FUNGAL SECONDARY METABOLITES.

2. CONTENTS
• Overview of fungal secondary metabolites
• Introduction
• Description
-culture isolation
-pigment extraction
-separation and purification of crude pigment
-structural determination of pigment
• Conclusion
• Reference

3. OVERVIEW OF FUNGAL
SECONDARY METABOLITES
Fungi produce an enormous array of secondary
metabolites someof them are :
1.Strobilurin (antifungal)
2.Gibberelin (growth hormone)
3.Herbicides (control weeds)
4.Mycotoxins (poisonous)
5.Insecticides (control insects)
6.Enzymes (protein)
7.Pigments (dyes)
8.Antibiotics (drugs)
9.Pharmacological drugs

4. INTRODUCTION
PIGMENT: The term pigment is derived from Latin word
Pigmentum means “colouring matter”.
 Pigment is an organic compound that gives a
characteristic color to microbes, plant or animal tissues
and is involved in vital processes.
Natural pigments are obtained from various sources
like plant, animals and microbes.
 Natural pigment are non toxic, non polluting and less
hazardous .

5. PIGMENT PRODUCED BY MICROBES
• Microbes can produce a large amount of stable
pigment such as anthraquinones, carotenoids,
flavonoids, quinines, tetrapirroles and rubramines.
• Microbial pigment production is growing due to
their natural character, medicinal properties and
nutritive value,production being independent of
season and safty to use.
• Fungi contain several anthraquinones compounds
and pigments such as delphinidin, melanin,
carotenoids and volatile organic compounds.

6. FLOW CHART OF FUNGAL SECONDARY METABOLITES: PIGMENT
EXTRACTION,PURIFICATION AND CHARACTERIZATION
Sample collection from different sources like soil, water etc.
Isolation of fungus for pigment production
Screening of crude pigment extract
Extraction of pigment from isolates by specific solvent like
acetone
Separation and purification of pigment using column
chromatography , TLC and HPLC
Characterization of the purified pigment by using FT-IR and
1HNMR Spectroscopy

7. DESCRIPTION
1.CULTURE ISOLATION
• Sample was collected from soil.
• A strain of Rhodotorula glutinis isolated by plating method
& its identity was confirmed.
• This was maintain on potato dextrose slants & stored at 4◦c.
• Then mass cultivation was carried out in fermentor to
achieve high cell densities for crude production of the
crude pigment.
• The inoculum of R.glutinis were grown on culture medium
Czapek dox at 29-32 ◦c for 48 hrs & placed on a rotatory
shaker(200rpm)& added to fermentor.
• Fermented media were filtered & its dry weight was stored
in freeze & it was used to determine the pigment

8. PIGMENT EXTRACTION
• Freezed dried red yeast (R. glutinis) was hydrolysed with
1N HCl in water bath at 70◦c for one & half hrs.
• After removal of excess acid by washing with water, the
cells were soaked overnight in acetone: methanol (1:1).
• The pigment was extracted with acetone until the entire
colour was removed from the cells.
• The acetone extracted were transferred to light
petroleum (40-60◦c) in a separating funnel & washed
throughly thrice with distilled water.
• The absorbance of the light petroleum phase was read at
474nm ( λmax) & the concentration of carotenoids
determined using the absorption coefficient.

9. SEPARATION AND PURIFICATION OF VARIOUS
FRACTION OF THE CRUDE PIGMENT EXTRACT
A. COLUMN CHROMATOGRAPHY
• The pigment from the Red yeast R.glutinis were extracted with
acetone ,transferred to petroleum ether & fractioned on
Magnesium Oxide Hyflo Super Cel.
• A major red coloured fraction was adsorbed on the column ,
while the other pigment were eluted by the developing solvents
(petroleum ether, ethyl ether &methanol).
• The yellow fraction was eluted first by petroleum ether ,
followed by ethyl ether & methanol (10:1). The 3rd fraction
which was retained in the column was then eluted with acetic
acid ethyl ether(1:10).

10. • The water soluble compounds were removed by water washes
& the ethyl ether was dried over anhydrous Sodium Sulphate.
• The fraction were saponified by adding Potassium hydroxide
methyl alcohol solution & heated for 5 min on a steam.
• Petroleum ether is added to the mixture , which was washed
free from water soluble materials. The petroleum ether
solution was then dried over anhydrous sodium sulphate.
• The red coloured fraction (for fraction 3)& the orange coloured
fraction (fraction 2)were taken to dryness in the rotatory flash
evaporator & then dissolved in choloroform & petroleum ether
respectively.

11. THIN LAYER CHROMATOGRAPHY (TLC) ANALYSIS & PURIFICATION OF
THE PIGMENT FRACTION
• TLC separation of the fraction from the crude extracted pigment of
R.glutinis was carried out using TLC plates (silica gel 60) using
petroleum ether : acetone as a mobile phase and determined their
Rf .
Table 1.Separation of absorbance spectral analysis of the fraction of crude
extract from R.glutinis
The Rf value resembles three carotenoids pigment viz.
that are torularhodin , torulene & beta carotene.
PIGMENT FRACTION Rf VALUE ABSORPTION MAXIMUM
( λmax)
Fraction 1.(yellow) 0.92 446
Fraction 2.(orange) 0.78 479
Fraction3.(red) 0.20 515

12. HIGH PRESSURISED LIQUID CHROMATOGRAPHY
(HPLC)
• After that final purification of the three different fraction were
checked by HPLC with Uv detection at 1575 using a reversed
phase –C18 column & isocratic elusion with
acetonitrile:tetrahydrofuran:water at a flow rate of 1.0ml/min.

13. 1: HPLC analysis of the three pigment fractions from R. glutinis

14. STRUCTURAL DETERMINATION /CHARACTERIZATION
OF PIGMENT PRODUCED BY ISOLATED R.glutinis
The structure of three purified fraction was determined using
Nuclear Magnetic Resonance (NMR) spectroscopy & Infrared
(FT-IR).
• NMR spectra of three sample were recorded. All measurement
were performed at 23◦c in Cdcl under Argon that passed twice
through an allumina mini column.
• Chemical configuration of the pigment was determined by infrared
spectroscopy.IR spectra recorded from 4000 cm-1 to 500 cm-1.

15. RESULTS OF FT-IR SPECTRUM
Fig. 2 A.: FT-IR spectra of pigment fractions from R. glutinis

16. • The FT-IR of fraction 1(yellow solution in petroleum ether)
in fig 2A . The bands at 2955 cm-1 & 2920 cm-1 are due
asymmetrical streching vibration of aliphatic CH3 and CH2
groups & bands at 2869 cm-1 & 2851 cm-1 are due to
symmetrical streching vibration of aliphatic CH3 and CH2
groups.
• The asymmetrical & symmetrical deformation vibrations
CH3 groups are due observed at 1463 cm-1 &1377 cm-1
respectively.
• Low intensity bands at 1713 cm-1 may be due to >c=o
group.

17. Fig. 2 B : FT-IR spectra of pigment fractions from R. glutinis

18. • The FT-IR spectrum of fraction 2 (red solution in benzene) in
fig.2B.the bands at 2954 cm-1 & 2921 cm-1 are due to asymmetrical
streching vibration of aliphatic CH3 and CH2 groups respectively.
• Broad bands centered around 2851 cm-1 indicates that there may
be four or more CH2 groups in a row.
• Low intensity bands at 3008 cm-1 and 1652 cm-1 indicate the
presence of an olefinic functional group. The band at 3008 cm-1 is
due to CH stretching vibration CH = CH and at 1652 cm-1 may be due
to C = C stretching vibration of the same group.
• Medium intensity band at 1738 cm-1 is due to >C= O group probably
an ester.
• The presence of a band at 1259 cm -1 which could be the C – O
stretching vibration of the ester.

19. RESULTS OF NMR SPECTRA

20. CONCLUSION
• The fungi Red yeast R.glutinis was isolated by plating method.
• Isolated pigment was extracted with acetone .
• Separation & purificaton of various fractions of the crude
extract was done by column chromatography , TLC , HPLC.
• Then structure of the purified fractions was determine by
NMR spectroscopy & IR. H-NMR spectra the isolated three
major fractions were identified: fraction 1 as –beta carotene,
fraction 2 as torulene and fraction 3 as torularhodin.
• Infrared spectra gives chemical configuration of the pigment.
It can be concluded from the study that fungi R.glutinis
can be a potensial source of pigment.