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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 22, No. (1) : 2020 : 7-14
© Global Science Publications
ISSN-0972-3005
ANTIBACTERIAL ACTIVITY AND GC-MS ANALYSIS OF
TRICHAPTUM BIFORME EXTRACT
YAKHLEF DJAMILA, DIB SOULEF AND FORTAS ZOHRA
Laboratoire de Biologie des Micro-organismes et Biotechnologie, Université Oran 1, Algeria
(Received 29 September, 2019; accepted 30 November, 2019)
Key words : Trichaptum biforme, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922
Extract, Soxhlet, GC-MS.
Abstract – In our study, mushrooms called Trichaptum biforme are harvested in west forest of Algeria in order
to research their antibacterial activity. Description of mushrooms is carried out by morphological and
microscopic methods. Extract is obteined by Soxhlet from the dried sporophores of Trichaptum biforme. This
extract is tested in vitro, in Petri dishes, on the growth of a Gram + and Gram- bacteria: Staphylococcus aureus
ATCC 25923 and Escherichia coli ATCC 25922. The results obtained showed a remarkable inhibitory effect of
the Trichaptum biforme extract on the growth of the two pathogenic bacterial strains tested. The antibacterial
activity of the extract towards the bacterial strains tested by the disc diffusion method has shown that this
extract is biologically bacteriostatic. Analysis of the extract by GC-MS allowed the identification of 30
components. Octodrine, caryophyllene oxide, phenolic compounds, fatty acids and fatty acids esters were
noted as key compounds in the Trichaptum biforme extract, which were perhaps causing the antibacterial
activity. Trichaptum biforme are are therefore interesting candidates for obtaining antibacterial bioactives
substances with therapeutic interest.
INTRODUCTION
Trichaptum biforme is a genus of mushrooms
belonging to basidiomycetes, without stipes and
with pores on the underside of the cap Gevry et al.,
2009.
Bioactive molecules have been reported in some
basidiomycetes species. These molecules are
antimicrobial, antioxidant, anti-tumor, cytostatic,
antiallergic, hypoglycemic, anti-inflammatory and
hepatoprotective (Wasser and Weis, 1999;
Lindequist et al., 2005).
Basidiomycetes are a poorly exploited source of
antimicrobial substances. Indeed, it has been
reported in the literature that they contain
molecules with antiviral, antibacterial and
antifungal activities (Wasser and Weis, 1999;
Lindequist et al., 2005; Colombo and Bosisio, 1996;
Iwu et al., 1999; Yamac and Bilgili, 2006; Alves et al.,
2012; Orion, 2013).
Our study focuses on the research of the
antibaterial activity of the extract of Trichaptum
biforme, a basidiomycete harvested in a west forest of
Algeria (M’sila forest in Oran).
MATERIALS AND METHODS
Study Site
Trichaptum biforme is harvested in the forest of
M’Sila, at 30 km west of Oran in Algeria (Fig. 1). This
forest has poor soils, with a clay-siliceous texture, or
sandy-clay-silty soils depending on the area (Aimé,
1991). Most of this forest is covered with cork oak
trees (Benazza-Bouregba et al., 2017). The
prospection is carried out on January with the
presence of foresters, after 3 days of rain, during the
winter period.
Fig. 1. Forest of M’sila in Oran (Algeria): a) Geographical
position (google earth, 2019) and b) general view.
8YAKHLEF ET AL
Mushrooms are harvested in a random manner,
without particular statistical methods. They are
carefully cleaned and placed in paper envelopes.
The mushroom Trichaptum biforme
The mushroom presents several characteristics
(Fig.2). Cap: 10 × 5 cm, concentric grooves felted
pale gray, often tinged with green, spread fan,
margin corrugated and lobed. Flesh: Pale brown or
purplish, hard. Hymenium (underside of the cap)
angular, purple to reddish-brown pore.
Basidiospores (8-10 x 2.5-3 m), are cylindrical.
After drying in the sun, in an aerated place,
mushrooms are crushed using a mechanical grinder;
the powder thus obtained is used for extraction by
the soxhlet.
10 g of dried fragments of Trichaptum biforme are
used for extraction with the soxhlet. Fragments are
introduced into a 500 mL round bottomed flask
containing 300 mL of solvent (methanol, ethanol 96°
or ethyl acetate). The body of the extractor
(containing the fragments) is surmounted by a
refrigerant. The solvent is vaporized and condensed
while remaining in contact with the fragments.
The extraction is carried out at 77 °C for 1h30min.
It is complete when the solvent becomes very
colored. The solvent is then removed with
Retavapor Buchi R200 (at 50°C) until brownish
residue (concentrated extract) which is recovered
using a dropper and then weighed.
Bacterial strains
Two bacterial strains are tested: Staphylococcus aureus
ATCC 25923 and Escherichia coli ATCC 25922. These
strains belong to the laboratory’s collection (LBMB).
Preparation of bacterial culture
3 to 5 well isolated colonies are taken from a
preculture of 24h and emulsified in a tube
containing 10 mLof nutrient broth. After 24h of
incubation at 37 °C, the tube of the bacterial
suspension is compared with the Mc Farland
standard 0.5 tube (108 CFU/mL) to adjust the
turbidity of the bacterial suspension. Evaluation of
antibacterial activity was determined using the disc
diffusion method (Perez et al., 1990).
In Vitro evaluation of the antibacterial activity
20 mL of Mueller-Hinton medium is poured in Petri
dishes (90mm in diameter). 1mL of standardized
bacterial inoculum (108UFC/ mL) is aseptically
spread on the surface of the medium using a swab.
One disc of filter paper (6mm in diameter) is
impregnated with mushroom extract and deposited
on the surface of the medium. The control disc is
impregnated with the extraction solvent (negative
control) and deposited on the surface of the
medium. The positive controls are standard
antibiotics: chloramphenicol and streptomycine at
30µg/disc. Three repetitions are carried out for each
bacterial strain tested.
The Petri dishes are left for a quarter of an hour at
room temperature so that the contents of the discs
diffuse into the medium. They are then sealed with
tape and incubated at 37 °C for 24h.
The sensitivity of the bacterial strains tested is
determined by measuring the diameters of the
inhibition zones in the two perpendicular directions
around the discs, in Petri dishes.
Statistical Analysis
The results were expressed as means followed by
the standard error (means ± SD). Excel (Microsoft
Corporation, USA) and Statistica (version 7.1) were
used for statistical analysis.
Gas Chromatography- Mass Spectrometry (GC-
MS) analysis
The changeable volatile compounds profiling of
methanol extract, ethanol extract and ethyl acetate
extract, from the mushroom Trichaptum biforme, was
measured using Gas chromatography-Mass
spectrometry (GC-MS). A quantity in the order of
milligrams of the dried crude extract was dissolved
in methanol. The method was described by (Hossain
and Rahman, 2011).
GC-MS analysis is performed on a Perkin Elmer
Clarus 500 equipped with an automatic injector. The
column used is a Restek Rxi 5ms (5% diphenyl / 95%
dimethylpolysiloxane) with a length of 60 meters in
Fig. 2. The mushroom Trichaptum biforme : a) Sporophores
of the mushroom on the trunk of cork oak, b) pores
on the underside of the cap of the mushroom, c)
basidiospores (G×1000).
Antibacterial Activity and GC-MS Analysis of Trichaptum biforme Extract 9
diameter 0.25 mm.
The analysis parameters are:
-Injector temperature: 220 °C.
-volume injection 1 L
-the carrier gas is helium with a flow rate of 1 mL
/ min.
-ramp 1, 10 °/ min to 150 °C.
-ramp 2, 3 °/ min up to 220 °C.
-Duration of the analysis 33 min.
-M / Z mass detector40-500 daltons.
-Ionization at 70 eV.
-temperature of the transfer line 250 °C.
-temperature of the source 220 °C.
In mass spectrometry, the ionization process used
is electron impact ionization; it is the most used
method for compounds that can go into the gaseous
state. This is particularly the case of organic
molecules. It consists in causing collisions between
the initial no-charge molecules and the electrons
obtained by thermo-ionic effect. In a shock, one of
the least retained electrons of the molecule is torn
off, which leads to an ion carrying a positive
elementary charge.
The identification of the separated substances
was done by comparing their mass spectra obtained
by GC-MS with those provided by the libraries:
wiley 9, Nist, AAFS0603, AAFSDrugs, NMStox1,
pflegerand mainlib.
RESULTS AND DISCUSSION
In vitro evaluation of the antibacterial activity
The extract of Trichaptum biforme had antibacterial
activity against both bacterial strains tested (Fig.3,
Table 1). These results are similar with those of
many authors who found strong antimicrobial
activity of several species of mushroom against
bacteria (Rosa et al., 2003).
Some authors reported that extracts of several
basidiomycetes (Cantharellus cibarius, Pleurotus
ostreatus) inhibit the growth of bacteria regardless of
their Gram (Bacillus subtillis ATCC 25922,
Staphylococcus aureus ATCC 25923, Escherichia coli
ATCC 25922 and Pseudomonas aeruginosa ATCC
27453) (Kalyoncu et al., 2010). This therefore joins
our results.
The extract strongly inhibits the growth of
Escherichia coli ATCC 25922 (28mm inhibition zone)
and Staphylococcus aureus ATCC 25923 (13mm
inhibition zone).
The Gram + bacterial strain tested (Staphylococcus
aureus ATCC 25923) is less sensitive than the Gram-
bacterial strain (Escherichia coli ATCC 25922). It is
well known that Gram- bacteria have a periplasmic
space and a thinner peptidoglycan layer that are
absent in Gram + bacteria and make them more
sensitive (Basile et al., 1998). In contrast, according to
(Balakumar et al., 2011), Gram +bacteria
(Staphylococcus aureus and others) are more sensitive
to extracts of mushroom species than Gram-
bacteria.
Analysis of the Trichaptum biforme extracts using
Gas Chromatography- Mass Spectrometry (GC-
MS)
The analyzes by GC-MS provided the
chromatograms illustrated respectively by Figs. 4, 5
and 6. These analyzes reveal the large number of
products contained in the extracts. This is due to the
extraction method, nature and selectivity of the
organic solvents used. Soxhlet has been shown to be
effective in driving a large number of volatile
constituents when solvents such as methanol, ethyl
acetate and ethanol are used.
Table 1. Diameters of inhibition zones of bacterial strains tested (values, including diameter of the filter paper disc (6.0
mm), are means±SD of three replicates).
Bacterial strains tested Mushroom Solvent (negative Positive reference
extract control : T) standards (30µg/disc)
Staphylococcus aureus ATCC 25923 13±2.08 6±0 27±1.5 (Chloramphenicol)
Escherichia coli ATCC 25922 28±2.51 6±0 19±1 (Streptomycine)
Fig. 3. Activity of the extract of Trichaptum biforme (EXT6)
on the growth of Staphylococcus aureus ATCC 25923
(left) and Escherichia coli ATCC 25922 (right), after
24h of incubation at 37°C on Mueller-Hinton
medium. Note the absence of an inhibition zone
around the control disc (T).
10 YAKHLEF ET AL
Identifications of the eluted compounds are
grouped in Table 2. Retention times and spectral
data are reported and compared with those of the
literature.
GC-MS analysis of Trichaptum biforme extract
showed a list of chemical compounds that includes
a very large number of substances with antibacterial
activity. According to Martchenko and Kim (2014),
Octodrine (C8H19N) inhibits the proliferation of
microbial growth, including growth of the genus
Candida and both Gram negative and Gram positive
Fig. 4. Chromatogram obtained by GC-MS for the methanol extract
Fig. 5. Chromatogram obtained by GC-MS for the ethyl acetate extract
bacteria. Many studies revealed that the chemical
analysis of plant extracts and essential oils with
antimicrobial activity show a remarkable presence
of an Sesquiterpene, caryophyllene oxide
(C15H24O)(Alsultan et al., 2016). Caryophyllene oxide
showed activity against Staphylococcus sp.,
Macrococcus caseolyticus, Enterococcus faecium and
Enterococcus faecalis (Xiong et al., 2013)
Also, it is noted the presence of phenolic
compounds with aromatic nucleus (C14H22O,
C13H16O2, C8H6O4, C6H2Cl2O2). These compounds
Antibacterial Activity and GC-MS Analysis of Trichaptum biforme Extract 11
Fig. 6. Chromatogram obtained by GC-MS for the ethanol 96°extract
Table 2. Main components present in the extracts of Trichaptum biforme identified on the basis of GC-MS analysis.
Compound name Molecular Chemical Retention Extraction
formula 2D structure time (min) solvent
(PubChem :
http:// pubchem.
ncbi.nlm.nih.gov)
4,7-Benzofuradione, 3-acetyl-3a,7a- C23H42O8Si418.105 Methanol
dihydro-2-methyl-3a,5,6,7a-tetrakis
[(trimethylsilyl)oxy]
Docosanoic acid, 1,2,3-propanetriyl ester C69H134O620.341 Methanol
Heneicosanoic acid C21H42O227.473 Methanol
Octodrine C8H19N 27.849 Methanol
Hexadecanoic acid, dodecyl ester C28H56O228.199 Methanol
Pentanoic acid, 10-undecenyl ester C16H30O231.495 Methanol
Oleic acid C18H34O236.915 Methanol
Hexanedioic acid, bis(2-ethylhexyl) ester C22H42O436.622 Methanol
1-tetradecene C14H28 15.569 Ethyl acetate
2,5-cyclohexadiene-1,4-dione, 2,6-dichloro C6H2Cl2O217.560 Ethylacetate
12 YAKHLEF ET AL
Table 2. Continued ...
Compound name Molecular Chemical Retention Extraction
formula 2D structure time (min) solvent
(PubChem :
http:// pubchem.
ncbi.nlm.nih.gov)
2-Decenal, (E)- C10H18O 17.805 Ethyl
Acetate
Phenol, 2,4-bis(1,1-dimethylethyl) C14H22O 18.585 Ethyl
Acetate
Cetene C16H32 20.381 Ethyl
Acetate
Methanone, (1-hydroxycyclohexyl)phenyl- C13H16O222.762 Ethyl
Acetate
(E,E)-1,12-dibromo-2,10-dodecadiene C12H20Br2 27.764 Ethyl
Acetate
Hexadecanoic acid, dodecyl ester C28H56O228.284 Ethyl
acetate
n-Heptadecanol- 1 C17H36O 28.739 Ethyl
Acetate
2-Fluoro-1-iodo-2-methylundecane C12H24FI 30.450 Ethyl
Acetate
Octadecanoic acid C18H36O231.925 Ethyl
Acetate
5-cholene, 3,24-dihydroxy C24H40O232.545 Ethyl
Acetate
2-phenanthrenecarboxaldehyde, 1,2,3,4,4a, C19H30O233.741 Ethyl acetate
4b,5,6,7,8,8a,9-dodecahydro-7-hydroxy-
2,4b,8,8-tetramethyl
Octadecanol C18H38O 34,366 Ethyl acetate
Caryophyllene oxide C15H24O 35.532 Ethyl acetate
Antibacterial Activity and GC-MS Analysis of Trichaptum biforme Extract 13
Table 2. Continued ...
Compound name Molecular Chemical Retention Extraction
formula 2D structure time (min) solvent
(PubChem :
http:// pubchem.
ncbi.nlm.nih.gov)
1-tetradecene C14H28 15.539 Ethanol 96°
Isopropylphosphonic acid, C13H28FO2P 20.101 Ethanol 96°
fluoroanhydryde-, decyl ester
D- Mannitol C6H14O624.132 Ethanol 96°
Cetene C16H32 24.717 Ethanol 96°
Pentadecanoic acid C15H30O226.208 Ethanol 96°
1,2-benzendicarboxylic acid C8H6O426.413 Ethanol 96°
n-hexadecanoic acid C16H32O228.314 Ethanol 96°
2-Fluoro-1-iodo-2-methylundecane C12H24FI 28.714 Ethanol 96°
9,12-octadecadienoic acid, C22H42O3Si 31.590 Ethanol 96°
18-(trimethylsiloxy)-, methyl ester
Octadecanoic acid C18H36O2 31.965 Ethanol 96°
Triacontane,11,20-didecyl- C50H102 33.366 Ethanol 96°
reflect a strong antibacterial potency (Rapior et al.,
1996; Rapior et al., 1997; Alsultan et al., 2016;
Khadhri et al., 2017).
CONCLUSION
The extract of Trichaptum biforme showed an
antibacterial activity against both bacterial strains
tested Escherichia coli ATCC 25922 and Staphylococcus
aureus ATCC 25923. This mushroom extract
exhibited bacteriostatic and bactericidal effect
against these pathogenic bacteria. GC-MS analyses
showed the presence of octodrine, caryophyllene
oxide, phenolic compounds, fatty acids (palmitic
14 YAKHLEF ET AL
acid, phthalic acide) and fatty acids esters and other
components that have been proposed to play an
important role in the antimicrobial actions. This
study revealed that Trichaptum biforme comprised
constituents with antibacterial effect, which is
considered important for application in the
pharmacological field.
ACKNOWLEDGMENTS
Authors are grateful to anonymous reviewers for
their valuable comments. They are also grateful to
Mr.Benabdesselam Mustapha and Mrs.Madouri
Imène for their help in GC- MS.
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