Embed Size (px)
Citation preview
Available online at www.sciencedirect.com
www.elsevier.com/locate/bcdf
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1
http://dx.doi.org/102212-6198 & 2014 El
nCorresponding aE-mail address: l
Antibacterial and antioxidant activities of aqueousextracts of eight edible mushrooms
Lu Rena,n, Yacine Hemara, Conrad O. Pereraa, Gillian Lewisb,Geoffrey W. Krissansenc, Peter K. Buchanand
aSchool of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland, New ZealandbSchool of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New ZealandcSchool of Medical and Health Sciences, The University of Auckland, Auckland 1005, New ZealanddLandcare Research, Private Bag 92170, Auckland, New Zealand
a r t i c l e i n f o
Article history:
Received 20 December 2013
Received in revised form
19 January 2014
Accepted 29 January 2014
Keywords:
Mushroom polysaccharides
Antibacterial
Antioxidant
Disk diffusion
Microdilution
FT-IR
.1016/j.bcdf.2014.01.003sevier Ltd. All rights rese
a b s t r a c t
Polysaccharides extracts of eight edible mushroom species, including five species collected
from New Zealand forests and parks, were tested for their ability to inhibit the growth of
five common bacterial strains. Antibacterial activity was assayed using the disc diffusion
and microdilution methods. An aqueous extract from Cordyceps sinensis inhibited the
growth of Bacillus subtilis and Streptococcus epidermidis with minimum inhibitory concentra-
tion (MIC) values of 938 and 469 μg/mL, respectively. A Pleurotus australis extract had the
same MIC of 469 μg/mL against S. epidermidis. Comparatively, the microdilution method
was more efficient and accurate than the disk diffusion method at measuring the
antimicrobial activity of high molecular weight polysaccharides. All polysaccharides
exhibited DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activities, with P. aus-
tralis having the highest antioxidant activity (EC50 of 4.03 mg/mL). Fourier transform
infrared (FT-IR) analyses indicated that some extracts contained α or β-conformations.
Their relative quantities of OH, evaluated by the ratios of OH group/CH group, did not
correlate with their scavenging activity based on EC50 values. Several of the mushroom
polysaccharide extracts investigated in this study have antibacterial and antioxidant
activities that warrant further study as potential dietary supplements to improve health
and well-being.
& 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The growing emergence of drug-resistant bacterial strains is aserious threat to the effective treatment of infections (Klein,Smith, & Laxminarayan, 2007). Drug resistance usually hap-pens after long-term misuse of antibacterial agents (Gao,Zhou, Huang, & Xu, 2003), and is a consequence of the
rved.
z (L. Ren).
acquisition of mutations in the bacterial genome and genesthat assist bacterial survival (Mazodier & Davies, 1991).Whilst it is impossible to prevent bacterial evolution, it isimportant to choose the most appropriate antibiotics and touse them appropriately to minimise the development ofdrug-resistant strains (Alves, Ferreira, Martins, & Pintado,2012). The application of antibiotics in animal feed has been
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 142
reduced as a measure to avoid the emergence of newantibiotic resistant strains. Regulatory restrictions on thesubtherapeutic use of antibiotics necessitates alternativemeans to restrain bacterial infections in poultry (Giannenaset al., 2010). There will forever be a need for novel antimicro-bial compounds to outwit bacteria and other pathogens(Zgoda & Porter, 2001).
Stress on the body due to aging, obesity, and detrimentrallifestyle choices is another significant health issue, whichoften takes the form of oxidative damage to tissues. Super-oxide radicals (O2
�), hydroxyl radicals ( �OH) and hydrogenperoxide (H2O2) damage DNA, impair enzymes and structuralproteins, and provoke uncontrolled chain reactions includinglipid peroxidation (Halliwell & Cross, 1994). The reactiveoxygen species (ROS) and oxygen-derived free radicals areknown to play key roles in carcinogenesis and cellulardegeneration which promote the development of cancer,cardiovascular and neurological diseases, cataracts, diabetes,and rheumatoid arthritis (Circu & Aw, 2010; Cadenas &Davies, 2000; Jeong et al., 2012). Almost all organisms havedefence systems to protect against free radical damage. Theactivity to scavenge radicals has been associated with a risein the antioxidant enzyme activities. These enzymes includesuperoxide dismutase (SOD) which catalyzes the dismutationof superoxide anion to hydrogen peroxide, catalase (CAT)which detoxifies hydrogen peroxides and converts lipidhydroperoxides to non-toxic substances, and glutathioneperoxidase (GPx) which maintains the levels of reducedglutathione (GSH) (Guo, Ji, & Ping, 2009). However, thesesystems are not sufficient to prevent damage, and henceantioxidants are employed to form a cooperative defencesystem. Antioxidants are defined as compounds that possessan ability to protect biological systems against the potentiallyharmful effects of processes or reactions that can causeexcessive oxidation (Krinsky, 1989). Many synthetic antiox-idants, such as butylated hydroxyanisole and butylatedhydroxytoluene, have side effects and are thought to beresponsible for liver damage and carcinogenesis (Grice,1988). As a result, natural antioxidants are preferred in foodapplications. Natural substances, such as vitamins A, C and E,carotenoids, flavonoids and other simple phenolic com-pounds, are proven to prevent oxidative damage and thusprotect the human body (Mackerras, 1995).
Numerous antimicrobial agents, including penicillin andgriseofulvin, have been isolated from micro-fungi. The richdiversity of different fungal species offers a potential sourceof new antibiotics (Yamac & Bilgili, 2006). Mushrooms thatpossess the macroscopic reproductive structures of a diverserange of basidiomycete fungi have been utilized for curativeand medicinal purposes since prehistoric times. Mushroomsare a veritable treasure-house of bioactives that displayantimicrobial, antitumorgenic, hypolipidemic, and hypogly-caemic properties (Venturini, Rivera, Gonzalez, & Blanco,2008). Two major groups of mushroom bioactives are thepolysaccharides and triterpenes (Ameri, Vaidya, & Deokule,2011). Polysaccharides are responsible for the rigidity andmorphological properties of the fungal cell wall (Wolff et al.,2008). Many display potent activity against common strainsof bacteria (Yamac & Bilgili, 2006; Bala, Aitken, Cusack, &Steadman, 2012; Zhu, Sheng, Yan, Qiao, & Lv, 2012). Fungi
produce polysaccharides, phenolics, and various metabolitesthat represent potential sources of novel natural antioxidants(Huang, Cai, Xing, Corke, & Sun, 2007; Cheung, Cheung, & Ooi,2003).
The current study sought to investigate antibacterialactivities of five New Zealand native mushrooms and threecultivated mushrooms. Paper disc diffusion and microdilu-tion techniques were employed to measure antibacterialactivity, and compared in order to determine which methodwas superior. The antioxidant activity of these mushroomextracts was also assessed by measuring their radical scaven-ging activity.
2. Materials and methods
2.1. Materials
Eight species of mushroom were selected for evaluation ofantibacterial and antioxidant activities. Four mushroom spe-cies were collected fresh from native podocarp forests aroundAuckland (New Zealand), namely Auricularia cornea Ehrenb.,Calvatia gigantea (Batsch) Lloyd, Hericium coralloides (Scop.)Pers., and Pleurotus australis (Cooke & Massee) Sacc. Ileodictyoncibarium Tul. & C.Tul. was collected on wood mulch fromurban gardens. With the exception of P. australis, the otherfour New Zealand species were reportedly known to Maori asedible mushrooms (Fuller, Buchanan, & Roberts, 2004). Twocultivated mushroom species, Hericium erinaceum (Bull.) Pers.and Lentinula edodes (Berk.) Pegler, were purchased as Asianimported dried products from a local supermarket. Cordycepssinensis was obtained as a powdered product purchased fromMedimushrooms (Dr. Alla's Cordyceps, New Zealand). Thefree-radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH) and potas-sium bromide (KBr) were purchased from Sigma (Darmstadt,Germany).
2.2. Extraction of polysaccharides
Water soluble substances were extracted and purified frommushrooms according to the method described by Wanget al. (1993) with additional modifications as shown inFig. 1. Mushroom fruiting bodies were frozen at �80 1C,lyophilized in a vacuum freeze dryer, and reduced to a finedried powder (20 mesh) (Vaz et al., 2011). Powdered samples(100 g) were extracted with 150 mL of 80% ethanol for 1 h toeliminate low molecular components such as mono- anddisaccharides, oligosaccharides, amino acids, lipids and somephenols. After filtration with a Whatman No. 4 paper, theresidue was subjected to aqueous extraction under agitation(150 rpm; magnetic stirrer) with hot water (1 L, 100 1C, 3 h).The aqueous suspension was centrifuged at 8400� g for15 min followed by filtration with Whatman No. 4 paper.The supernatant was concentrated to 10 mL under vacuum at55 1C and then added to 5 volumes of ethanol to induceprecipitation at 4 1C overnight (Xie et al., 2010). The precipi-tated polysaccharides were collected after centrifugation asabove. The obtained precipitate was again dissolved in dis-tilled water (100 mL) and dialyzed through a DEAE cellulosetube (molecular weight cut-off of 12,000 Da) against distilled
Mushroom fruiting bodieMushroom fruiting bodies
LyophilisationGrind
Mushroom powdersMushroom powders
Extraction with 80% EtOH
FiltrationFiltration(Whatman paper No.4)(Whatman paper No.4)
ResidueResidue
FiltrateFiltrate
Extraction with water (1L,100oC, 3h)Centrifugation (8,400 × g, 15min)
ResidueResidue
FiltrateFiltrate
Concentration under vacuum (55oC)Precipitation with ethanol (5 × V)Centrifugation (8,400 × g, 15min)
PrecipitatePrecipitate
HydrationDialysis with DEAE cellulose tube (72h)Concentration & lyophilisation
Mushroom polysaccharidesMushroom polysaccharides
FiltrationFiltration( Whatman paper No.4)( Whatman paper No.4)
Fig. 1 – Flow-chart of the preparation of crude mushroompolysaccharides.
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1 43
water for 72 h to remove low molecular weight carbohydratematerials. After concentration under vacuum at 55 1C, thecontents of the dialysis tubes were frozen at �80 1C andlyophilized in a vacuum freeze dryer to obtain crude poly-saccharides. The extracts were dissolved in sterile water tomake up two stock solutions at concentrations of 3 and15 mg/mL, which were heated at 90 1C for 3 h to completethe hydration and then vortexed thoroughly.
2.3. Bacteria
Five species of bacteria were assessed for susceptibility togrowth inhibition by the polysaccharide fractions. Theyincluded the three Gram-positive strains Bacillus subtilis,Enterococcus faecalis, Staphylococcus epidermidis, and the twoGram-negative strains Enterobacter aerogenes and Escherichiacoli 916. These bacteria were obtained from the School ofBiological Sciences, the University of Auckland, where theywere stored at �801 C.
2.4. Antibiotics
Two antibiotics, penicillin (Austral, Australia) and gentamicin(Deisenhofen, Germany), were purchased from Sigma. Theywere dissolved in sterile water to obtain stock solutions at1.2 mg/mL and 50 mg/mL concentrations, respectively.
2.5. Antibacterial activity
The antibacterial activity of the polysaccharide extracts wasdetermined using both the agar disc diffusion method andsingle microdilution assay. Agar disc diffusion was performedaccording to the method reported by Yamac and Bilgili (2006)with modifications as given below. Bacteria were culturedovernight, counted with a haemocytometer and adjusted to108 CFU/mL. An aliquot of l00 μL of bacterial suspension wasspread on the surface of nutrient agar with a sterile glass rod.The filter paper disks (6 mm diameter) that had been pre-viously impregnated with 10 μL of the mushroom extractsolutions (3 or 15 mg/mL) were laid on the inoculated media.Each petri dish received 6 paper disks. The petri dishes werekept at 4 1C for 2 h for diffusion of the metabolites followed byincubation at 37 1C for 24 h. Antibacterial activity was eval-uated by measuring the diameter of the clear inhibition zonearound each disk. Antibiotics were used as positive controlsand 0.9% saline solution was used as a negative control.
The susceptibility of bacteria to extracted polysaccharideswas also detected using the single microdilution methoddescribed by Casaril, Kasuya, and Vanetti (2011) with addi-tional modifications. In each well of a 96-well plate, 100 μL ofnutrient broth, 50 μL of mushroom extract solution (15 mg/mL) and 50 μL of bacterial suspension (108 CFU/mL) weremixed. After incubation at 37 1C for 24 h, the absorbance at750 nm of each incubation was read with a microplate reader(EnSpires Multimode Plate Reader, Finland). The antibacterialactivity was measured by comparing the optical densities ofwells containing the same extracts and controls. The percen-tage inhibition of bacterial growth by the polysaccharides wascalculated using the equation below:
Inhibition ð%Þ ¼ ½AB–ðABE�AEÞ�=AB � 100
where AB is the absorbance of the bacterial control withoutadding polysaccharides; ABE is the absorbance of the mixturecontaining the bacterial suspension and polysaccharideextract solution; and AE is the absorbance of the correspond-ing polysaccharide extract solution.
2.6. Minimum inhibitory concentration (MIC)
The polysaccharides exhibiting antibacterial activity werefurther studied to determine their MIC values by applyingthe microdilution method of Zgoda and Porter (2001) withsome modifications as given. Eight two-fold serial dilutionswere carried out using sterile water to prepare the polysac-charides at concentrations ranging from 7.5 to 0.12 mg/mL.A 100 mL aliquot of sterile distilled water was initially placedinto each well in a 96-well microplate followed by a 100 mLaliquot of polysaccharide stock solution of (15 mg/mL), andmixed thoroughly with a multi-channel pipetter. Then, 100 mLof the mixed solution was used for downstream serial
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 144
dilutions in consecutive wells. For the inoculation, 100 mL ofeach microbial suspension (108 CFU/mL) was added into eachwell. The last well, without bacteria, in each row served asthe negative control. The two antibiotics, penicillin andgentamicin, which were used as the positive controls, werediluted in the same manner starting with initial concentra-tions of 1.2 mg/mL and 50 mg/mL, respectively. After incuba-tion at 37 1C for 24 h, the growth of the bacteria was evaluatedby measuring the absorbance at 750 nm with an automatedmicroplate reader. MIC values were the lowest concentrationof a polysaccharide or antibiotic showing inhibition of thegrowth of the bacteria. The MIC values were obtained bycomparing the absorbance values of solutions containingbacteria and mushroom extracts at different concentrationswith that of the control (bacteria alone) without extracts sincethere was a direct relationship between the values of absor-bance and the growth of bacteria.
2.7. Antioxidant activity
The antioxidant assay was carried out in a 96-well microplateusing an EnSpire Microplate Reader according to the methodof Vaz et al. (2011), and modified as follows. Four concentra-tions of the extracted aqueous polysaccharide solutions wereprepared, starting with a 10 mg/mL solution and then dilutingit to 5, 2.5, and 1.25 mg/mL. In each well, one of the differentconcentrations of the extract (30 μL) and aqueous methanolicsolution (80:20, v/v, 270 μL) containing DPPH radicals (1.2�10�4 M) were added. The reaction was allowed to proceed for60 min in the dark. The reduction of DPPH radicals wasevaluated by measuring the absorption at 515 nm. Radicalscavenging activity (RSA) as evidenced by DPPH discoloura-tion is calculated using the equation below:
RSA¼ ½ADPPH–ðAM�AEÞ�=ADPPH � 100
where ADPPH is the absorbance of the DPPH solution; AM is theabsorbance of the mixture consisting of both DPPH andextract at a particular concentration; and AE is the absorbanceof the corresponding polysaccharide extract solution.
2.8. FT-IR spectroscopy
Two milligrams of freeze-dried extract were mixed with200 mg of KBr to make a pellet. The absorption spectra ofthe pellets were measured with a FT-IR and FT-FIR spectro-photometer (Spectrum 400, Perkin Elmer, USA) from 4000 to400 cm�1 at 0.2 cm/s according to Jouraiphy et al. (2008).
2.9. Statistical analysis
Statistical analysis was carried out using IBM SPSS statisticssoftware (Version 21, IBM Corp., 2012). The EC50 value stoodfor the effective concentration at which 50% of the radicalswere scavenged, which was calculated using a regressionprobit model. Also, one-way analysis of variance (ANOVA)was conducted for the comparison of mean values, whichwere further separated using Tukey's honestly significantdifference test.
3. Results and discussion
3.1. Antibacterial activity of the polysaccharide extractsbased on disc diffusion and single dilution methods
The polysaccharide extracts of eight mushroom extracts werescreened for antibacterial activity against five bacteria usingboth the disc diffusion and single microdilution methods.The results are shown in Table 1. Overall, the measurementof the growth inhibitory activities of the extracts and anti-biotics was not totally consistent between the two assays. Allthe mushroom extracts at the two concentrations of 3 and15 mg/mL failed to demonstrate antibacterial activity by thedisc diffusion method. Nonetheless in the single microdilu-tion method, antibacterial activity was shown by C. sinensisagainst B. subtilis and S. epidermidis, and by P. australis againstS. epidermidis. In contrast, antibiotics were more effective ingenerating inhibition zones around the paper discs. PenicillinG (1.2 mg/mL) only showed growth restriction of S. epidermi-dis, producing the largest clear zones on the agar. Gentamicinat the concentration of 50 mg/mL was more effective againstnearly all the bacteria except for E. faecalis. The smallestinhibition zones with an average diameter of 9 mm wereproduced by gentamicin on agar inoculated with the Gram-negative strain E. aerogenes. Of the results from the singlemicrodilution method, almost all tested bacteria were sus-ceptible to the two antibiotics, with the only exception beingpenicillin G having no observable effect on B. subtilis.
B. subtilis and S. epidermidis, which were inhibited by themushroom extracts, are Gram-positive strains, whereas thetwo Gram-negative strains were resistant to the mushroomextracts. The sensitivity of Gram-positive bacteria to mush-room extracts agrees with previous studies (e.g., Barros,Baptista, Estevinho, and Ferreira (2007), Venturini et al.(2008), Yamac and Bilgili (2006)). An explanation for thisphenomenon is that unlike Gram-positive bacteria, Gram-negative bacteria have an outer membrane and periplasmicspace surrounding the cell wall. The inner leaflet of the outermembrane consists of phospholipids, whereas the outerleaflet is mainly comprised of lipopolysaccharides (Holst &Müller-Loennies, 2007). Lipopolysaccharides are constructedfrom three parts: a proximal hydrophobic lipid A region, acore oligosaccharide region connecting a distal O-antigenpolysaccharide region to lipid A. Also, the lipopolysaccharidesmolecule contains six or seven covalently linked fatty acidchains. As a result, the asymmetric bilayer of the bacterialouter membrane forms lipid-like cell walls and serves as anefficient barrier against rapid penetration by antibiotics,chemotherapeutic agents (Cohen, 2004), and polysaccharidesform various mushroom species, such as Agaricus silvicola,Clitocybe nebularis, Tricholoma equestre (Venturini et al., 2008).The periplasmic space majorly contains peptidoglycan, alsoknown as murein. The peptidoglycan sacculus represents arigid layer that determines the form of the bacterial cell.Apart from peptidoglycan, the periplasmic space containsvarious smaller molecules, such as mono- and oligosacchar-ides, and amino acids. The high concentration of all thesesubstances makes the periplasmic space as a gel-like matrixwith some holes (Holst, Moran, & Brennan, 2009). Further, the
Tab
le1–Res
ultsof
inhibition
ofth
egrow
thof
bacteria
bym
ush
room
extrac
tsusingth
ediscdiffu
sion
andsinglem
icro
dilution
method
s.
Method
Bac
terium
Polysa
ccharideex
trac
tCon
trol
AC
CG
CS
HC
HE
ICLE
PASa
line
Penicillin
GGen
tamicin
Discdiffu
sion
Bacillu
ssu
btilis
––
––
––
––
––
19En
teroba
cter
aerogene
s–
––
––
––
––
–9
Escherichiacoli91
6–
––
––
––
––
–10
Enterococcus
faecalis
––
––
––
––
––
–
Stap
hylococcus
epidermidis
––
––
––
––
–31
15
Singlemicro
dilution
B.su
btilis
––
þ–
––
––
––
þþþ
E.ae
rogene
s–
––
––
––
––
þþ
þE.
coli91
6–
––
––
––
––
þþ
þE.
faecalis
––
––
––
––
–þþ
þþþ
þS.
epidermidis
––
þþþ
––
––
þþ
–þ
þþ
þ
WhereAC¼Auricularia
cornea
;CG¼Calva
tiagiga
ntea
;CS¼Cordy
ceps
sine
nsis;HC¼Hericium
clathroide
s;HE¼Hericium
erinaceu
s;IC
¼Ile
odictyon
ciba
rium;LE
¼Lentinula
edod
es;PA
¼Pleu
rotusau
stralis
.In
the
discdiffu
sion
method,th
eva
lues
areth
ediameters(m
m)ofinhibition
zones
.Theresu
ltsforth
e3an
d15
mg/mLco
nce
ntrationsareth
esa
me.
Inth
esinglemicro
dilution
method,an
tiba
cterial
activity
isba
sedon
inhibition
perce
ntage
s:low
activity
(þ,r
35%),moderateac
tivity
(þþ,
435
%an
dr
70%),an
dhigh
activity
(þþþ
,470
%).Noan
tiba
cterialac
tivity
“�”.
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1 45
enzymes within the periplasmic space are capable of break-ing down foreign molecules introduced from outside (Duffy &Power, 2001). The periplasmic space is thus crucial forosmotic stability and also acts as a protective barrier (Holstet al., 2009). In contrast, the absence of an outer membrane inGram-positive bacteria results in the absence of a membrane-bound periplasm. Instead, they contain multilayered pepti-doglycan covalently substituted with the anionic polymersteichoic acid or teichuronic acid. Gram-positive walls possessa large water capacity and ability to retain large amounts ofproteins, lipoglycans and cations, due to this thick, hydro-philic, porous structure (Hancock, 2002). All these propertiesmake Gram-positive walls more permeable than those ofGram-negative. Therefore, Gram-positive bacteria are likelyto be more vulnerable to the attack of mushroompolysaccharides.
C. sinensis is one of the most valuable traditional Chinesemedicines. Its given Chinese name is “Dong-Chong-Xia-Cao”which means “Winter-worm summer-grass”. As an entomo-pathogen, it parasitises larvae of certain Lepidoptera species(Ng & Wang, 2005). Wild C. sinensis is a rare and endangerednatural species, found only in a few isolated regions of highplateaus 3500–5000 m above sea level in western China(Wang et al., 2007). Kuo, Chang, and Cheng (2007) reportedthat polysaccharides from C. sinensis promoted immunomo-dulatory activity by enhancing CD11b expression, phagocy-tosis, and release of cytokines such as tumor necrosis factoralpha (TNF-α), and interleukin (IL)-6. Phagocytosis is theprimary means to eliminate free-living microorganisms fromblood and tissue fluids (Lee & Lee, 2005).
To the best of our knowledge, this is the first study toreveal that polysaccharides isolated from P. australis haveantibiotic activity against bacteria. P. australis used in thisstudy was collected from forests near Auckland. The genusPleurotus has about 20 species (Kirk, Cannon, Minter, &Stalpers, 2008), which are known commonly as oyster mush-rooms since their pileus or cap is shell-like and their stipe iseccentric or lateral (Shoji, 2000). Like C. sinensis, the poly-saccharides extracted from certain Pleurotus species werereported to have antitumor and immunomodulatory activ-ities (Wang, Hu, Liang, & Yeh, 2005), particularly β-(1,3)- andβ-(1,6)-glucans (Wolff et al., 2008). However, the antibacterialmechanism as well as other biochemical mechanisms sup-porting those therapeutic activities remain largely undefined.The possible antibacterial mechanisms of polysaccharides inprevious studies might provide some clues. For instance, ithas been suggested that the antibacterial activity of chitosanis mainly due to ionic surface interaction. The positivelycharged chitosan interacts with many negatively chargedmicrobial cell components, such as cell wall, DNA and RNA,which results in an increase in cell wall permeability andintracellular components leakage (Je & Kim, 2006). Also, it caninhibit the mRNA and protein synthesis via the penetrationof chitosan into nuclei of the microorganisms (Sudarshan,Hoover, & Knorr, 1992). Additionally, chitosan is able to forman external barrier, chelating metals and provoking thesuppression of essential nutrients to microbial growth(Roller & Covill, 1999). These mechanisms were also proposedfor the antibacterial activities of a polysaccharide isolatedfrom Streptomyces virginia H03 (He, Yang, Yang, & Yu, 2010).
3750 1875 938 469 234 117 59 0
Abs
orba
nce
(nm
)
0.00
0.05
0.10
0.15
0.20
0.25
0.30Extract from C. sinensis against B. subtilisExtract from C. sinensis against S. epidermidisExtract from P. australis against S. epidermidisControl of B. subtilisControl of S. epidermidis
Extract (μg/mL)
Fig. 2 – Effect of mushroom extracts at differentconcentrations on bacterial growth. The absorbances ofbacterial suspensions were recorded as the mean7standarderror (n¼4), and are directly proportional to the number ofbacteria.
Extract (mg/mL)0 2 4 6 8 10 12
Rad
ical
sca
veng
ing
activ
ity (%
)
0
20
40
60
80 A. corneaC. giganteaC. sinensisH. coralloidesH. erinaceumI. cibariumL. edodesP. australis
Fig. 3 – DPPH radical scavenging activity of mushroomextracts. (n¼4.).
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 146
Here we have demonstrated that polysaccharides from C.Sinensis and P. Australis can directly inhibit the growth ofcertain bacteria. Nonetheless, more studies are required toclarify the antibacterial mechanisms of mushroompolysaccharides.
Even though our extract from L. edodes had no effect on thegrowth of the bacteria investigated, Venturini et al. (2008)reported that an aqueous extract of this mushroom inhibitedthe growth of Vibrio parahaemolyticus, Yersinia enterocolitica,Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, andClostridium perfringens. However, it did not inhibit E. coli, inagreement with our results. Other mushrooms in the currentstudy that failed to inhibit the growth of all five bacteriaincluded H. erinaceum and the four native mushrooms,A. cornea, C. gigantea, H. coralloides, and I. cibarium.
Several reasons can be suggested to explain the differencein the results obtained with the two methodologies tomeasure antibacterial activity. The disc diffusion method isvery dependent on the diffusion ability of the test substances.The polysaccharides used in this study were biopolymerswith high molecular weights, which diffuse poorly into agar(Romberger & Tabor, 1971). Davidson and Parish (1989)reported that differences in the antimicrobial activitybetween different types of extracts were caused by the abovelimitation of the diffusion agar method, and considered thisapproach inappropriate for testing partially or completelyhydrophobic compounds. Thus, the results herein reliedupon how readily the compounds diffused in a hydrophilicenvironment, and indicate that the bioactives are of highmolecular weight. Therefore, the microdilution method hasadvantages over the disc diffusion in respect of measuringthe antibacterial properties of fungal polysaccharide poly-mers. Note that as the crude polysaccharides investigatedhad not been fully purified, trace amount of non-polysaccharide compounds might exist in the mixtures. Theeffect of these compounds, even if they are in trace amounts,on bacteria cannot be ruled out. Thus, further work onpolysaccharide fractions purified by chromatographic meth-ods should be performed on the most active fractions infuture work.
3.2. Minimum inhibition concentration of extracts
The microdilution assay was performed to determine the MICvalues of those extracts that demonstrated antibacterialactivities (Table 1). MIC values were determined as 938 μg/mL for C. sinensis against B. subtilis; and 469 μg/mL for both C.sinensis and P. australis against S. epidermidis (Fig. 2). Thegrowth inhibitory effects of the polysaccharide extracts wereconcentration dependent, in accord with an earlier study byHe et al. (2010). The standard reference antibiotics were morepotent, having lower MIC values against these two bacteria.Gentamicin was effective against B. subtilis with a MIC of0.39 μg/mL. Penicillin G had MICs of 37.50 and 1.56 μg/mLagainst S. epidermidis, respectively (results not shown). TheMIC values of mushroom extracts in our study were on asimilar scale with those reported by Yoon, Eo, Kim, Lee, andHan (1994). In their study, the aqueous extract from thecarpophores of Ganoderma lucidum showed an MIC of 750 μg/mL against Micrococcus luteus. Although the MIC values of
mushroom extracts investigated in the present study aremuch higher than the reference antibiotics, and consideringthey were crude mixtures rather than pure substance, theymay have some potential for development as novelantibiotics.
3.3. Antioxidant activity
All investigated mushroom extracts showed concentration-dependent scavenging activity of DPPH radicals (Fig. 3).At 10 mg/mL, the scavenging percentages of four extracts(A. cornea, C. gigantea, C. sinensis, and L. edodes) was less than40%. A. cornea demonstrated the lowest ability to reduce DPPHat 26%. The maximum scavenging percentage for L. edodeswas 36% at 5 mg/mL, and was largely unchanged at 10 mg/mL. The congeneric H. coralloides and H. erinaceum showedsimilar scavenging behaviour at the concentrations tested.For example, at 1.25 mg/mL their scavenging percentage was
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1 47
approximately 27%; and at 10 mg/mL they were 55% and 64%,respectively. P. australis had the greatest scavenging activityacross all concentrations. P. australis exhibited the mostpotent antioxidant activity with an EC50 of 4.03 mg/mL. I.cibarium was the next most potent with an EC50 of 5.78 mg/mL, followed by H. erinaceum and H. coralloides EC50s of 5.82and 7.19 mg/mL, respectively.
The DPPH radical scavenging assay is a widely acceptedmodel to assess free radical-scavenging activity (Naik et al.,2003). The assay avoids side reactions such as metal ionchelation and enzyme inhibition, which complicate assaysbased on laboratory-produced free radicals such as hydroxylradical and superoxide anion (Amarowicz, Pegg, Rahimi-Moghaddam, Barl, & Weil, 2004). The ability of antioxidantsto scavenge DPPH is attributed to their hydrogen donatingactivity (Liu, Jia, Kan, & Jin, 2013). Reactive oxygen andnitrogen species are related to the pathophysiology of a widerange of diseases. Oxidative damage to DNA is able to triggercarcinogenesis (Ajith & Janardhanan, 2007). It has beensuggested that the antitumor and immunomodulating activ-ities of polysaccharides are largely related to their antiox-idant properties (Russell & Paterson, 2006).
3.4. FT-IR spectra
FT-IR spectra were obtained to investigate the molecular proper-ties of mushroom polysaccharides, and to evaluate correlationsbetween their structural characteristics and bioactivity. Asshown in Fig. 4, all mushroom extract spectra showed typicalcarbohydrate patterns, with strong and broad absorption near3400 cm�1 (A. cornea: 3400.1 cm�1, C. gigantea: 3388.6 cm�1, C.sinensis: 3389.0 cm�1, H. coralloides: 3390.6 cm�1, H. erinaceum:3369.7 cm�1, I.cibarium: 3400.1 cm�1, L. edodes: 3389.5 cm�1, P.australis: 3389.4 cm�1); which indicates the presence of OHgroups. Other regular features of polysaccharides were identifiedin the FT-IR spectra of all extracts. The weak peaks at �2922cm�1 are interpreted to be due to CH2 stretching and bendingvibrations (Kozarski et al., 2012). Carbonyl groups (C¼O) showtwo bands: an asymmetrical stretching band around 1650 cm�1
and a weak symmetric stretching band near 1400 cm�1 (Casu,Scovenna, Cifonelli, & Perlin, 1978). The 1200–1000 cm�1 regionis dominated by sugar ring vibrations overlapping with stretch-ing vibrations of (C–OH) side groups and (C–O–C) glycosidic bondvibration at around 1150 cm�1. The peaks near 1374 cm�1
correspond to C–H in-plane bending vibration. The characteristicstretching band around 1080 cm�1 is curved by the signal of apyranose form of the O-substituted residue (C–O) (Mathlouthi &Koenig, 1986; He, Ru, Dong, & Sun, 2012).
Apart from the common carbohydrate characteristics,several distinct structural traits were observed for the differ-ent extracts. The signal at 1733 cm�1 found in the extracts ofA. cornea is indicative of a carboxylic ester band. The adsorp-tion peaks around 1247, attributed to C–C band stretchingvibrations from a ketone sugar, are evident in extracts of A.cornea, H. coralloides, H. erinaceum, L. edodes, and P. australis. Ingeneral, an adsorption band at 1155 cm�1 is characteristic ofβ-glycosidic linkages, the band at around 1024 cm�1 indicat-ing (C–O) stretching, and the shoulder at 890 cm�1 alsoindicating β-glycosidic linkages. Nonetheless, the bands near920 and 1032 cm�1 are indicative of the α-linkage (Kozarski
et al., 2012). Considering these criteria, the extracts from A.cornea, C. gigantea, and C. sinensis consisted of β-glycosidiclinkages, while those of I. cibarium, L. edodes, and P. australiscomprised both α and β-glycosidic conformations. The amidebands at around 1636 and 1411 cm�1 represented someresidual protein in the polysaccharide mixtures. A smallamount of protein was observed in all the extracts investi-gated, which may reflect proteoglycan content.
The structural characteristics of the polysaccharides, such asmolecular weight, monosaccharide composition, configurationand type of glycosidic linkage, were found to modulate andaffect their antioxidant activities (Luo et al., 2010; Lo, Chang,Chiu, Tsay, & Jen, 2011). Higher antioxidant activity was evidentin lower molecular weight polysaccharides or those with a β-configuration in the pyranose form. Polysaccharides with a highrhamnose content were likely to exhibit potent antioxidanteffects (Luo et al., 2010). Two glycosyl linkage types of simplesugars, including the glucose 1-6 linkage and arabinose 1-4linkage, were implicated in the scavenging of DPPH radicals (Loet al., 2011). In a study involving G. lucidum, G. applanatum,Trametes versicolor, and L. edodes, it was found that the con-formation of the polysaccharides was more important for DPPHradical scavenging activity than monosaccharide composition(Kozarski et al., 2012).
3.5. Relationship between the relative quantity of OH andantioxidant activity
The reaction of antioxidants to scavenge DPPH radical iscaused by the hydrogen donating ability of these compounds(Shimada, Fujikawa, Yahara, & Nakamura, 1992). Polysac-charides have been proved for this ability, where the hydroxylgroup of the monosaccharide unit can donate hydrogen toreduce DPPH radical (Yang, Zhao, Prasad, Jiang, & Jiang, 2010).It is also known that the scavenging ability of polysaccharidesrelies on the concentration of available hydroxyl groups (Keret al., 2005). In order to evaluate the relationship between theradical scavenging ability and the quantity of hydroxyl group,an identical amount of each extract (2 mg) was used to makeKBr pellets. The peak heights of the OH groups from the FT-IRspectra were measured and compared. The single peakheight measurement proved to be unreliable, hence the peakheight ratio was calculated using a peak height of anothergroup in the same spectra. In the present study, the CH groupat around 1370 cm�1 was chosen as it is characteristic ofpolysaccharide structure and present in all spectra analysed.The resulting ratios of OH group/CH group peak heights,representing the relative quantities of OH groups in thedifferent mushroom crude extracts, were compared to eval-uate correlation with radical scavenging activity. The ratiosare given in Table 2. The ratios for the C. gigantea and H.erinaceus extracts were not significantly different from eachother (p40.05), and there were also no significant differencesbetween the ratios for extracts from C. sinensis, H. coralloides,and H. erinaceum (p40.05). Extracts derived from A. cornea hadthe lowest ratio, which was consistent with the antioxidantactivity test where these extracts had the lowest radicalscavenging activity. The highest ratio was recorded forextracts from H. coralloides (Table 2), whereas P. australis hadthe highest antioxidant activity EC50 value (Fig. 3). Thus, the
4500 4000 3500 3000 2500 2000 1500 1000 500 0
Abs
orba
nce
Wavenumbers (cm-1)
3400
.1
2928
.0
1637
.7
1375
.7
1076
.0
A. cornea
3368
.6
2924
.9
1655
.4
1369
.7
1079
.3
C. gigantea
3389
.0
2924
.9
1650
.7
1367
.3
1079
.2
C. sinensis
3390
.6
2920
.2 1646
.1
1372
.0
1077
.4H. coralloides
3369
.7
2920
.2
1648
.6
1381
.4
1076
.4
H. erinaceum
3400
.1
2925
.7
1625
.5
1369
.7 1074
.6
I. cibarium33
89.5
2922
.6
1650
.8
1372
.0
1078
.7
3389
.4
2925
.0
1641
.4
1372
.0
1078
.7
L. edodes
P. australis
Fig. 4 – FT-IR spectra of mushroom extracts.
Table 2 – Ratio of OH group/CH group peak heights in FT-IR spectra of mushroom extracts.
Fungus A. cornea C. gigantea C. sinensis H.coralloides
H. erinaceum I. cibarium L. edodes P. australis
Peak heightsratio (OH/CH)
1.9270.01 A 3.5870.10 B 3.8870.23C 4.0170.08C 3.7670.08 BC 2.2970.04 D 2.7070.03 E 3.1870.03 F
Values are mean7standard deviation, n¼4.Means followed by different letters are significantly different at po0.05, ANOVA, Tukey.
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 148
ranking of OH content did not exactly correlate with radicalscavenging activity. The results suggest that apart from theavailable amount of hydroxyl group, other physicochemicalproperties, such as molecular weight, monosaccharide
composition, configuration and the type of glycosidic linkage,should also be considered as important factors to signifi-cantly affect the scavenging ability of polysaccharides, ashighlighted in previous studies (Luo et al., 2010; Lo et al.,
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1 49
2011). Further research is needed to correlate the physico-chemical properties of mushroom extracts with their anti-oxidant activity.
A single analytical method may not reveal the totalantioxidant capacity of a group of compound, since differentantioxidants may function by different mechanisms. A vari-ety of reactive oxygen species (ROS) can be generated in thehuman body. The superoxide radical which is produced as abyproduct of metabolic processes, and respiration is regardedas the primary ROS. Superoxide radicals can react with othermolecules to produce secondary ROS, including hydroxylradicals, hydrogen peroxide and singlet oxygen, eitherdirectly or via enzyme or metal-catalyzed processes (Valkoet al., 2007). These various ROS can induce oxidative damageto lipids, proteins and DNA. A diverse array of antioxidantswith varying abilities to prevent chain initiation, bind transi-tion metal ion catalysts, decompose peroxides, prevent con-tinued hydrogen abstraction, and reduce capacity and radicalscavenging ability have been employed to prevent or reversetissue damage (Liu, Wang, Xu, & Wang, 2007). The DPPHradical scavenging assay used in the present study is only apreliminary research tool to illustrate the antioxidant proper-ties of mushroom polysaccharides. Other methods arerequired to assess additional types of antioxidant activity ofthese mushroom extracts may possess.
4. Conclusion
The polysaccharides extracted from C. sinensis inhibited thegrowth of B. subtilis and S. epidermidis, whereas the P. australisextract restricted the growth of S. epidermidis. Further testingof those polysaccharides for antibiotic properties should beconsidered. The results confirmed that Gram-negative bac-teria were more resistant to the growth inhibitory effects offungal polysaccharides. Further work is required to under-stand the molecular basis of the antibacterial activity of thepolysaccharides. The microdilution method was more reli-able than the disc diffusion method in detecting antibacterialactivity. The latter approach was confounded by the inabilityof high molecular weight to diffuse sufficiently within agar.The microdilution assay is efficient and convenient for high-throughput screening of organic extracts.
Polysaccharides for all eight mushroom species showedDPPH radicals scavenging activities. P. australis exhibited thehighest antioxidant activity with an EC50 of 4.03 mg/mL. FT-IRanalyses indicated that extracts from A. cornea, C. gigantea,and C. sinensis contained β-glycosidic linkages, while thosefrom I. cibarium, L. edodes, and P. australis exhibited both α-and β-glycosidic conformations. The OH content of theseextracts, evaluated by determining the ratios of OH group/CH group peak heights, did not correlate with antioxidantactivity EC50 values, suggesting that other chemicophysicalfactors of polysaccharides, in particular structural properties,might have more effect on antioxidant activity. The antibac-terial and antioxidant activities detected here warrant inves-tigation for their potential to improve human health, andapplication as dietary supplements.
r e f e r e n c e s
Ajith, T. A., & Janardhanan, K. K. (2007). Indian medicinalmushrooms as source of antioxidant and antitumor agents.Journal of Clinical Biochemistry and Nutrition, 40, 157–162.
Alves, M. J., Ferreira, I. C. F. R., Martins, A., & Pintado, M. (2012).Antimicrobial activity of wild mushroom extracts againstclinical isolates resistant to different antibiotics. Journal ofApplied Microbiology, 113, 466–476.
Amarowicz, R., Pegg, R. B., Rahimi-Moghaddam, P., Barl, B., &Weil, J. A. (2004). Free-radical scavenging capacity andantioxidant activity of selected plant species from theCanadian prairies. Food Chemistry, 84, 551–562.
Ameri, A., Vaidya, J. G., & Deokule, S. S. (2011). In vitro evaluationof anti-staphylococcal activity of Ganoderma lucidum,Ganoderma praelongum and Ganoderma resinaceum from Pune,India. African Journal of Microbiology Research, 5, 328–333.
Bala, N., Aitken, E. A. B., Cusack, A., & Steadman, K. J. (2012).Antimicrobial potential of Australian macrofungi extractsagainst foodborne and other pathogens. Phytotherapy Research,26, 465–469.
Barros, L., Baptista, P., Estevinho, L. M., & Ferreira, I. C. F. R. (2007).Bioactive properties of the medicinal mushroom Leucopaxillusgiganteus mycelium obtained in the presence of differentnitrogen sources. Food Chemistry, 105, 179–186.
Cadenas, E., & Davies, K. J. A. (2000). Mitochondrial free radicalgeneration, oxidative stress, and aging. Free Radical Biology &Medicine, 29, 222–230.
Casaril, K. B. P. B., Kasuya, M. C. M., & Vanetti, M. C. D. (2011).Antimicrobial activity and mineral composition of shiitakemushrooms cultivated on agricultural waste. Brazilian Archivesof Biology and Technology, 54, 991–1002.
Casu, B., Scovenna, G., Cifonelli, A. J., & Perlin, A. S. (1978).Infrared spectra of glycosaminoglycans in deuterium oxideand deuterium chloride solution: Quantitative evaluation ofuronic acid and acetamidodeoxyhexose moieties.Carbohydrate Research, 63, 13–27.
Cheung, L. M., Cheung, P. C. K., & Ooi, V. E. C. (2003). Antioxidantactivity and total phenolics of edible mushroom extracts. FoodChemistry, 81, 249–255.
Circu, M. L., & Aw, T. Y. (2010). Reactive oxygen species, cellular redoxsystems, and apoptosis. Free Radical Biology & Medicine, 48, 749–762.
Cohen, G. N. (2004). Microbial biochemistry (pp. 7–10)Dordrecht:Springer7–10.
Davidson, P. M., & Parish, M. E. (1989). Methods for testing theefficacy of antimicrobials. Food Technology, 43, 148–155.
Duffy, C. F., & Power, R. F. (2001). Antioxidant and antimicrobialproperties of some Chinese plant extracts. International Journalof Antimicrobial Agents, 17, 527–529.
Fuller, R. J. M., Buchanan, P. K., & Roberts, M. (2004). Maoriknowledge of fungi/Matauranga o nga harore. In E. H. C.McKenzie (Ed.), The Fungi of New Zealand/Ngā Harore o Aotearoa,vol. 1. Introduction to Fungi of New Zealand (pp. 81–118). HongKong: Fungal Diversity Press.
Gao, Y. –H., Zhou, S., Huang, M., & Xu, A. (2003). Antibacterial andantiviral value of the genus Ganoderma P. Karst. species(Aphyllophoromycetideae): A review. International Journal ofMedicinal Mushrooms, 5, 235–246.
Giannenas, I., Tontis, D., Tsalie, E., Chronis, E. F., Doukas, D., &Kyriazakis, I. (2010). Influence of dietary mushroom Agaricusbisporus on intestinal morphology and microflora compositionin broiler chickens. Research in Veterinary Science, 89, 78–84.
Grice, H. C. (1988). Safety evaluation of butylated hydroxyanisolefrom the perspective of effects on forestomach andoesophageal squamous epithelium. Food and ChemicalToxicology, 26, 717–723.
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 150
Guo, C. Y., Ji, S. Z., & Ping, C. X. (2009). Modulatory effect ofGanoderma lucidum polysaccharides on serum antioxidantenzymes activities in ovarian cancer rats. CarbohydratePolymers, 78, 258–262.
Halliwell, B., & Cross, C. E. (1994). Oxygen-drived species: Theirrelation to human disease and environmental stress.Environmental Health Perspectives, 102, 5–12.
Hancock, I. C. (2002). Bacterial cell walls: An overview. In M.Sussman (Ed.), Molecular medical microbiology (pp. 33–45). SanDiego: Academic Press.
He, F., Yang, Y., Yang, G., & Yu, L. J. (2010). Studies on antibacterialactivity and antibacterial mechanism of a novel polysaccharidefrom Streptomyces virginia H03. Food Control, 21, 1257–1262.
He, J. –Z., Ru, Q. –M., Dong, D. –D., & Sun, P. –L. (2012). Chemicalcharacteristics and antioxidant properties of crude watersoluble polysaccharides from four common ediblemushrooms. Molecules, 17, 4373–4387.
Holst, O., Moran, A. P., & Brennan, P. J. (2009). Overview of theglycosylated components of the bacterial cell envelope. In A. P.Moran (Ed.), Microbial glycolipids: Structures, relevance andapplications (pp. 1–13). Amsterdam: Elsevier Inc.
Holst, O., & Muller-Loennies, S. (2007). Microbial polysaccharidesstructures. In J. P. Kamerling, G. -J. Boons, Y. C. Lee, A. Suzuki,N. Taniguchi, & A. G. J. Voragen (Eds.), Comprehensiveglycoscience. From chemistry to systems biology, 1 (pp. 123–179).Amsterdam: Elsevier Inc.
Huang, W. Y., Cai, Y. Z., Xing, J., Corke, H., & Sun, M. (2007).A potential antioxidant resource: Endophytic fungi frommedicinal plants. Economic Botany, 61, 14–30.
Je, J. Y., & Kim, S. K. (2006). Chitosan derivatives killed bacteria bydisrupting the outer and inner membrane. Journal ofAgricultural Food Chemistry, 54, 6629–6633.
Jeong, E. M., Liu, M., Sturdy, M., Gao, G., Varghese, S. T., Sovari, A. A.,et al. (2012). Metabolic stress, reactive oxygen species, andarrhythmia. Journal of Molecular and Cellular Cardiology, 52, 454–463.
Jouraiphy, A., Amir, S., Winterton, P., El Gharous, M., Revel, J. -C.,& Hafidi, M. (2008). Structural study of the fulvic fractionduring composting of activated sludge–plant matter:Elemental analysis, FTIR and 13C NMR. Bioresource Technology,99, 1066–1072.
Ker, Y. B., Chen, K. C., Chyau, C. C., Chen, C. C., Guo, J. H., Hsien, C. L.,et al. (2005). Antioxidant capability of polysaccharidesfractionated from submerge-cultured Agaricus blazei Mycelia.Journal of Agricultural and Food Chemistry, 53, 7052–7058.
Kirk, P. M., Cannon, P. F., Minter, D. W., & Stalpers, J. A. (2008).Ainsworth & Bisby's Dictionary of the Fungi. Wallingford, UK: CABInternational.
Klein, E., Smith, D. L., & Laxminarayan, R. (2007). Hospitalizationsand deaths caused by methicillin-resistant Staphylococcusaureus, United States, 1999–2005. Emerging Infectious Diseases,13, 1840–1846.
Kozarski, M., Klaus, A., Niksic, M., Vrvic, M. M., Todorovic, N.,Jakovljevic, D., et al. (2012). Antioxidative activities andchemical characterization of polysaccharide extracts from thewidely used mushrooms Ganoderma applanatum, Ganodermalucidum, Lentinus edodes and Trametes versicolor. Journal of FoodComposition and Analysis, 26, 144–153.
Krinsky, N. I. (1989). Antioxidant functions of carotenoids. FreeRadical Biology and Medicine, 7, 617–635.
Kuo, M. –C., Chang, C. –Y., & Cheng, T. L. (2007).Immunomodulatory effect of exo-polysaccharides fromsubmerged cultured Cordyceps sinensis: Enhancement ofcytokine synthesis, CD11b expression, and phagocytosis.Applied Microbiology Biotechnology, 75, 769–775.
Lee, Y., & Lee, T. S. (2005). Enhancement in ex vivo phagocyticcapacity of peritoneal leukocytes in mice by oral delivery ofvarious lacticacid-producing bacteria. Current Microbiology, 50,24–27.
Liu, C., Wang, C., Xu, Z., & Wang, Y. (2007). Isolation, chemicalcharacterization and antioxidant activities of twopolysaccharides from the gel and the skin of Aloe barbadensisMiller irrigated with sea water. Process Biochemistry, 42,961–970.
Liu, J., Jia, L., Kan, J., & Jin, C. - H. (2013). In vitro and in vivoantioxidant activity of ethanolic extract of white buttonmushroom (Agaricus bisporus). Food and Chemical Toxicology, 51,310–316.
Lo, T. C. -T., Chang, C. A., Chiu, K. -H., Tsay, P. -K., & Jen, J. -F.(2011). Correlation evaluation of antioxidant properties on themonosaccharide components and glycosyl linkages ofpolysaccharide with different measuring methods.Carbohydrate Polymers, 86, 320–327.
Luo, A., He, X., Zhou, S., Fan, Y., Luo, A., & Chun, Z. (2010).Purification, composition analysis and antioxidant activity ofthe polysaccharides from Dendrobium nobile Lindl. CarbohydratePolymers, 79, 1014–1019.
Mackerras, D. (1995). Antioxidant and health. Fruits andvegetables or supplements. Food Australia, 47, 3–23.
Mathlouthi, M., & Koenig, J. L. (1986). Vibrational spectra ofcarbohydrates. Advances in Carbohydrate Chemistry andBiochemistry, 44, 7–89.
Mazodier, P., & Davies, J. (1991). Gene transfer between distantlyrelated bacteria. Annual Review of Genetics, 25, 147–171.
Naik, G. H., Priyadarsini, K. I., Satav, J. G., Banavalikar, M. M.,Sohoni, D. P., Biyani, M. K., et al. (2003). Comparativeantioxidant activity of individual herbal components used inAyurvedic medicine. Phytochemistry, 63, 97–104.
Ng, T. B., & Wang, H. X. (2005). Pharmacological actions ofCordyceps, a prized folk medicine. Journal of Pharmacy andPharmacology, 57, 1509–1519.
Roller, S., & Covill, N. (1999). The antifungal properties of chitosanin laboratory media and apple juice. International Journal FoodMicrobiology, 47, 67–77.
Romberger, J. A., & Tabor, C. A. (1971). The Picea abies shoot apicalmeristem in culture. I. Agar and autoclaving effects. AmericanJournal of Botany, 58, 131–140.
Russell, R., & Paterson, M. (2006). Ganoderma—A therapeuticfungal biofactory. Phytochemistry, 67, 1985–2001.
Shoji, O. (2000). Influence of wood species on the sawdust-basedcultivation of Pleurotus abalonus and Pleurotus eryngii. Journal ofWood Science, 46, 175–179.
Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992).Antioxidant properties of xanthan on the antioxidation ofsoybean oil in cyclodextrin emulsion. Journal of AgriculturalFood and Chemistry, 40, 945–948.
Sudarshan, N. R., Hoover, D. G., & Knorr, D. (1992). Antibacterialaction of chitosan. Food Biotechnology, 6, 257–272.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., &Telser, J. (2007). Free radicals and antioxidants in normalphysiological functions and human disease. InternationalJournal of Biochemistry & Cell Biology, 39, 44–84.
Vaz, J. A., Barros, L., Martins, A., Santos-Buelga, C., Vasconcelos,M. H., & Ferreira, I. C. F. R. (2011). Chemical composition ofwild edible mushrooms and antioxidant properties of theirwater soluble polysaccharidic and ethanolic fractions. FoodChemistry, 126, 610–616.
Venturini, M. E., Rivera, C. S., Gonzalez, C., & Blanco, D. (2008).Antimicrobial activity of extracts of edible wild and cultivatedmushrooms against foodborne bacterial strains. Journal of FoodProtection, 71, 1701–1706.
Wang, D., Xu, M., Zhu, H. T., Chen, K. K., Zhang, Y. J., & Yang, C. R.(2007). Biotransformation of gentiopicroside by asexualmycelia of Cordyceps sinensis. Bioorganic & Medicinal ChemistryLetters, 17, 3195–3197.
Wang, G. Y., Zhang, J., Mizuno, T., Zhuang, C., Ito, H., Mayuzum, H., etal. (1993). Antitumor active polysaccharides from the Chinese
B i o a c t i v e C a r b o h y d r a t e s a n d D i e t a r y F i b r e 3 ( 2 0 1 4 ) 4 1 – 5 1 51
Mushroom Songshan Lingzhi, the fruiting body of Ganodermatsugae. Bioscience Biotechnology and Biochemistry, 57, 894–900.
Wang, J. C., Hu, S. H., Liang, Z. C., & Yeh, C. J. (2005). Optimizationfor the production of water-soluble polysaccharide fromPleurotus citrinopileatus in submerged culture and its antitumoreffect. Applied Microbiology and Biotechnology, 67, 759–766.
Wolff, E. R. S., Wisbeck, E., Silveira, M. L. L., Gern, R. M. M., Pinho,M. S. L., & Furlan, S. A. (2008). Antimicrobial and antineoplasicactivity of Pleurotus ostreatus. Applied Biochemistry andBiotechnology, 151, 402–412.
Xie, J. H., Xie, M. Y., Nie, S. P., Shen, M. Y, Wang, Y. X., &Li, C. (2010). Isolation, chemical composition andantioxidant activities of a water-soluble polysaccharide fromCyclocarya paliurus (Batal.) Iljinskaja. Food Chemistry, 119,1626–1632.
Yamac, M., & Bilgili, F. (2006). Antimicrobial activities of fruitbodies and/or mycelial cultures of some mushroom isolates.Pharmaceutical Biology, 44, 660–667.
Yang, B., Zhao, M. –M., Prasad, K. N., Jiang, G. –X., & Jiang, Y. –M.(2010). Effect of methylation on the structure and radicalscavenging activity of polysaccharides from longan(Dimocarpus longan Lour.) fruit pericarp. Food Chemistry, 118,364–368.
Yoon, S. Y., Eo, S. K., Kim, Y. S., Lee, C. K., & Han, S. S. (1994).Antimicrobial activity of Ganoderma lucidum extract alone andin combination with some antibiotics. Archives of PharmacalResearch, 17, 438–442.
Zgoda, J. R., & Porter, J. R. (2001). A convenient microdilutionmethod for screening natural products against bacteria andfungi. Pharmaceutical Biology, 39, 221–225.
Zhu, H. –J., Sheng, K., Yan, E. -F., Qiao, J. –J., & Lv, F. (2012).Extraction, purification and antibacterial activities of apolysaccharide from spent mushroom substrate. InternationalJournal of Biological Macromolecules, 50, 840–843.
