Ganoderma lucidum (Leyss.: Fr) Karst, a mushroom-like fungus is one of the most famous traditional Chinese medicinal herbs. It received wide popularity as a healthy food and medicine in the Far East for more than 2000 years because of its high medicinal value. One of very interesting aspects of G. lucidum's performance is antimicrobial effect due to the extracts derived from this mushroom, which contain bacteriolitic enzyme, lysozyme and acid protease. The effects of these extracts depend on their composition extraction mode and refining. Bioactive components isolated from several G. lucidum's strains showed different effects on the investigated microorganisms. In some cases, the influence was very intensive, with inhibitory or stimulating effect, while some of them did not show any influence on the investigated microorganisms.Ganoderma lucidum (Leyss.: Fr) Karst je jedna od najvažnijih tradicionalnih kineskih gljiva. Prema pisanim podacima starim više od 2000 godina koristi se na Dalekom istoku kao zdrava hrana i lekovita gljiva. Jedan od vrlo interesantnih aspekata korišćenja gljive Ganoderma lucidum je i upotreba ove gljive kao antimikrobnog sredstva, zahvaljujući izolovanim ekstraktima koji sadrže bakteriolitičke enzime, lizozime i kisele proteaze. Efekti ovih ekstrakata zavise od njihovog sastava, načina ekstrakcije i prečišćavanja. Bioaktivne komponente izolovane iz nekoliko sojeva gljive Ganoderma lucidum pokazale su različite efekte na ispitivane mikroorganizme. U nekim slučajevima uticaj ekstrakata je bio vrlo intenzivan, inhibišući ili stimulišući, dok neki od njih nisu pokazali nikakav uticaj na ispitivane mikroorganizme

Klaus, Anita

Nikšić, Miomir

Zbornik Matice srpske za prirodne nauke

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Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad,¥ 113, 219—226, 2007UDC 582.28:579.6A n i t a K l a u s, M i o m i r N i k š i ãFaculty of Agriculture, University of Belgrade,Nemanjina 6, 11080, BelgradeINFLUENCE OF THE EXTRACTS ISOLATEDFROM GANODERMA LUCIDUM MUSHROOMON SOME MICROORGANISMSABSTRACT: Ganoderma lucidum (Leyss.: Fr.) Karst, a mushroom-like fungus is oneof the most famous traditional Chinese medicinal herbs. It received wide popularity as a he-althy food and medicine in the Far East for more than 2000 years, because of its high medi-cinal value. One of very interesting aspects of G. lucidum's performance is antimicrobial ef-fect due to the extracts derived from this mushroom, which contain bacteriolitic enzyme,lysozyme and acid protease. The effects of these extracts depend on their composition,extraction mode and refining. Bioactive components isolated from several G. lucidum's stra-ins showed different effects on the investigated microorganisms. In some cases, the influen-ce was very intensive, with inhibitory or stimulating effect, while some of them did notshow any influence on the investigated microorganisms.KEY WORDS: antimicrobial effect, extraction, extracts, Ganoderma lucidumINTRODUCTIONFor more than 2000 years, Ganoderma lucidum has been regarded as apopular folk medicine in the Far East, used to treat various human diseases,such as hepatitis, hypertension, hypercholesterolemia, gastric cancer and manyothers. Due to its ability to cure many different diseases it received names like“Elixir of life", “Food of Gods", “Mushroom of the Universe". Its intracellularand extracellular polysaccharides showed inhibition of the growth of severaltypes of cancer cells (S o n e et al., 1985, W a n g at al., 2002, Z h a n g etal., 1994). Besides, it also produces many oxygenated triterpenes, especiallyganoderic acid, with various biological functions such as cytotoxicity to hepa-toma cells, inhibition of histamine release, inhibition of cholesterol synthesisand absorption, stimulation of platelet aggregation, and inhibition of thrombininduced platelet aggregation (S h i a o et al., 1994).Antimicrobial drugs derived from different kinds of microorganisms havelong been used for prophylactic and therapeutic purposes. However, using thesame antibiotic for a long period may cause the resistance of microorganisms219to that antibiotic. So, the investigation of influences of different kinds ofpolysaccharides derived from mushrooms on microorganisms and their effectson host's immune system are very important today. Such compounds would beexpected to function by mobilising the body's humoral immunity to ward offviral, bacterial, fungal and protozoal infections resistant to the current antibio-tics (S m i t h et al., 2002).MATERIALS AND METHODSMaintenance of microorganismsAntimicrobial effects of the extracts of G. lucidum on certain strains ofmicroorganisms were investigated:1. Escherichia coli ATCC2. Bacillus cereus ATCC3. Staphylococcus aureus ATCC4. Salmonella enteritidis — Faculty of Agriculture collection5. Proteus mirabilis — Faculty of Agriculture collection6. Saccharomyces cerevisiae ATCC7. Aspergillus niger — Faculty of Agriculture collectionATCC — American Type Culture Collection, Rockville, MarylandBacterial strains of E. coli, B. cereus, S. aureus, S. enteritidis and P. mi-rabilis were maintained on nutrient agar. The slant was inoculated and incuba-ted at 37°C for 24 h, than stored at 4°C.Investigated yeast S. cerevisiae and mould A. niger were maintained onmalt agar. The slant was inoculated and incubated at 30°C, 48h for yeasts andat 25°C, 7 days for mold, than stored at 4°C.Preparation of microorganismsBacterial strains of E. coli, B. cereus, S. aureus, S. enteritidis and P. mi-rabilis were inoculated in nutrient broth and incubated at 37°C for 24 h, toreach the concentration of 106 cells/ml.Yeast S. cerevisiae and mould A. niger were inoculated in malt broth andincubated at 30°C for 24 h for yeast and at 25°C for 24 h for mold, to reachthe concentration of 106 cells/ml.Used strains of Ganoderma lucidumFor this experiment ten different extracts, derived from different strains ofG. lucidum were used. Strains Gl-I, K1 were isolated naturally from Serbianwoods, strains Gl-7 and Gl-349 were taken from the Research Plant Internatio-nal collection, Holland and strain Gl-K originated from China.220Hot extraction of bioactive compounds from driedGanoderma lucidum mushroomPowdered tissue (1—9 g) was washed with 96% ethanol (300 ml), thanfiltered and dried in vacuum (at 40°C for 60 min) up to getting powder. Driedfiltercake was mixed with deionized water (300 ml) and glucans were extrac-ted by autoclaving at 120°C for 20 min. Material was cooled down and centri-fuged (10000 rpm, at 4—9°C for 10 min). Supernatant was mixed with 2 vol.96% ethanol and left at 4°C untill precipitate was formed. After centrifuge(10000 rpm, at 4—9°C for 10 min) the collected pellet were dried in vacuum(at 40°C for 60 min) and the powder was dissolved in Tris buffer 0.01 M (50ml). The suspension was dialyzed for 24 h at room temperature. Dialyze is ne-cessary for refining because low-molecular weight molecules will pass throughthe membrane in solution, while high-molecular weight molecules, b-glucanwill stay inside the membrane. After dialyzing the content was centrifuged(10000 rpm, at 4—9°C for 10 min) and 2 vol. 96% ethanol was added to su-pernatant and left at 4°C for a couple of hours. To remove supernatant centri-fugation (10000 rpm, at 4—9°C for 10 min) was repeated and the pellets weredried in vacuum (at 40°C for 60 min). The dried pellets were dissolved in PBSand used for further examination on microorganisms.Room temperature extraction of water-soluble bioactive compoundsfrom dried mushroom Ganoderma lucidumPowdered tissue (10 g) was mixed with water (300 ml) and steered onmagnetic stirrer at room temperature for 24 h. After filtration, the supernatantwas removed and (NH4)2SO4 was added to 90% saturation. Centrifugation(10000 rpm, at 4—9°C for 10 min) was done and Tris 0.01 M was added tothe pellets for dialyzing (at room temperature for 24 h). This is the way toobtain the lectins which will stay within the membrane and will be separatedby the centrifugation (10000 rpm, at 4—9°C for 10 min). The obtained drylectins were dissolved in PBS and used for the investigation of their influenceon the observed microorganisms.Influence of the extracts on microorganismsPetri dishes were inoculated with 0.2 ml suspension of certain microor-ganism strains, and overlayed with 20 ml of medium. For bacterial growth,Mueller Hinton agar was used, and for yeast and mould, malt agar was used.Three filter disks (Schleicher & Schuell), 6 mm in diameter were placed oneach agar, and 10 ml of appropriate mushroom extract was added. Blind probecontained only PBS, without any mushroom extract. The bacteria were incuba-ted at 37°C for 24 h, the yeast was incubated at 30°C for 48 h, and the mouldwas incubated at 25°C for 7 days.After incubation, inhibition zones around the filter disks were measured.221All experiments were performed in duplicate, for three times. The analy-sis of variance test (P < 0.05) was used to determine the statistical signifi-cance.RESULTS AND DISCUSSIONExamination showed that the bioactive compounds derived from G. luci-dum mushroom have had some influence on the observed microorganisms.PBS did not show any influence on the observed microorganisms.The results of influence of ten different extracts derived from G. lucidummushroom on the microorganisms are shown in the Table 1.Tab. 1 — Influence of different extracts derived from Ganoderma lucidum on microorganismsMicroorganismsInhibition or stimulation zone in diameter (mm)Mushroom extract1 2 3 4 5 6 7 8 9 10Salmonellaenteritidis2.66 2.66 — 1 2.66 3 — 2 3 2.66Escherichia coli 2.33 1.33 — — 2 2 — — 3 2.5Bacillus cereus 14.67*• 15*• 14.67*• 14*• 14.33*• 13.33*• 12*• 14*• 14*• 14*•Proteus mirabilis 16*• 16*• 15*• 16*• 16*• 15.33*• 15*• 14*• 15*• 14.33*•Staphylococcusaureus— — — — — — — — — —Saccharomycescerevisiae— — — — — — — — — —Aspergillus niger — — — — — — — — — —* stimulation of growth— with no influence on growth• significant difference was found between the treatment and the control (p < 0.05)All investigated extract were derived by water extraction and alcohol pre-cipitation, except the extract number 8 which was derived by hot waterextraction.The extracts derived from different strains and parts of fruitbodies of G.lucidum mushroom were used. These extracts were labeled with numbers1—10:1. Extract derived from the fruiting body of G. lucidum2. Extract derived from the spore broken cell walls of G. lucidum Gl-K3. Extract derived from the powdered fruiting body of G. lucidum K14. Extract derived from the micro powdered fruiting body of G. lucidumGl-K5. Extract derived from the G. lucidum Gl-K hypha6. Extract derived from the G. lucidum Gl-K powdered spores7. Extract derived from the fruiting body of G. lucidum Gl-I by waterextraction at room temperature2228. Extract derived from the fruiting body of G. lucidum Gl-I by hotwater extraction9. Extract derived from the fruiting body of G. lucidum-710. Extract derived from the fruiting body of G. lucidum-349It was established that the extracts derived from G. lucidum Gl-K pow-dered spores (Fig. 4) and fruiting body of G. lucidum-7 had significantly (PTon bacteria S. enteritidis. The extracts derived from the fruiting body of G. lu-cidum Gl-I, the spore broken cell walls of G. lucidum Gl-K, the G. lucidumGl-K hypha and the fruiting body of G. lucidum-349 (Fig. 2) showed reducedinhibition effects. The extracts derived from the powdered fruiting body of G.lucidum K1 and from the fruiting body of G. lucidum Gl-I by water extractionat room temperature did not influence the growth of bacteria S. enteritidis.The strongest inhibition effect on the growth of bacteria E. coli was ob-served in the case of the extract derived from the fruiting body of G. luci-dum-349 (Fig. 1), and the extract derived from the fruiting body of G. lucidumGl-I, while the extracts derived from the powdered fruiting body of G. luci-dum K1, the micro powdered fruiting body of G. lucidum Gl-K, the fruitingbody of G. lucidum Gl-I by water extraction at room temperature and the frui-ting body of G. lucidum Gl-I by hot water extraction, did not influence thegrowth of bacteria E. coli.All investigated extracts derived from different kinds of G. lucidum mush-room showed significantly strong stimulating effects (P < 0.05) on the growthof bacteria B. cereus (fig. 5 and 6). The strongest effect showed the extract de-rived from the spore broken cell walls og G. lucidum Gl-K. The most reducedstimulating effect showed the extract derived from the fruiting body of G. luci-dum Gl-I by water extraction at room temperature.All investigated extracts had significantly strong stimulating effects (P <0.05) on the growth of bacteria Proteus mirabili. The extracts derived from thefruiting body of G. lucidum Gl-I, the spore broken cell walls of G. lucidumGl-K, the micro powdered fruiting body of G. lucidum Gl-K and G. lucidumGl-K hypha (Fig. 3) were the strongest. The lowest stimulating effect showed223Fig. 1 — Inhibitory effect of extractderived from the fruiting body ofGanoderma lucidum-349 on the growthof bacteria Escherichia coliFig. 2 — Inhibitory effect of extractderived from the fruiting body ofGanoderma lucidum-349 on the growthof bacteria Salmonella enteritidisthe extract, derived by hot water extraction, from the fruiting body of G. luci-dum Gl-I.The investigated extracts did not show any effect on the growth of bacte-ria S. aureus, yeast S. cerevisiae, and mould A. niger.CONCLUSIONSG. lucidum, one of the oldest salutary remedy known for more than 3000years, became a subject of interest in many contemporary science researchingpapers. Numerous experiments showed different possibilities of using of ex-tracts derived from this mushroom in various disease treatments, improvingimmune system function which results in improving the general condition ofan organism. One of the possible ways of utilising these extracts is their actionon microorganisms which endanger human and animal health very often. Stan-dard procedures in repression of harmful microorganisms are applications ofantibiotics, but too much usage of antibiotics, and the ability of microorga-224Fig. 3 — Stimulating effect of extractderived from Ganoderma lucidum Gl-Khypha on the bacteria Proteus mirabilisFig. 4 — Inhibitory effect of extractderived from Ganoderma lucidum Gl-Kpowdered spores on the bacteriaSalmonella enteritidisFig. 5 — Stimulating effect of extractderived from Ganoderma lucidumon the bacteria Bacillus cereusFig. 6 — Stimulating effect of extractderived from Ganoderma lucidum Gl-K,by hot water extraction, on thebacteria Bacillus cereusnisms to develop resistance to them, result in their decreased effects on micro-organisms. On the other hand polysaccharides derived from mushrooms be-came very interesting due to their influence on immune system and on micro-organisms, that can be used in a struggle against them. In this work, the in-fluence of some polysaccharides derived from G. lucidum mushroom on seve-ral microorganisms was investigated. In some cases the examined extractsshowed very intensive influence, inhibitory or stimulating, while some of themdid not show any influence on the examined microorganisms.We believe that this investigation is of a current interest and should becontinued with more microorganisms and numerous chemically defined frac-tions derived from the extracts of G. lucidum mushroom.REFERENCESK l a u s, A. (2004): Proizvodnja i bioaktivna svojstva gljive Ganoderma lucidum, magi-starski rad, Univerzitet u Beogradu, Poljoprivredni fakultet, Beograd.S h i a o, M. S., L e e, K. R., L e e, J. L., C h e n g, T. W., (1994): Natural productsand biological activities of the Chinese medicinal fungus, ACS Symp. ser. 547Food Phytochemicals for Cancer Prevention II, Washington DC: American Che-mical Society, 342—354.S m i t h, R o w a n and S u l l i v a n (2002): Medicinal mushrooms: Their therapeuticproperties and current medical usage with special emphasis on cancer treatments,University of Strathclyde.S o n e, Y., O k u d a, R., W a d a, A., K i s h i d a, A., M i s a k i, A. (1985): Structu-res and antitumor activities of the polysaccharides isolated from fruiting body andthe growing culture of mycelium of Ganoderma lucidum, Agric. Biol. Chem., 49:2642—50.W a n g, G. Y. Y., K h o o, K. H., C h e n, S. T., L i n, C. C., W o n g, C. H., L i n, C.H. (2002): Studies on the Immuno-Modulating and Antitumor Activities of Gano-derma lucidum (Reishi) Polysaccharides: Functional and Proteomic Analyses of aFucose-Containing Glycoprotein Fraction Responsible for the activities, Bioorga-nic & Medicinal Chemistry 10: 1057—1062.Z h a n g, J., W a n g, G., L i, H., Z h u a n g, C., M i z u n o, T., I t o, H., M a y u -z u m i, H., O k a m o t o, H., L i, J. (1994): Antitumor Active Protein-containingGlycans from the Chinese Mushroom Songshan Lingzhi, Ganoderma tsugae Myce-lium, Biosci. Biotech. Biochem., 58 (7): 1202—1205.225UTICAJ EKSTRAKATA IZOLOVANIH IZ GQIVEGANODERMA LUCIDUM NA NEKE MIKROORGANIZMEAnita Klaus, Miomir NikšiãPoqoprivredni fakultet, Univerzitet u Beogradu,Nemawina 6, 11080 Beograd, SrbijaRezimeGanoderma lucidum (Leyss.: Fr.) Karst je jedna od najvaÿnijih tradicional-nih kineskih gqiva. Prema pisanim podacima starim više od 2000 godina ko-risti se na Dalekom istoku kao zdrava hrana i lekovita gqiva. Jedan od vrlointeresantnih aspekata korišãewa gqive Ganoderma lucidum je i upotreba ovegqive kao antimikrobnog sredstva, zahvaqujuãi izolovanim ekstraktima kojisadrÿe bakteriolitiåke enzime, lizozime i kisele proteaze. Efekti ovih eks-trakata zavise od wihovog sastava, naåina ekstrakcije i preåišãavawa. Bioak-tivne komponente izolovane iz nekoliko sojeva gqive Ganoderma lucidum poka-zale su razliåite efekte na ispitivane mikroorganizme. U nekim sluåajevimauticaj ekstrakata je bio vrlo intenzivan, inhibišuãi ili stimulišuãi, dokneki od wih nisu pokazali nikakav uticaj na ispitivane mikroorganizme.226

Uticaj ekstrakata izolovanih iz gljive Ganoderma lucidum na neke mikroorganizme

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Uticaj ekstrakata izolovanih iz gljive Ganoderma lucidum na neke mikroorganizme

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