Wood-rotting basidiomycete, Laetiporus sulphureus (Bull.: Fr.) Murill., also known as chicken of the woods, is known for its nutritional value. In this study, aqueous extract obtained from L. sulphureus was investigated for its antimicrobial properties using microdilution in vitro assay. Plant, animal and human pathogens, as well as food spoilage agents, were tested. Aqueous extract obtained from L. sulphureus showed strong activity against the tested microorganisms in a dose dependent manner. Considering that there is a constant emerging of pathogen resistance to the known synthetic agents, there is an undeniable need for new therapeutical drugs and preservatives in food industry. Thus, these results that indicate activity of natural products may be of practical use.Gljiva mrke truleži, Laetiporus sulphureus (Bull.: Fr) Murill., poznata i kao šumsko pile, već je poznata po svojoj nutritivnoj vrednosti. U ovoj studiji ispitivana je antimikrobna sposobnost vodenog ekstrakta L. sulphureus, mikrodilucionom in vitro metodom. Biljni, životinjski i humani patogeni, kao i prouzrokovači kvarenja hrane su testirani. Vodeni ekstrakt dobijen od L. sulphureus pokazao je snažnu aktivnost na testirane mikroorganizme uz doznu zavisnost. S obzirom da postoji konstantni porast rezistentnosti patogena na poznate sintetičke agense, potreba za novim terapeutskim lekovima i konzervansima u prehrambenoj industriji je neophodnost. Stoga, rezultati koji ukazuju na aktivnost prirodnih produkata mogu biti od velikog praktičnog značaja.Projekat ministarstva br. 14304

Glamočlija, Jasmina

Nikolić, Miloš

Soković, Marina

Stojković, Dejan

Ćirić, Ana M.

Šiljegović, Jovana D.

English

 Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

299
UDC 632.4:582.475
DOI: 10.2298/ZMSPN1120299S
Ј o v a n a  D .  Š i l j e g o v i ć ,  D e j a n  S .  S t o j k o v i ć , 
M i l o š  M .  N i k o l i ć ,  J a s m i n a  M .  G l a m o č l i j a , 
M a r i n a  D .  S o k o v i ć ,  A n a  M .  Ć i r i ć
Institute for Biological Research “Siniša Stanković“, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia
ANTIMICROBIAL ACTIVITY OF AQUEOUS EXTRACT 
OF LAETIPORUS SULPHUREUS (BULL.: FR.) MURILL 
ABSTRACT: Wood-rotting basidiomycete, Laetiporus sulphureus (Bull.: Fr.) Murill., 
also known as chicken of the woods, is known for its nutritional value. In this study, aqueous 
extract obtained from L. sulphureus was investigated for its antimicrobial properties using 
microdilution in vitro assay. Plant, animal and human pathogens, as well as food spoilage 
agents, were tested. Aqueous extract obtained from L. sulphureus showed strong activity 
against the tested microorganisms in a dose dependent manner. Considering that there is a 
constant emerging of pathogen resistance to the known synthetic agents, there is an undeni-
able need for new therapeutical drugs and preservatives in food industry. Thus, these results 
that indicate activity of natural products may be of practical use. 
KEY WORDS: antimicrobial activity, aqueous extract, Laetiporus sulphureus
INTRODUCTION
Researches in the past decade suggest that significant number of people 
is involved in various forms of alternative medicine. Estimates for using com-
plementary and alternative medicine in adults worldwide have increased in 
the last twenty years (B a r n e s et al., 2004.). The main reason for this lies in 
the fact that many people are to a certain extent dissatisfied with the conven-
tional treatment (A s t i n, 1998). Also, constant emerging resistance of bacte-
rial and fungal species, known for the ailments they cause in humans, to com-
mercial synthetic medicines is probably the most important reason why people 
turn to alternative medicine. While the use of herbs is rather common in the 
Western hemisphere, medicinal use of mushrooms, which has a long tradition in 
the Asian countries, has been slightly increased in Europe since the last decades 
(L i n d e q u i s t et al., 2005). Although there have been extensive researches 
on the use of medicinal mushrooms, their true potential is yet to be revealed.
Excessive use of antibiotics and antimycotics in the treatment of infec-
tious diseases caused by human pathogenic microorganisms led to multiple 
Зборник Матице српске за природне науке / Proc. Nat. Sci, Matica Srpska Novi Sad,
№ 120, 297—303, 2011
300
drug resistance. This motivated scientists to search for new substances with 
antimicrobial activity in natural products. Plants and mushrooms turned out 
to be an excellent source of novel chemotherapeutic agents (K a r a m a n et 
al., 2003). A number of compounds that have been proved to posses signifi-
cant antimicrobial activities were isolated from polypore fungi. They provide 
a rich variety of active secondary metabolites and polysaccharides. Polysac-
charides emerged as an important class of bioactive substances, and many 
medicinal and therapeutic properties are attributed to them (A l q u i n i et al., 
2004). Trametes versicolor, Laetiporus sulphureus and Ganoderma lucidum 
are just some of the known mushrooms with this potential. This fact alone 
made them good candidates for critically needed new antibiotics and antimy-
cotics (Z j a w i o n y, 2004). 
Laetiporus sulphureus (Bull.: Fr.) Murill is a wood-rotting basidiomycete, 
growing on several tree species and producing shelf-shaped fruit bodies with 
a bright yellow fleshy margin. This recognizable pigmentation along with the 
fruit body form is responsible for the trivial name under which this fungus is 
known, and that is sulfur shelf (W e b e r et al., 2003).
Even though it is known as a source of active compounds, and is widely 
used as food among Anatolian people, literature reports on antimicrobial ac-
tivity of L. sulphureus extracts are scarce (Z j a w i o n y, 2004; T u r k o g l u 
et al., 2007). 
The potential barrier to everyday use of medicinal mushrooms as therapy 
is the manner in which the mushroom is consumed. Most of the researches on 
fungi as potential antimicrobial agents are based on ethanolic and methanolic 
extracts of the fungal fruit body (T u r k o g l u et al., 2007; B a r r o s et al., 
2007). Consumption of products on this basis is of no practical use.
Therefore, the aim of the present study is to evaluate the antimicrobial 
potentials of aqueous extracts of L. sulphureus fruit bodies on several micro-
organisms of medicinal importance. This would perhaps allow daily use of 
mushrooms that are available in various forms.
MATERIALS AND METHODS
Mushroom
Fruiting bodies of L. sulphureus were collected in the woods near Belgrade. 
Identification and classification were carried out and all specimens were depos-
ited at the Mycological Laboratory, Department of Plant Physiology, Institute 
for Biological Research “Siniša Stanković”, Belgrade, Serbia. Mature basidio-
carps of L. sulphureus, were collected in Salix alba in 2009. Fresh mushrooms 
were randomly divided, dried in an oven at 40 °C before analysis. Dried mush-
room material was grounded to a fine powder with blender. The sample (30 mg) 
was dissolved in 1ml of distilled water and left for 24 h at room temperature. 
The aqueous solution was then centrifuged at 5000 rpm for 10 minutes; the 
supernatant was poured off and used in the in vitro assay.
301
Antimicrobial activity
Antibacterial activity
The following Gram negative bacteria were used: Escherichia coli (ATCC 
35210), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhimurium 
(ATCC 13311), and Enterobacter cloacae (human isolate), as well as the follow-
ing Gram positive bacteria: Listeria monocytogenes (NCTC 7973), Bacillus 
cereus (clinical isolate), Micrococcus flavus (ATCC 10240) and Staphylococcus 
aureus (ATCC 6538). The organisms were obtained from the Mycological 
Laboratory, Department of Plant Physiology, Institute for Biological Research 
“Siniša Stanković”, Belgrade, Serbia. The antibacterial assay was carried out by 
modified microdilution method (D a o u k  et al., 1995; H a n e l  and R a e t h e r 
1988; E s p i n e l – I n g r o f f , 2001) in order to determine the antibacterial 
activity of compounds tested against the human pathogenic bacteria.
The bacterial suspensions were adjusted with sterile saline to a concen-
tration of 1.0 x 105 CFU/ml. The inocula were prepared daily and stored at 
+4°C until use. Dilutions of the inocula were cultured on solid medium to 
verify the absence of contamination and check the validity of the inoculum.
Antifungal activity
For the antifungal bioassays, seven fungi were used: Aspergillus ochraceus 
(ATCC 12066), Aspergillus fumigatus (plant isolate), Aspergillus niger (ATCC 
6275), Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 
36839), Penicillium ochrochloron (ATCC 9112) and Trichoderma viride (IAM 
5061). The organisms were obtained from the Mycological Laboratory, De-
partment of Plant Physiology, Institute for Biological Research “Siniša Stan-
ković”, Belgrade, Serbia. 
The micromycetes were maintained on malt agar and the cultures were 
stored at + 4 °C and sub-cultured once a month (B o o t h, 1971). In order to 
investigate the antifungal activity of the tested aqueous extract, a modified 
microdilution technique was used (D a o u k  et al., 1995; H a n e l  and R a e -
t h e r , 1988; E s p i n e l – I n g r o f f , 2001.). The fungal spores were washed 
from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 
80 (v/v). The spore suspension was adjusted with sterile saline to a concentra-
tion of approximately 1.0 x 105 CFU in a final volume of 100 μl per well. The 
inocula were stored at +4 °C for further use. Dilutions of the inocula were 
cultured on solid malt agar to verify the absence of contamination and check 
the validity of the inoculum.
Microdilution test
The minimum inhibitory, bactericidal and fungicidal concentrations 
(MICs, MBCs and MFCs, respectively) were determined using 96-well mi-
crotitre plates. The bacterial suspension was adjusted with sterile saline to a 
concentration of 1.0 x 105 CFU/ml. The microplates were incubated for 24 h at 
302
37°C. The lowest concentrations without visible growth (at the binocular mi-
croscope) were defined as concentrations that completely inhibited bacterial 
growth (MICs). The MBCs were determined by serial sub-cultivation of 2 μl 
into microtitre plates containing 100 μl of broth per well and further incubation 
for 72 h. The lowest concentration with no visible growth was defined as the 
MBC value, indicating 99.5% killing of the original inoculum. Streptomycin 
was used as a positive control (1 mg/ml DMSO). 
Minimum inhibitory concentration (MIC) determinations for fungi were 
performed using 96-well microtiter plates. The microplates were incubated for 
7 days at 25 °C. The lowest concentrations without visible growth (at the bin-
ocular microscope) were defined as MICs. 
The fungicidal concentrations (MFCs) were determined by serial subcul-
tivation of 2 μl into microtiter plates containing 100 μl of broth per well and 
further incubation for 5 days at 25 °C. The lowest concentration with no vis-
ible growth was defined as MFC indicating 99.5% killing of the original in-
oculum. Commercial fungicide ketoconazole was used as positive control 
(1mg/1ml DMSO).
RESULTS 
The antimicrobial effect of aqueous extract of L. sulphureus was tested 
against four species of Gram negative bacteria, four species of Gram positive 
bacteria, and seven species of fungi. The extract generally exhibited better 
antifungal than antibacterial activity (Figure 1). Among the tested bacteria, L. 
sulphureus extract strongly inhibited M. flavus and L. monocytogenes, with 
MIC values of only 1.50 mg/ml. As for the remaining bacterial strains, in the 
tested concentrations (0.15-1.50 mg/ml) aqueous extract showed activity, but 
insufficient to be considered as inhibitory. Antibiotic streptomycine (1mg/1ml 
DMSO) was used as the positive control. In the tested concentrations, strepto-
mycine showed inhibitory activity in the range of 0.63-12.50 x 10-3 mg/ml, and 
bactericidal activity on the tested bacteria in the range of 1.25-25.00 x 10-3 mg/ml. 
The bacteria most resistant to the effect of streptomycine was proved to be 
L. monocytogenes, which could be of practical use, because it was also proved 
to be the most susceptible to the effects of extract of L. sulphureus. 
DISCUSSION
Presented results of antibacterial activity are consistent with the results 
for M. flavus published by T u r k o g l u et al. It proved to be the most suscep-
tible species, though it should be noted that in the mentioned study agar-well 
diffusion method was used, and the extract was methanolic (T u r k o g l u et 
al., 2007). 
It should be mentioned that there are no previous reports on the antifun-
gal activity of extract of L. sulphureus. In the present study, aqueous extract 
303
showed excellent fungicidal activity against all the tested fungi, with 0.30 mg/ml 
as MFC value. In comparison with antifungal activity of antimycotic ketoco-
nazole (1mg/ml DMSO), the tested extract showed lower activity, but still good 
enough to be considered as a promising antifungal agent. Ketoconazole exhib-
ited inhibitory activity against all the tested fungi in the range of 0.63-25.00 x 10-3 
mg/ml, and fungicidal activity in range of 1.25-25.00 x 10-3 mg/ml.
Resistance to antibiotics and antifungal agents is emerging in a wide variety 
of organisms, and multiple drug resistant organisms pose a serious threat to the 
treatment of infectious diseases. Hence, medicinal mushrooms, or mushroom 
derived antimicrobial substances must receive proper attention, especially 
when they include a harmless basis, which is water in this case.
Terpenes, lectins, polysaccharides etc. are known to have an effect on bac-
terial cytoplasmatic membrane, making it vulnerable (L i n and C h o u, 1998; 
Y a n g et al., 2002). L. sulphureus is rich source of these compounds, which 
are potentially responsible for its antimicrobial activity.
C o w a n (1999) reported that the most active components are generally 
water-insoluble, but O l e n n i k o v et al. (2009) determined the existence of 
water-soluble polysaccharides in L. sulphureus that could be bioactive princi-
ples playing an important role in the activity of mushroom (C o w a n, 1999; 
O l e n n i k o v et al., 2009). It is expected that low polarity organic solvents 
would yield more active extracts, but at the same time organic solvent is what 
makes these extracts unsafe and impractical for everyday use (C o w a n , 1999). 
Fig. 1 – Antibacterial and antifungal effect of L. sulphureus extract in comparison to 
streptomycin and ketoconazole
304
Reports on antimicrobial activity of L. sulphureus are very scarce, and 
this is the first report on the antibacterial and antifungal activity of aqueous 
extract of this mushroom.
Results of this study confirm once more the true potential of natural 
products as antimicrobial agents. L. sulphureus presents a valuable source in 
the fight against the tested pathogens, as a sole therapy, which requires further 
clinical trials, or in combination with other chemotherapeutical agents.
ACKNOWLEDGMENTS
This study was supported by the Ministry of Science and Environmental 
protection, Grant # 143041.
REFERENCES
A l q u i n i ,  G . ,  C a r b o n e r o ,  E .  R . (2004): Polysaccharides from the fruit bodies of 
the basidiomycete L. sulphureus. FEMS Microbiology Letters, 230 (1): 47-52.
A s t i n ,  J .  A . (1998): Why patients use alternative medicine: results of a national study. The 
Journal of American Medical Association, 279 (19): 1548-1553.
B a r n e s ,  P.  M . ,  P o w e l l - G r i n e r ,  E . ,  M c F a n n ,  K . ,  N a h i n ,  R .  L . (2004): 
Complementary and alternative medicine use among adults: United States, 2002. Adv. 
Data, (343): 1-19.
B a r r o s ,  L . ,  C a l h e l h a ,  R .  C . ,  V a z ,  J .  A . ,  F e r r e i r a ,  I .  C .  F .  R . , 
B a p t i s t a ,  P . ,  E s t e v i n h o ,  L . (2007): Antimicrobial activity and bioactive com-
pounds of Portuguese wild edible mushrooms methanolic extracts. Eur Food Res Technol 
225: 151-156.
B o o t h ,  C . (1971): Fungal Culture Media. In: (Eds. J. R. Norris and D. W. Ribbons), Meth-
ods in Microbiology, IV, 49-94. Academic Press, London and New York.
C o w a n ,  M .  M . (1999): Plant products as antimicrobial agents. Clinical Microbiolgy re-
view, 12 (4): 564-582.
D a o u k ,  K .  D . ,  D a g h e r ,  M .  S . ,  S a t t o u t ,  J .  E . (1995): Antifungal activity of 
the essential oil of Origanum syriacum L. Journal of Food Protection, 58: 1147-1149.
E s p i n e l - I n g r o f f  (2001): Comparison of the E-test with the NCCLS M38-P method for 
antifungal susceptibility testing off common and emerging pathogenic filamentous fungi. 
Journal of Clinical Microbiology, 39: 1360-1367.
H a n e l ,  H . ,  R a e t h e r ,  W. (1988): A more sophisticated method of determining the fun-
gicidal effect of water-insoluble preparations with a cell harvester, using miconazole as 
an example. Mycoses, 31: 148-151.
L i n ,  J .  Y. ,  C h o u ,  T .  B . (1984): Isolation and Characterization of a lectin from edible 
mushroom, Volvariella volvacea. The Journal of Biological Chemistry, 96 (1): 35-40.
L i n d e q u i s t ,  U . ,  T i m o ,  J .  H . ,  J u l i c h ,  W.  D . (2005): The pharmacological 
potential of mushrooms. ECAM, 2 (3): 285-299.
305
O l e n n i k o v ,  D .  N . ,  A g a f o n o v a ,  S .  V. ,  B o r o v s k i i ,  G .  B . ,  P e n z i n a , 
T .  A . ,  R o k h i n ,  A .  V. (2009): Water-soluble endopolysaccharides from the fruit-
ing bodies of L. sulphureus (Bull.: Fr.) Murr. Applied Biochemistry and Microbiology, 
45 (5): 536-543.
Tu r k o g l u ,  A . ,  D u r u ,  M .  E . ,  M e r c a n ,  N . ,  K i v r a k ,  I . ,  G e z e r ,  K . (2007): 
Antioxidant and antimicrobial activities of L. sulphureus (Bull) Murill. Food Chemistry 
101: 267-273.
W e b e r ,  R .  W.  S . ,  M u c c i ,  A . ,  D a v o l i ,  P . (2003): Laetiporic acid, a new polyene 
pigment from the wood-rotting basidiomycete L. sulphureus (Polyporales, Fungi). Tet-
rahedron letters 45: 1075-1078.
Ya n g ,  B . K . ,  K i m ,  D .  H . ,  J e o n g ,  S .  C . ,  D a s ,  S . ,  C h o i ,  Y.  S . ,  S h i n , 
J .  S . ,  S o n g ,  S .  C . ,  S o n g ,  C .  H . (2002): Hypoglycemic effect of a Lentinus 
edodes exo-polymer produced from a submerged mycelial culture. Bioscience Biotech-
nology and Biochemistry, 66 (5): 937-942.
Z j a w i o n y ,  J .  K . (2004): Biologically active compounds from Aphyllophorales (Polypore) 
fungi. Journal of Natural Products, 67: 300-310.
АН ТИ МИ КРОБ НА АК ТИВ НОСТ ВО ДЕ НОГ ЕКС ТРАК ТА 
L. SULPHUREUS (BULL.: FR.) MURILL
Јо ва на Д. Ши ље го вић, Де јан С. Стој ко вић, Ми лош М. Ни ко лић, 
Ја сми на М. Гла моч ли ја, Ма ри на Д. Со ко вић, Ана М. Ћи рић
Ин сти тут за би о ло шка ис тра жи ва ња „Си ни ша Стан ко вић”, 
Бу ле вар Де спо та Сте фа на 142, 11060 Бе о град, Ср би ја
Резиме
Гљи ва мр ке тру ле жи, Laetiporus sulphureus (Bull.: Fr) Murill., по зна та и као 
шум ско пи ле, већ је по зна та по сво јој ну три тив ној вред но сти. У овој сту ди ји ис пи -
ти ва на је ан ти ми кроб на спо соб ност во де ног екс трак та L. sulphureus, ми кро ди-
лу ци о ном in vitro ме то дом. Биљ ни, жи во тињ ски и ху ма ни па то ге ни, као и про у з-
ро ко ва чи ква ре ња хра не су те сти ра ни. Во де ни екс тракт до би јен од L. sulphureus 
по ка зао је сна жну ак тив ност на те сти ра не ми кро ор га ни зме уз до зну за ви сност. 
С об зиром да по сто ји кон стант ни по раст ре зи стент но сти па то ге на на по зна те 
син те тич ке аген се, по тре ба за но вим те ра пе ут ским ле ко ви ма и кон зер ван си ма у 
пре храм бе ној ин ду стри ји је нео п ход ност. Сто га, ре зул та ти ко ји ука зу ју на ак тив-
 ност при род них про ду ка та мо гу би ти од ве ли ког прак тич ног зна ча ја. 


Antimicrobial activity of aqueous extract of Laetiporus sulphureus (Bull.: Fr.) Murill

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Antimicrobial activity of aqueous extract of Laetiporus sulphureus (Bull.: Fr.) Murill

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