ? ? ? ? ?
Staphyloccocus aureus - - ? ? ? ? ? ?
C control, - clear, ? trouble
Fig. 2 Agar dilution minimum inhibitory concentration (MIC) of polyphenols on a E. fecalis, b K.pseudomonas and c E. coli the growth of which was inhibited at 1/50
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effect of polyphenols, which is 1/25 for E. coli (Gram-negative) which is the most
resistant strain and 1/50 for E. fecalis Gram (?) and Klebsilla pseudomonas (Gram-
positive) (Fig. 3).
Discussion
Following these results, polyphenols have shown very interesting antibacterial
characteristics on the microorganisms tested. Several authors who have shown that
polyphenols inhibit mycelial development of strains and their antimicrobial power
[46–48].
Phenolic compounds are also characterized by very strong protein cross-linking
and protein-denaturizing activity, and other studies have reported that different
bacterial species (Gram-positive and Gram-negative) exhibit different sensitivities
towards phenolic compounds and phenolic acids in which our extract is rich [49,
50].
Due to their acidic side chain, phenolic acids are much less polar. This property
might facilitate the transport of these molecules across the cell membrane, which
might be related in turn to the stronger inhibitory effect of phenolic acids. On the
other hand, they are known to interact with membrane lipids by a neutralization of
the membrane’s electric potential following penetration of the molecule. A similar
effect could occur in the bacterial cell membrane affecting their energy metabolism.
Indeed, the antibacterial activity of polyphenols can be explained by the
mechanism of toxicity of flavonoids against microorganisms, by, for example, non-
specific interactions such as hydrogen bonding setting with proteins or cell wall
enzymes, chelation of metal ions, inhibition of bacterial metabolism, or sequestra-
tion of substances necessary for the growth of bacteria [51].
Their activity is probably due to their ability to complex with extracellular and
soluble proteins and to complex with bacterial cell walls, as is the case for quinones.
More lipophilic flavonoids may also disrupt microbial membranes [52, 53].
Flavonoid compounds also exhibit inhibitory effects against multiple viruses.
Numerous studies have documented the effectiveness of flavonoids such as
swertifrancheside, glycyrrhizin, and chrysin against human immunodeficiency virus
(HIV) [54]. According to several studies, the synergistic interactions between
Table 5 Bactericidal activity (MBC)
Germs 1/10 1/25 1/50 Control
Gram (-) Echerichia.coli - - ? ?
Proteus sp. - - ?
Klebsilla pseudonomas - - – ?
Pseudomonas aeroginosa - - ?
Gram (?) Enteroccus fecalis - - - ?
Staphyloccocus aureus - - ?
- inhibits (MBC), ? persists
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different compounds may be the origin of the much more pronounced activity than
that expected for the major compounds [55, 56].
Phenolic compounds (phenolic acids, tannins, and flavonoids) form a group of
the most important phytochemical polyphenols [53, 57]. The majority of the
pharmacological effects of polyphenols are mainly due to their phenolic group. In
fact, the location and number of hydroxyl groups on the phenolic compounds
increases the toxicity against microorganisms [20]. The bactericidal activity arises
from the synergistic effect of constituents such as the total polyphenols, flavonoids,
and tannins. Consequently, we note that the inhibition of bacterial growth depends
on two factors: the bacteria used, and the nature of the product tested [54, 58].
Comparing the disk diffusion method and agar dilution, the disk diffusion
method is simple and inexpensive to test sensitivities, and any zone of inhibition
should be obtained to determine the MIC category sensitivity. Therefore, it is
recommended that the agar dilution method is used for MIC determination for
isolates with a zone diameter of 20 mm or more than 26 mm [59].
Conclusion
The extraction of polyphenols from OMW provided an important yield of 1.367 %.
With the wealth of vegetable phenolics, the biological effectiveness of polyphenols
against pathogenic strains is related to their chemical composition, by the functional
groups of the major compounds, and possible synergistic effects between
components. These preliminary results can be supplemented by other more detailed
studies (toxicological tests, mode of delivery, cost, etc.). To exploit the antibacterial
properties, the nature of the chemical structures and their proportions play a role.
This efficiency often reduces the activity of the major compounds, or those likely to
be active.
Fig. 3 Agar dilution minimum bactericidal concentration (MBC) of polyphenols against the mostsensitive strains
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