Isolation and Activity Assement of Lentinan Metabolite from Shiitake

Miscelium(Lentinus edodes) and Effetcs on Candida albicans Growth

Sukmawati, Dalia. Universitas Negeri Jakarta

daliasukmawati@yahoo.com

Abstract

This research is aimed to isolate and asses the activity of lentinan metabolite from Shiitake
miscelium (L. edodes ). Sample is taken from fruit body using streak plate method (Gores
method). The lentinan metabolite is produced by precipitating the supernatan of Shiitake
miscelium using 70% etanol. Macroscopic and microscopic sign of isolat are being observed.
The research through few steps : fungi isolation, isolat renewal, fementation, lentinan
metabolite extraction and potention test as anti-C.albicans. The result is the lentinan
metabolite can block the C.albicans growth by creating clear zone around the paper disc.
The concetration of the lentinan metabolite used is this research are 100 μg/ml, 500 μg/ml,
1000 μg/ml , 1500 μg/ml dan 2000 μg/ml, and the average of resistance zone that created
are 0,445 mm, 0,555 mm, 0,8225 mm , 0,835 mm and 1,6505 mm. The conclusion is the
lentinan metabolite of L.edodes that produced by still fermentation has the the anti-
C.albicans properties.

Keywords: Shiitake, still fermentation,clear zone, anti-C.albicans

Introduction

Many researches have been conducted recently, to find new alternative source of
food which have not only medical effects, but also accessible, cheap and easy to produce,
and safe to consume. Most of these reseaches were conducted in developing countries
which have limited land for agriculture. As a result , one of many alternative sources of food
which has the same criterias as above is mushroom (Chang & Miles, 1998).
In many countries, particularly in European countries, mushroom is collected and
can be used directly as food. Mushroom is used as additional ingridient in soup, stirfries and
tea. Mushroom can be considered as an alternative for high protein sources after meat and
milk. (Breene, 1990) Mushroom contains amino acid such as sistein and metionin. Fresh
mushroom has 3-21% carbohydrate, and 3-35% fiber of dry weight. Instead of containing
mineral, mushroom also contains some vitamins.(Breene,1990)
Edible mushroom and curing capability comes from Basidiomycota class. Species
that used mostly is Lentinus (Lentinula) or known as Shiitake (Chang & Miles, 1989). It has
been reported that Lentinus has several roles in human health : immunomodulator, anti-
cancer capability, lowering the cholesterol and hipertention, liver function protection, anti-
diabetes, anti-virus and anti-microbacteria (Ooi & Liu, 1999). One of active metabolites in
lentinus is called Lentinan, a polisacharide in form of ß-D- glukans with heterosacharides
chains containing xylose, mannose, galactose (Feng et al., 2001) which functioned as
immunomodulator.
Problems that occur in developing countries is infection disease especially caused by
fungus (Dhanutirto, 1987). Disease that caused by khamir and fungus can be candidiasis
and aspergilosis (Georgopapadakou, 1998). Lennette (1985) reported that C. parapsilosis,
C. tropicalis, C. albicans, dan C. Guilliermondii cause endocarthritis and candidiasis. One of
khamir species that caused fungal infections is C. albicans (Didomenico, 1999). It is normal
to find Candida albicans in human body for example, C. albicans can be find in digestive
system, upper respiratory tract (Georgopapadakou, 1998). Some anti-khamir products have
been produced commercially such as : amphotericin B, fluconazole, echinocandins which
have resistance activity opposite to C. albicans (Georgopapadakou, 1998).
One of lentinan potention to produce anti-khamir C. albicans has not been
researched yet. The aims of this research is to discover its ability to produce anti-Khamir
metabolite.
Research Methods

Material and Method:

Microtube 1 ml (Eppendrof), Micropipet + tips, Erlenmeyer bulb, vortex mixer, sentrifuge,
rotary evaporator, freeze dryer, incubator, spektrophotometer UV-VIS.
Materials:
Alcohol 70%, KH2PO4, MgSO4, CaCl, KCl, PDBFeSO4, MnSO4, ZnSO4, CuSO4, ekstrak
yeast extract, pepton, glucose, DNS, enzim  1-3 glucanase, standar glucan , liquid nitrogen.
Growth medium are Potato Dextrose Agar, Difco (PDA) dan Yeast Malt Agar, Difco (YMA).
Microorganism that used in this research are shiitake fungi (Lentinus edodes) and dalah C.
albicans (UICC Y-29).
Methods:

The methods of this research can be devided into five stages:

a. Miscellium isolation of Shiitake (Lentinus edodes)

Isolation was performed using aseptic technic. Isolated part was taken from uppest
part of shiitake’s body. The body tissue is taken and innoculated using PDA medium, and
were incubated in room temperature for about one week.

b. Purification of Isolate
Purification of isolate were checked in two different ways : macroscopically and
microscopicallly. Identification and characterization of fungi were performed using
identification key from Domch et al (1995).

c. Miscellium fermentation of Shiitake mushroom

Fermentation medium using (g/l): glucose 39gr, pepton 1gr, yeast extract 2gr,
K2HPO4 1gr, MgSO4 0,2gr, (NH4)2SO4 5gr. Sterilization in autoklaf 1210C, 2 atm, for 15
minute and optimum pH 6,0. 2 ml Shiitake miscellium suspension was innoculated in
500mL Erlenmeyer bulb containing 100mL medium. The supernatan was being sentrifuge in
100rpm and was incubated in room temperature for 1 month.

Fig 1. Fermentation of L.edodes in medium Potato Dextrosa Broth (PDB) 30 days incubation
 d. Lentinan metabolite extraction from miscellium fermentation.
Supernatan that produced by shaker and still fermentation are separated from the
miscellium The next stage is filtration using Whatman paper to obtain supernatan of
miscellium. Crude extract collected from filtration was minced. Then crude extract was
soaked in ethanol with ratio 5:1. After that we evaporate the filtrat on 40 celc deg and
vacuum pressure to get the lentinus extract (Mizuno, 1999)

Fig 2. Precipitation on miscellium L.edodes and bold extract of fermentation L.edodes

Fig 3. Lentinan extraction methods of Lentinus edodes miscellium fermentation (Mizuno, 1999)
e. Anti-khamir assesment of Lentinan exctract
Cylinder Plate Assay Method (Levinson & Jawetz, 1992) was used to asses anti-
khamir activity of lentinan extract. 10 ml YMA medium that contains khamir cells suspension
with cell density 5,1- 6,65 107 sel/ml, poured in petry dish. Sterile cylinder glass put on
harden medium, and every cylinder we added 0,25ml fungi filtrat. Resistance zone
observation was performed after 18 hour. Resistance zone calculated by measuring the
total diameter of resistance zone and substracted by cylinder diameter. The calculation is
done three times on 2 petry dish containing C. albicans on YMA medium .
Result

Morfology of Lentinus edodes are the colony is circular with diameter of 6.5-8.6 cm,
white in colour, slow growth. Microscopically, shiitake has septated hypha with clamp
connection.As a result of fermentation we obtained shiitake micelium grew on the whole
surface of Potato Dextrosa Broth (PDB) media.The supernatan is in the liquid form with
brown colour.

Lentinant extract that produced form still fermentation has the anti-khamir activity to
C. albicans.

Concentration (ppm) Diameter (mm) resistance zone on

C.albicans

100 0,445

500 0,555

1000 0,8225

1500 0,835

2000 1,6505

Table 1. Resistance zone of Lentinan on C.albicans

Supernatan contains anti-khamir metabolite, indicated by clear zone formed on the

C.albicans innoculated medium. According Tomita (1985), effects of anti-khamir which
shown by clear zone forming is one of screening methods that can be done to select
microbe which potential producing anti-microbe metabolite. Positive result fungi isolat
L. edodes shows that fungi colony growth is optimum to produce anti-C.albicans metabolite.
Secondary metabolite produced at the end of exponential phase, after fungi through the lag
phase and ends the exponential phase when the nutrition is decreasing (Fedrichi, 1993).
 Fig 4. Resisting activity of L.edodes on C.albicans growth

Conclusion

1. Lentinan metabolite from L.edodes can be obtained by using still fermentation method

2. Lentinan metabolite has antikhamir efect to C.albicans growth

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