Fungal Secondary

Metabolites الفطريه األيوضالثانويهمازن الماجستير طالب

سلمان سليم

The FungiFungi are one of the largest group of

organisms , world wide distributed and play a vital role in ecosystems and as one of the most important tool in biotechnology

Fungi & BiotechnologyBIOTECHNOLOGY means “ Use of Fungi to

produce, develop, improve, remove or sustain of a particular thing which can be useful for man and nature “

The most important use of fungi is the production of Secondary Metabolites

Such as Antimicrobial Bioactive Substances that can be beneficial for Medical therapy

The economic significance of Fungi

Fermentation technologyEnzymes production technology Production of acids and chemicalsCultivation of fungi for protein Food processing by fungi (Bread, cheese)Fungi can be used in many applications

in (industry, agriculture, medicine , and environment)

Production of Bioactive compounds (Antibiotics)

Secondary metabolites

Organic compounds , with low molecular weight ,which are not essential for fungal growth but their natural production have certain significances.

Why secondary metabolites are produced?They are chemical compounds

produced by many fungi growing on substrates. They play a role in competition ,antagonism and self-defense mechanisms against other living organisms to allow the fungus to occupy the niche and utilize the food

Production of SECONDARY METABOLITES

Compounds produced during the stationary and decline phases of the fungal growth

They are vary in structures and biosynthetic pathways . So they differ among the fungal species and isolates

Fungal metabolites structures

Fungal secondary metabolites can be obtained by growing fungi in cultures under certain conditions (Penicillin is best example)

gs Types of Fungal secondary

metabolites 1. Strobilurin (antifungal) 2. Gibberellins (growth Hormons) 3. Herbicides (control weeds) 4. Mycotoxins (poisneous) 5. Insecticides ( control insects) 6. Enzymes (proteins) 7. Pigments (dyes) 8. Antibiotics (drugs)

9. Pharmacological drugs

AntibioticsAntibiotics: Are chemical substances produced

by fungi which has the capacity to inhibit the growth of / and or even kill other microorganisms . The action of an antibiotic is a selective in nature .

History of ANTIBIOTICS discovery(1928) Alexander Flimmings discovered

the Penicillin(1935) Prontosil, the first sulfa drug

was discovered by Gerhard Domagk(1943) Andrew Moyer, industrial

production of Penicillin(1943) Selman Waksman discovered

the Streptomycin from soil Bacteria

(1955) Tetracycline was patented by Lioyd Conover

(1957) Nystatin was patented(1981) Smith Kline Beckham patented

AmoxillinSince then many antibiotics have been

found and still continued to discover more…….

Antibiotic can be divided according their bioactivity into:

Antifungal antibiotics

Antibacterial antibiotics

Antiviral antibiotics

Antitumoral antibiotics

Mechanism of antibiotic action

The aim of present study 1. Screening of local fungi isolates for

antimicrobial bioactive compounds production.

2. Isolation and purification of the bioactive compounds using specific techniques

3. Examining the optimal conditions (Temp, pH, media , C, N source and some other factors) for a mass production of these compounds in batch cultures.

4. Characterization of the purified fungal extracts by using

HNMR and GC Mass.5. Testing the inhibition activity of these

compounds against a selected pathogenic microorganisms (Bacteria, Candida, Dermatophytes and other fungi).

Most recent publications on bioactive metabolites by fungi Last decade several published papers on this aspect: For Examples

Anke et al. (2004, 2006) from GermanyLindquist et al. (2005, 2006) from GermanyAbad et al. (2007) from SpainStamets P. (2007) from USAJohnathan et al. (2010) South AfricaMuhsin et al (2011) from Iraq

Pohanka ( 2006 ) PhD. Thesis (Sweeden)Roberts (2004). PhD. Thesis (Australia)Khalaf KT (2008) PhD. Thesis (Iraq)

Materials and methods

1. Sample collection and fungal isolation: Sample are collecting from different sources (soil , water ,

air , rhizospher , rhizoplane ) for fungal isolation Isolation, purification and identification of fungi on solid

mediaScreening of the inhibition bioactivity of each fungal species

against the selected pathogenic microorganisms including: A. Bacteria B. Candida C. Dermatophytes D. Other

fungi by using fungal discs on solid media.2. Fungal cultures Extraction: The selected fungal isolates will be grown in specific

liquid culture(using batch large bottles) under shaking condition for two

week incubation period at 25 C

3 .Antimicrobial bioactivity assay

Filter paper discs (0.6 mm) after being sterilized by autoclave are socked in each fungal crude extract solution for 5 min.,

Filter paper discs with extracts are placed on the surface of agar medium in Petri-dishes streaked with 0.2 ml of bacterial suspension of Escherichia coli and Staphylococcus aureus, Candida albicans, Microsporum gypseum, Fusarium oxysporum.

Plates are incubated at 37 ◦C for 24 hr, an appearance of inhibition zones around the filter paper disc indicating the bioactivity of crude metabolites of the tested fungal isolates (Casals, 1979).

4. Extraction of the fungal filtrates Five discs (0.5 mm diam.) are cut from the axenic fungal

culture of each isolate by using a cork borer and amended into PD liquid medium in 500 ml flasks (with triplicates) and incubation at 25 ◦C for 2 weeks on a rotary shaker.

Fungal cultures are filtered on Whatman No 1 filter paper and the pH adjusted at 3 for each fungal filtrate.

Filtrate is extracted in ethyl acetate (1:1 vol) by using separating funnel. The organic layer is collected by dehydration of water by using Na2SO4. The filtrate is filtered again and place in Petri dishes then leave to be dried at room temperature.

100 ug of the dried extract is dissolved in 1 ml ethanol as stock extract solution to be used for further experiments.

5 .Minimal inhibitory concentration testThe MIC values will be determined by the

standard serial dilution assay (McGinnis, 198o) .

Different concentrations of the crude fungal extract are used. Filter paper discs (0.5 mm) are soaked in each extract concentration and will be tested against each selected microorganisms on solid medium. The appearance of inhibition zone around the disc indicates an inhibitory action .

6.Cytotoxicity test Cytotoxicity of the fungal crud extracts was

examined by using human RBC following a previously described method ( Xian-

guo and Ursula, 1994).7 .Chemical analysis of fungal crude extractsFungal culture extracts will be chemically analyzed

for alkaloids, phenols, amino acids, flavenoides and Tannins according to following the described method

8. Purification of fungal extractsCrud extract will purified by using TLC and HPLC

technique.

9.Identification and characterization of the purified extract

By using GC –Mass and HNMR technique

10.Optimal Production of Bioactive metabolites

Experimentation will be conducted to examine the optimal production of the bioactive compounds in batch liquid cultures using:

1. Effect of Temperature 2. Effect of pH3. Effect of Media4. Effect of carbon and nitrogen sources (4

sources)5. Effect light and dark period6. Effect microelements (eg Zn, Fe)

11. Comparison of the bioactivity of the studied compounds with some commercial antibiotics.

12. If the allows, some other works will be included

Selected fungi for this studyWe try to investigate some fungi

belong to Deuteromycetes in particularRhizoctonia and TrichodermaFor the following reasons:1. No research so far dealt with these

fungi elsewhere2. These fungi are highly competitive

against other soil and plant fungi 3. Easy to grow in culture and handle

with