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BINDING PROPERTY OF AFLATOXIN BY WILD


YEAST AND THEIR IDENTIFICATION

A Dissertation Submitted To
P G Department of Biotechnology
Sri Dharmasthala Manjunatheshwara College
Ujire, Dakshina Kannada
Affiliated to Mangalore University, Mangalore

In Partial Fulfillment of Requirement
For The Award of the Degree of
MASTER OF SCIENCE
IN
BIOTECHNOLOGY


Submitted by
JAHNAVI S.P
Reg.No. 071640105


Under the Guidance of
DR. ANU APPAIAH K.A, M. Sc., Ph.D.
Work Carried Out at



Department of Food Microbiology
Central Food Technological Research Institute
Mysore - 570020, INDIA
2008 - 2009






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Dr. ANU APPAIAH. K.A, M.Sc., Ph.D.
Scientist, Food Microbiology Department
CFTRI, Mysore 570 020
E-mail:- anuapps@yahoo.com
Date: 13-01-2009
CERTIFICATE

This is to certify that the project work, entitled Binding
Property of Aflatoxin by Wild Yeast and their Identification to be
submitted to the Dept. of Biotechnology, Sri Dharmasthala
Manjunatheshwara College, Ujire (Affiliated to Mangalore
University) in partial fulfillment of the requirement for the award of the
degree of Master of Science in Biotechnology, is a record of original
research work done by Ms. Jahnavi. S.P., under my guidance and
supervision at the Department of Food Microbiology, Central Food
Technological Research Institute, Mysore during June and July, 2008.




Dr. ANU APPAIAH. K.A
Guide



Dr . S. Umesh Kumar
Head,
Dept . of Food Mi cr obi ol ogy,
CFTRI , Mysor e -570020.




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DECLARATION

I, Ms Jahnavi S.P., hereby declare that the project work entitled
Binding property of Aflatoxin by wild yeast and their Identification
submitted to Sri Dharmasthala Manjunatheshwara college, Ujire
(Affiliated to Mangalore University), in partial fulfillment of the
requirement for the award of the degree of Master of Science in
Biotechnology, is a record of original research work carried out by me
under the supervision and guidance of Dr. Anu Appaiah K.A, Scientist,
Food Microbiology Department, Central Food Technology Research
Institute (CFTRI) Mysore, India.
I further declare that the work carried out in this project has not
been submitted previously for the award of any degree, diploma or any
other similar title.


Signature of the Student.


Jahnavi S.P




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ACKNOWLEDGEMENT

I express my heartfelt gratitude to my guide Dr. Anu Appaiah
K.A, Scientist, Food Microbiology Department, CFTRI, Mysore, for his
constant encouragement, critical suggestions and guidance extended to
me throughout my work.
My sincere thanks to Dr.V. Prakash , Director; Dr.M.C. Varadaraj,
Head HRD and Dr. S. Umesh Kumar, Head of Food Microbiology
Department, CFTRI, Mysore, for permitting me to undertake the project
work at CFTRI, Mysore.
I would like to thank Dean and Management, SDM college Ujire and
Dr. Maruthi K.R, Head of the Department , Biotechnology, SDM
College, Ujire, for granting me the opportunity to carry out the project at
CFTRI, Mysore.
I express my immense gratitude to Dr.Prakash M Halami, Scientist,
Food Microbiology Department, CFTRI, Mysore for his support and
guidance extended to me throughout my work.
I express my earnest gratitude to Dr. Vijayendra, Scientist, Food
Microbiology Department for his words of encouragement.
I also thank Mr. Akmal Pasha of Food Control and Infestation
Control Department for his cooperation in performing TLC.
My sincere thanks to Mr. Mukund of CIFS, CFTRI for helping me
work with HPLC and LC/MS. I would also like to extend my gratitude to
Mr. Anbalagan of CIFS, CFTRI for his cooperation in performing
Scanning Electron Microscopy.
My special thanks to Mr. Deepak M.B and Mrs Sowmya .V for
their guidance and support to finish my project work.
I would like to express my sincere gratitude to Mrs. Vanajakshi.V for
her encouragement. I would like to thank Mrs.Usha Rani, Ms. Snigdha


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Mohandas, Mr. Venkateshwaran, MrAnbarasu, Mr. Somshekar, Mr.
V. Badrinath for helping me during my project work.
I also thank Dr. Suryaprakash shenoy, Mr. Keshav Hegde
Korse, Dr. Harish B.G, Mrs Prarthana J, Mrs Smitha Rosario,
lecturers, Dept of Biotechnology, SDM College, Ujire for their
encouragement.
I am very much indebted to my beloved parents and sisters for their
encouragement.
I also thank my colleagues Miss. Vandana Thammaiah, Mr
Naveen Kumar, Miss Rashmi E, Mrs Swarna Gowri for their kind
support to complete my project.
Last but not the least I thank all those who have directly or
indirectly helped me in completing my project work successfully.



JAHNAVI S.P







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CONTENTS

Sl. No PARTICULARS PAGE No.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 2-8

3. OBJECTIVES 9

4. MATERIALS AND METHODS 10-21

5. RESULTS AND DISCUSSION 22-45

6. SUMMARY AND CONCLUSION 46-48

7. REFERENCES 49-52



















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LIST OF TABLES

Table 1: Differentiation of Aflatoxin in various foods

Table 2: Characteristics of Aflatoxin

Table 3: Different components used in PCR reaction mixture

Table 4: PCR cycle parameters

Table 5: Restriction digestion reaction mixture

Table 6: Cultures selected for aflatoxin test

Table 7: Potential for aflatoxin binding by yeast cultures

Table 8: Aflatoxin binding of selected yeast isolates at various time intervals

Table 9: Aflatoxin binding capacity of various yeast isolates

Table 10: Concentration of Aflatoxin B2 (mg/100ml) adsorbed by yeast cultures

Table 11: LC/MS Results at different incubation time

Table 12: Binding capacity of aflatoxin to yeast cells as indicated by MS data





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LIST OF FIGURES

Fig 1: Structures of Aflatoxins B1, B2, G1 and G2

Fig 2: Structures of Aflatoxins M1, M2, B2a and G2a

Fig 3(a): Chromatogram of sample No 18 P4

Fig 3(b): Chromatogram of sample No 18 S4

Fig 3(c): Chromatogram of Standard sample

Fig 4(a): MS data of standard sample

Fig 4(b): MS data of sample No 37 P4

Fig 4(c): MS data of sample No 37 S4

Fig 4(d): MS data of negative control sample

Fig 5(a): Phylogenetic tree of sample No 5

Fig 5(b): Phylogenetic tree of sample No18

Fig 5(c): Phylogenetic tree of sample No 37








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LIST OF PLATES
Plate 1: Aspergillus flavus grown on PDA media
Plate 2: Ground nuts infected by A flavus
Plate 3: TLC plate showing fluorescent spots
Plate 4: Scanning electron micrograph of culture code 5 (size= 4.82.18 m
magnification 10 K
Plate 5: Scanning electron micrograph of Yeast culture code 16 (size= 2.241.8
m magnification 20 K)
Plate 6: Scanning electron micrograph of culture code 18 (size= 3.061.25 m
magnification 10 K)
Plate 7: Scanning electron micrograph of culture code 18 (size=3.06 m 1.25
m magnification 15 K)
Plate 8: Scanning electron micrograph of Yeast culture code 29(size=61.8 m
magnification 10 K)
Plate 9: Scanning electron micrograph of Yeast culture code 29(size= 5.2 1.9m
magnification 10 K)
Plate 10: Scanning electron micrograph of Yeast culture code 30
(size= 3.12 0.3 m magnification 10 K)
Plate 11: Scanning electron micrograph of Yeast culture code 30
(size= 2.92 0.78 m magnification 20 K)
Plate 12: Scanning electron micrograph of Yeast culture code 37
(size= 3.87 2.25m magnification 10 K)
Plate 13: Scanning electron micrograph of Yeast culture code 40
(size= 5 2.8m magnification 10 K)
Plate 14: Band separation for isolated yeast DNA in agarose gel
Plate 15: PCR analysis of 18s rRNA gene of Yeast
Plate 16: Restriction pattern of Hae 111 and Alu 1 from the yeast cultures
Plate 17: Restriction pattern of Hae 111 of 18s rRNA
Plate 18: Restriction pattern of Alu 1 of 18s rRNA



11


















INTRODUCTION























12


INTRODUCTION

Aflatoxin are produced by certain fungal species including Aspergillus flavus
and Aspergillus parasiticus. These are highly carcinogenic polyketide
metabolites.(Jiang et al., 2005). Aflatoxins are an extremely toxic group of
mycotoxins that occur as natural contaminants in agricultural oil seed products such
as corn, cotton, peanut etc. Aflatoxins causes various acute and chronic intoxications
in humans and animals in addition to causing liver cancer. Dietary exposure to
aflatoxins in the developing countries is extensively reviewed (Williams et al ., 2004).
There are many types of Aflatoxins such as AflB1, AflB2, AflG1,AflG2 etc. AflB1
and AflB2 have the charecteristic of blue fluorescence. And AflG1 and Afl G2 have
the charecteristic of blue green fluorescence (Singh et al., 1991)
Biological decontamination of mycotoxins using microorganisms is one of the
well known strategies for the management of mycotoxins in foods and feeds . Among
the different potential by decontaminating microorganisms , different species of yeast
represent unique groups. These are also used in food fermentation and preservation .
Feeding of Saccharomyces cerevisiae to poultry showed beneficial effects against
aflatoxin induced toxicities (Stanley et al.,1993).
Aim of the present investigations is to screen different species of wild yeast
isolated from various sources for their binding ability and to further characterize the
binding in selected strains in terms of binding conditions and their identification.










13











REVIEW OF LITERATURE









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REVIEW OF LITERATURE

In 1959 a very singular event occurred which initiated the international
interest which now exists in mycotoxins. This was the deaths of several thousand
Turkey poults and other poultry on farms in East Anglia and, because of the
implications for the Turkey industry and the manufacture of pelletted feed which
supported it , a considerable effort was put into understanding the etiology of this
major outbreak of what was initially referred to as turkey X disease. Although the
name implies a disease such as a viral infection , it was shown that the birds had been
poisoned by a contaminant in the pelletted feed. The contaminant, which was called
Aflatoxin, fluoresced intensely under ultra-violet light and was shown to be produced
by the mould Aspergillus flavus growing on the groundnuts.
Aflatoxin is a mycotoxin produced by certain fungal species, mainly by
Aspergillus flavus and Aspergillus parasiticus Because of their pronounced toxicity
and extreme carcinogenicity in many animal species, aflatoxins have been and
continue to be extensively investigated. These toxins are named after the fungus
producing them ie
A. flavus. A from the genus name Aspergillus fla from the species name flavus
added to toxin to give the name Aflatoxin . These are highly carcinogenic polyketide
metabolites (Jiang et al., 2005). Polyketides are structurally diverse class of secondary
metabolites produced by certain species of fungi. The accumulation of mycotoxins in
foods and feeds represent a major threat to human and animal health as they are
responsible for several chronic health risks including immunosuppression, cancer
induction,digestive, blood and nerve defects. Mycotoxins negatively impact
agriculture and associated industries in different ways and the economic consequences
of mycotoxin contamination are profound. Regulations have been established in most
countries world wide to protect consumer health and ensure fair practices in food
trade ( Bandopadhyay et al., 2007).
Aspergillus flavus usually grow as saprophyte on plant debries of many crop
plants left on and in the soil. They are distributed worldwide with a tendency to be
more common in countries with tropical climates that have extreme ranges of rain fall,
humidity and temperature. Colonies of A flavus are green yellow to green.


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Taxonomy:
Class: Ascomycotina
Group: Plectomycets
Order: Perisporiales
Family: Eurotiaceae
Genus: Aspergillus
Species: flavus

Conidial heads bearing sterigmata or phialides, which consists of asexual
spores, charecterises the Genus Aspergillus; head is placed in conidiophore like
hyphae placed on a foot cell. The sterigmata may be arranged in one row (uniseriate)
or two rows (biseriate) in short, conidiophore termination in a vesicle. Sterigmata
arising in one or two series from upper part or entire surface of the vesicle.

Occurrence:
A flavus is universally distributed in the environment and aflatoxin
contamination in fods and feeds has been detected in all parts of the world . Aflatoxin
have been found in following commodities.

Table 1: Differentiation of Aflatoxin in various foods
Aflatoxin M1 has been found in milk of lactating animals that injest AFB1 with feed.
Oil seeds Groundnut, cotton seed, copra, sunflower and soy
bean
Crude vegetable oils Groundnut, olive and coconut
Grains Maize, sorghum, rice wheat, barley, millet, oats,
peas, beans and lentils
Tree nuts Pistachio and brazil nuts, almonds, walnuts and
filbert,
Tubers Potato, sweet potato and cassava
Fruits Figs, apple, peaches, areca nuts and plums


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TRUCTURAL DIVERSITY OF AFLATOXINS
Structurally, aflatoxins contain a coumarin nucleus fused to bifuran either a
pentanone (AFB1 and AFB2) or a six membered lactone (AFG1 and AFG2).

Table 2: Characteristics of Aflatoxin
AFLATOXIN
MOLECULAR
FORMULA
MOLECULAR
WEIGHT
MELTING
POINT
UV-
ABSORBTION
(362-363nm)
FLUORESCENC
E EMISSION
B1 C
17
H
12
O
6
312 268-269 21,800 425
B2 C
17
H
14
O
6
314 286-289 23,400 425
G1 C
17
H
12
O
7
328 244-246 16,100 450
G2 C
17
H
14
O
7
330 247-240 21,000 450
M1 C
17
H12O
7
328 299 19,000 425
M2 C
17
H
14
O
7
330 293 - -
B2a C
17
H
14
O
7
330 240 20,400 -
G2a C
17
H
14
O
8
346 190 18,000 -
R0 C
17
H
16
O6 314 230-234 14,100 425
B3 C
16
H
14
O
6
302 233-234 9,700 -
GM1 C
14
H
12
O
8
344 276 12000 -
P1 C
16
H
10
O
6
298 >320 14900 -

The molecular formula indicated that aflatoxins AFB1 and AFG1 respectively.
Hydroxylated aflatoxin derivatives called AFM1 and AFM2, were reported in the
milk of cows fed toxic rations. AFGM1, a hydroxylated derivative of AFG1 was
isolated from A flavus cultures. AFB2a and AFG2a are 2-hydroxy aflatoxins and
were reported under several names by various scientists as AFB1 hemiacetal , and
hydroxydihydro aflatoxin B1. 6 methoxy -7 (2hydroxy ethyle) difurocouramin has
been called as parasiticol or AFB3. Reduction of the pentanone of AFB1 by
microorganisms yielded aflatoxicol(AFL). Animals also metabolize AFB1 with
ammonia produced AFD1 and AFD2.



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Fig. 1: Structures of aflatoxins B
1
, B
2
, G
1
and G
2




Fig. 2: Structures of aflatoxins M
1
, M
2
, B
2A
and G
2a
.




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CHEMICAL PROPERTIES OF AFLATOXINS
ALKALI
In alkaline solutions, hydrolysis of lactone moiety occurs. This is a reversible
reaction, because recyclisation will occur following acidification.

MINERAL ACIDS
Mineral acids like nitric acid, sulphuric acid and phosphoric acid convert
AFB1 and AFG1 to AFB2a and AFG2a respectively. This is due to acid catalysed
addition of water across the double bond in the furan ring. Treatment with acetic
anhydride and hydrochloric acid will show the same reaction with acetoxy derivative.

TOXICITY OF AFLATOXINS
Many animals are susceptible to acute toxic effects of aflatoxins. The more
susceptible species of mammals and birds to AFB1 are rabbit and ducklings. Mice are
quit resistant to AFB1 .AFB1 and AFG1 were more toxic to ducklings, rats and fish
than either AFB2 or AFG2 with AFB1 being the most toxic. AFM1 was established to
be as less toxic. AFB2a and AFG2a are relatively non toxic and AFP1 is less toxic
than AFB1.

CARCINOGENICITY
Aflatoxin B1- classified as carcinogenic substance, Group 1B (IARC 1993)-
has been the most regulated mycotoxin worldwide (FAO 1997). Carcinogenic effects
of aflatoxins have been documented in several species: rat, mouse, trout, duck and
monkey. Aflatoxins are suspected to have a role in human cancers as well. Most
frequently the target organ is the liver. In some circumstances primary tumours have
also been observed in the kidney of animal given AFB1. Aflatoxins first must be
enzymatically activated by P450 enzymes in liver to 2,3 epoxide, which then
functions as the proximate carcinogen by intercalating into DNA and alkylating the
guanine residue. AFB1 has been detected in respiration ducts which result in lung
tumour with bronchiolar epithelium being the major site. Reason for the cancer by
aflatoxins is the mutation in 249
th
codon of p53 tumour suppressor gene at third base
of codon (G T transversion corresponding to the conversion of aminoacid arginine to
serine ). The carcinogenic potency of aflatoxin is in the order of AFB1>AFG1>AFB2.


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BIOLOGICAL DETOXIFICATION
Specific lactic acid bacterial strains and yeast strains efficiently remove
aflatoxin from solution by physical binding to cell wall polysaccarides and
peptidoglycan. Biological decontamination of aflatoxins using microorganisms is one
of the well known strategies for the management of it in foods and feeds (Bejaoui
et al., 2004).
Among the different potential decontaminating microorganisms, different
species of wild yeasts represent unique groups .

YEAST
Yeast is a model eukaryote (Cryer et al., 1998). Yeast are a group of
eukaryotic microorganisms classified in the kingdom Fungi, with about 1,500 species
described (Kurtzman, 2007) they dominate fungal diversity in the oceans (Klis et al.,
1994). Most reproduce asexualy by budding, although few do by binary fission .
Yeasts are unicellular, although some species of yeast forms many become
multicellular through the formation of a string of connected budding cells known as
Pseudohyphae or false hyphae as seen in most moulds (Kurtzman, 2006).Yeast size
can vary greatly depending on the species , typically measuring 3-4m in diameter,
although some yeast can reach over 40m (Walker, 2002).
Wild yeast are the naturally existing yeast in the air, on vegetation or blowing
around in the air. One of the most common characteristics of wild or indigenous
yeasts is their low resistance to alcohol. Gilliland (1967) defined Wild Yeast as
any yeast which is not deliberately used and under full control Standard brewing
yeast Sacharomyces cerevisiae is only one of 400 different species of the genus
Saccaromyces. There are also many hundreds of different strains of the species
Saccharomyces cerevisiae each with different brewing characteristics. A wild yeast
could therefore be a different strain of Saccaromyces cerevisiae but is more
commonly one of several other genera of yeasts example- Candida , Brettanomyces,
Hansenula , Kloeckera and Pichia.
All yeasts have certain properties in common which make them potentially
serious contaminants of brewing yeast and/ or beer; They are relatively tolerant of low
pH and alcohol concentration.


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The useful physiological properties of yeast have led to their use in the field of
Biotechnology. Fermentation of sugars by yeast is the oldest and largest application of
this technology. On 24
th
April 1996 S cerevisiae was announced to be the first
eukaryot to have its genome, consisting of 12 million base pairs, fully sequenced as a
part of the genome project (William, 1996). At the time it was the most complex
organism to have its full genome sequenced and took 7 years and the involvement of
more than 100 laboratories to accomplish (Complete DNA sequence of yeast, 2007).




















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OBJECTIVES

Following objectives are taken up for the present study:
To screen different isolates of wild yeast from various sources for their
Aflatoxin binding ability.
To characterize the aflatoxin bound in selected yeast isolates.
To identify the type of aflatoxins which the cultures are able to bind.
Identification of screened cultures by 18s rRNA sequencing.










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MATERIALS AND METHODS















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MATERIALS AND METHODS

SUBCULTURING OF Aspergillus flavus :
PDA (Himedia Mumbai)

PDA media was prepared by dissolving 300g of Potato, 20g of Dextrose
and15g of agar in 1000ml distilled water and pH was adjusted to 5 0.2.

Methodology:
A loopfull of inoculum was taken and streaked on PDA slants under aseptic
conditions
These slants were incubated at 30 C for 2 dayes

PRODUCTION OF AFLATOXIN (Davis et al., 1980):
Groundnut (infected with Aspergillus flavus) 10g
Chloroform 100ml
Water 10ml

Methodology:
10 g of groundnut was partially crushed and uniformly spread on a petriplate
covered with wet tissue.
To the plate 2ml of fungal suspension(A flavus) was added.
Incubated it for 3 days at 30 C
The infected groundnuts were crushed using a pestle and mortar
Crushed groundnuts were weighed
For every 10g of material , 10ml of water and 100ml of chloroform was added
and kept in rotary shaker for 30minutes
Filtered and the filtrate was passed through a bed of anhydrous sodium
sulphate to remove the moisture
Concentrated under vacuum using rotavapour
The residue was dissolved in 100l of Chloroform



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OLUMN SEPARATION:
Column 15 10 mm
Silica gel 60-120 mesh size
Chloroform
Anhydrous Sodium sulphate
Glass wool
Hexane
Anhydrous ether
Methanol

Methodology
A ball of glass wool was placed in the bottom of chromatographic column.
Anhydrous sodium sulphate was added to give a base to silica gel
Sides of the column was washed with chloroform to disperse silica gel, A
slurry of silica gel in chloroform was added to the column
When rate of setting slows down small volume of chloroform was drained out
to aid setting, leaving a column of 5-7 cm of chloroform above silica gel
Slowly 15g of anhydrous sodium sulphate was added to the column
5ml of sample was added to the column
Eluted at maximum flow rate with 20ml hexane followed by 20ml anhydrous
ether . The elute was discarded
Aflatoxin was eluted with 20ml methanol-chloroform mixture in a ratio of
3:97
The elutant was evaporated to dryness using nitrogen gas

TREATING AFLATOXIN WITH WILD YEAST:
Isolates collected from various ecological niche was collected from the culture
collection of the laboratory. 40 isolates were taken for the study.

Microbial media and growth conditions:
For the cultivation and maintenance of yeast cultures , SDA was used. The
yeast cultures were incubated at room temperature for 24 hours.
SDA media (Himedia Mumbai)


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SDA media was prepared by dissolving 10g of Peptone , 40g of Dextrose, 16g
of Agar in 1000ml of distilled water and the final pH was set to 5.6.

Methodology:
A loopfull of inoculum was taken and streaked on SDA slants under
aseptic conditions
These slants were incubated at room temperature for 2-3 days

SAMPLE PREPARATION FOR BINDING PROPERTY OF AFLATOXIN
WITH YEAST ISOLATES:
Chloroform extract of aflatoxin 100L
1xPBS buffer 100l
Culture suspension 100l
Distilled water 100l
Chloroform

Methodology
0.1 ml of chloroform extract of aflatoxin was taken after partial purification
0.1 ml of PBS buffer was added to the extract
Yeast culture suspension was prepared by adding 1000l of sterile water to
loopfull of inoculum, whose optical density was nearly 2.0
0.1ml of culture suspention was added to the eppendroff containing 0.1ml of
Aflatoxin and 0.1ml of PBS buffer
These tubes were incubated for 30 minutes at room temperature
Pellet and supernatant were separated
To the eppendroff containing pellet and 0.1ml of distilled water was added ,
vortexed
To both tubes containing supernatant and pellet 0.1ml of chloroform was
added
Vortexed and lower chloroform layer was separated and collected
The pellet was washed twice with Chloroform. The chloroform fraction were
poured
0.5ml of each sample was used for TLC analysis


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THIN LAYER CHROMATOGRAPHY (Walter et al.,1980):

Methodology:
TLC plates were prepared with the help of TLC spreader (CAMAG instrument
Switzerland). Glass plates of 20 X 20 cm were cleaned with hexane to remove grease
and fatty substances. 13g/plate silica gel was taken in 500mL glass stoppered
Erlenmeyer flask . Water in the ratio of 1:2 (w/v) was added to the silica gel and was
shaken vigorously for 1-2 minutes. Freshly prepared slurry was poured into the
applicator and coated on TLC plate (300m thick). The plates were allowed to dry
for 1-2 hours at room temperature. Plates were activated for 1 hour at 100 C in a hot
air oven and stored in a desicator until use. Samples were developed with a solvent
system of chloroform:acetone ( 9:1 ratio). Plates were observed under UV for
flurescence spots.

BINDING PROPERTY ASSAY OF AFLATOXIN WITH YEAST ISOLATES:

1x PBS buffer
1X PBS buffer was prepared by dissolving 140Mm sodium chloride, 3Mm
KCl, 8Mm Na
2
HPO
4
, 2Mm KH
2
PO
4
. And pH was set to 7.4. 80ml deionized water
added and stirred until they dissolve completely. The pH was adjusted to 7.4 with 1 N
HCl. The final volume was adjusted to 1 liter.
YEPD Broth
YEPD Broth was prepared by dissolving 20g of Peptic digest of animal tissue,
10g of Yeast extract and 20g of dextrose in 1000 ml distilled water.

Methodology:
100ml of YEPD broth was taken in 250 ml flask, autoclaved
A loopfull of culture was inoculated into the broth under aseptic conditions
The inoculated flask was kept on a orbital shaker (200 rpm) at room
temperature for 48 hours
Fermented broth was centrifuged at 600rpm for 10-15 minutes
pellet was washed with 10ml of 1xPBS buffer
Cell suspension was prepared in PBS buffer with a cell density of 10
8
cells
(OD <2)


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To 0.1ml of cell suspension , 0.1ml of toxin extract was added along with
1ml PBS buffer
The samples were incubated at different intervals of time ie, 0,10, 20, 30
minutes, 1 hour, 2 hour and 4 hour
After appropriate incubation 0.1ml of sample was withdrawn
Centrifuged at 6000 rpm for 10 minutes
The pellet and supernatant were separated and individually washed with
0.1ml of chloroform twice.Chloroform fraction was collected
5l of sample was loaded to the TLC plate , developed and observed under
uv light at 360nm
Chloroform fraction was used for HPLC and Mass spectrometry

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) :

High performance liquid chromatography is an optional technique for ultimate
separation and quantitation. It allows us to use a very small particle size for the
column packing material giving a much greater surface area for interactions between
the stationary phase and the molecule which allows a much better separation of the
components of the mixture ( clark, 2007).

Materials required:
The concentrations of aflatoxins were determined by HPLC. SCL-10 AVP Shimadzu
amino acid analyzer was used with fluorescence detection. A reverse phase C-18
column (15 cm and 150mm) was used with a flow rate of 1ml min
1
.
Water: Acetonitrile: Methanol were used as eluent. Standard Aflatoxin was
prepared in the concentration of 1mg/ml.

Methodology:
Sample was prepared by nitrogen flushing of chloroform extract which was
used for TLC
Dissolve the residue in 50l of methanol
20l of above sample was injected to the column


28

Solvent used was a mixture of water , acetonitrile and methanol in a ratio
of 6:3: 1
Flow rate was 1ml/min
Fluorescence was detected at excitation wavelength of 350nm and
emission wavelength 460nm (Walter et al .,1980)

MASS SPECTROMETRY (Ventura et al., 2004):
Aflatoxin which were analysed by liquid chromatography were further
subjected to mass spectrometry single quadruple using an electrospray ionization
(ESI) sourse (LC-MS) in order to avoid derivatization. In mass spectrometry atoms
can be deflected by magnetic fields provided the atom is first turned into an ion.
Electrically charged particles are affected by magnetic field although electrically
neutral ones are not.

Methodology
It was performed in electrospray ionization source (ESI) and operating in the
positive mode. Nitrogen was used as the collision gas
Capillary voltage is 35kv , cone voltage 100kv, source block temperature 80
C evaporation temperature 180C
Solvent gas 475L/h, cone gas 50L/h
Low mass resolution and high mass resolution was 15
Ion energy 0.5 extractor is 7
Rf lens 0.5 and electron multiplyer voltage 650v

SCANNING ELECTRON MICROSCOPY:
The selected yeast cultures were prepared for scanning electron microscopy
visualization.(MC Dougall.et al., 1994 and Ramesh et al., 2000 )

1x PBS buffer
glutaraldehyde 2.5%
Absolute alcohol


29

Methodology
1ml of the yeast culture sample grown for 24 hour in YEPD was
centrifuged at 8000rpm for 10 minutes
Supernatant was decanted and to the pellet , 1x PBS buffer was added and
vortexed for 2-3 times
Supernatant was decanted and to the pellet 2.5% gluteraldehyde was added
and mixed thoroughly . These tubes were incubated at room temperature
overnight and Centrifuged
The pellet was passed through a gradient of ethanol from 10% to 100%
The sample was placed on coverslips and dried. These cover slips with
samples were kept in dessicator until analysis

A carbon coated membrane was placed on an aluminium disc and the sample
was smeared on the lining . An inert metal (gold) was coated using a sputter coater
(Cool Sputter Coat System , Model 5001, England) on the sample and this was placed
inside the SEM (435 V. P., Leo Electron Microscopy Ltd ., UK) at 20Kv. A tungsten
electron gun releases a beam of electrons (primary electrons) that passes through
magnetic lenses scanning the surface of the sample . A beam of electrons (secondary
electrons ) is ejected from the surface of the sample that is received by the electron
amplifier . The amplified image was detected by photo detector and transferred to the
computer screen.

ISOLATION OF YEAST DNA (Hoffman and Wiston., 1987)

The yeast DNA was isolated for further studies.
yeast lysis buffer
Ingredients g/l
Triton x-100 4ml
10% SDS 20ml
5M NaCl 4ml
0.5M EDTA4ml 400l
1M Tris (pH 8) 2ml
Above components are dissolved in 200 ml distilled water and pH was set to
8 by adding TE buffer. The final volume is made up to 200ml.


30

Methodology :
10ml of yeast were grown overnight to saturation in YEPD broth at 30C
The cells were centrifuged for 10 minutes at 8000rpm and it was resuspended
in 0.5 ml of sterile distilled water
In the following order, 200l yeast lysis buffer and 20l of proteinase k were
added
The above mixture was kept in water bath at 55 C for 2-3 hours
After incubation , phenol: chloroform: isoamyl alcohol were added in a ratio
of 25:24:1
The mixture was centrifuged at 10,000 rpm for 10 minutes . Supernatant was
collected (250l)
To the above solution , approximately 750l of ice cold isopropyl alcohol in a
ratio 1:3 was added
20l of 3M sodium acetate was added
The mixture was were kept overnight at refrigerated condition
The microfuge was centrifuged at 10,000rpm for 10minutes , and pellet
collected
The pellet was washed with 500l of 70% ice cold ethanol
The pellet was air dried and resuspended in 20l sterile wate

AGAROSE GEL ELECTROPHORESIS:
It is a method used in biochemistry and molecular biology to separate DNA
strands by size. This is achieved by pulling negatively charged DNA molecules
through an agarose matrix with an electric field . Shorter molecules move faster than
larger ones.

Agarose
1xTAE buffer
Marker dye(bromophenol blue)
ethydium bromide (1l/ml)
electrophoresis unit




31

Methodology:
100ml of 0.8% agarose gel was prepared by dissolving (boiling) 0.8g of
agarose in 100ml of 1x TAE buffer . The gel was cooled and poured into
the boat containing the comb and the gel was allowed to solidify.
After the solidification of the gel , samples containing the DNA were
loaded into the wells along with 2l of marker dye (bromophenol blue)
(5l of each sample in a well)
Then the samples were electrophoresis at 50v for 1 to 1 hour
The gel was stained with ethydium bromide (1l/ml) for 1 minute in dark
The gel was then observed and analysed using the uv gel documentation
system

POLYMERASE CHAIN REACTION:
PCR was done to amplify the targeted kb 16s r RNA gene of selected isolates.
The reaction was carried out in the thermocycler gene Amp PCR system 9700 (Perkin
elmer) using standard protocol (Sambrook and Russel, 2001)

PCR reaction components:
Template DNA
16s rRNA gene specific primers
Farward primer BSF(1:10)
Reverse primer BSR(1:10)
Primer(5to 3 direction) Expected size of the amplicon
BSF 5

GCATATCAATAAGCGGAGGAAAAG 3


BSR 5 GGTCCGTGTTTCAAGACGG 3
Taq DNA polymerase (Bangalore Genei , India)

10x reaction buffer
Ingredients Amount
Tris buffer 1 00mM
KCl 500mM
MgCl
2
15mM
Gelatin 0.1%
pH 9


32

Nuclease free water (Millipore water)
dNTP mix (10 mM of each dNTP)
The quantity and concentration of different components in the PCR reaction mixture
are as follows;

Table 3. Different components used in PCR reaction mixture:
Components Volume(l) Final concentration
Nuclease free water 1:10 dilution
10x reaction buffer 2.5
dNTP mix(10Mm) 5
Taq DNA polymerasre 0.3
Primer 2 (1:1) 1:10 dilution
Template DNA 2 1:10 dilution
Total volume 2.5

Table 4.PCR cycle parameters:

Parameters Temparature ( C) Time
Initial denaturation 95 3 min
35 cycles
Denaturation 94 40 sec
Annealing 50 40 sec
Synthesis 72 1 min , 20 sec
Final extention 72 5 min

Methodology:
Master mix was prepared by mixing all components except template DNA
and sterile water
The master mix was then added to the PCR tubes containing template
DNA and sterile water
The contents of the tubes were mixed by brief spin of microcentrifuge




33

PCR product analysis:
The PCR product was analysed by using 1.5% agarose gel electrophoresis
A 5l aliquote of the amplified PCR product was taken , mixed with 2l of
loading dye and loaded on to the well
The size of the yeast amplicon was confirmed by comparing with 2kb
ladder(Bangalore Geni), which was used as a molecular size marker
Electrophoresis was carried out at 100v till the dye reaches of the gel
The gel was stained , destained and the amplicon band were observed
under uv- transilluminator

RESTRICTION DIGETION:
Restriction mapping involves the size analysis of restriction fragments
produced by several restriction enzymes both individually and in combination.
Comparision of the leanth of fragments obtained allows their relative positions within
the DNA fragment to be deduced.( R.Rapley et al. 1998)

Enzyme 1l
Alu I
Hae
Buffer 2l (10x assay buffer)
Template (DNA or PCR product) 7 or 5l
MQ water 12 l
Agarose gel 2%

Table 5. Restriction digestion reaction mixture
Amount Volume (l)
AluI 1
Hae 1
Buffer 2
PCR product 5
Water 12
Total volume 25



34

Methodology:
For restriction digestion, 1l of enzyme either Alu I or Hae III was used
To that 2 l of 10x assay buffer was added with 5l of template
For mixture of AluI and Hae III , 0.5 l of each enzyme with 2 l of buffer
and template were used and 15 l of sterile water is added
The mixture was kept in water bath at 37C for 2-4 hours
Restriction digestion was analysed in 2% agarose gel through
electrophoresis

PCR PRODUCT PURIFICATION:
(HiPurA Himedia. Mumbai)
PCR product 5 vol
PCR binding solution 50l
Mini preparation spin column
wash solution 700l
Elution buffer 30l

Methodology:
Add 5 volume of PCR binding solution (SPB) to 1 volume of the PCR sample
and mix well by pipetting. It is not necessary to remove the mineral oil
For example, add 50 l of PCR binding solution to 100l PCR sample
Place the mini preparation spin column in a 2 ml collection tube provided
with the kit.
Apply the PCR sample and PCR binding solution mixture to the spin column
. centrifuge for 1 minute at 13000 rpm
Discared the flow through and replace the column in the same collection tube
Centrifuge for 1 min at 13000 rpm to remove excess ethanol
Transfer the Miniprep spin column to a clean collection tube and pipette 5l
of elution buffer to the center of the spin column and centrifuge for 1 minute
at 13000 rpm
Alternatively for increased DNA concentration , 30 l elution buffer was
added to the center of the spin column . Incubated at room temperature for 1 min and
then centrifuged for 1 min at 13000 rpm
The PCR amplification product was now present in the elute is ready for
immediate use . Alternatively for future use stored at -20C or lower


35














RESULT AND DISCUSSION
















36

RESULT AND DISCUSSION


Plate 1. Aspergillus flavus grown on PDA media

Work was undertaken to study the binding capacity of yeast. A flavus was
collected from laboratory culture collection. Well grown culture was added on liquid
inoculum to damaged diswalled groundnuts. The inoculated cultures were incubated
at 32C for 3-4 days. Infected groundnuts were extracted for Aflatoxin with
chloroform and concentrated. The toxin was used for aflatoxin binding test.


Plate 2. Ground nuts infected by A flavus

SUBCULTURED YEAST ISOLATES:
The cultures were isolated from various ecological niche , purified and
preserved in the culture collection of the lab as shown in Table 6.


37

Table 6: Cultures selected for aflatoxin test
SL NO
CODE AS PER THE
CULTURE COLLECTION
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
115 WL iii 9
706
SL1 (6) 63
571
H
560
706
CPI (2) 23
563
CF10
CL(I) 32
530
CF22
C P ( 1 ) V Pa
140
CF 20
CF 17
528
157
525
523
chI (2)38
575
217
546
Surface 6
519
500
509
146
607
527
CF9
SLI72 (8)
Garcinia wine
151
514
CF23
CLII (4) VS
421


38

POTENTIAL FOR AFLATOXIN BINDING BY YEAST ISOLATES:
Cultures drawn from the yeast culture collection was treated with aflatoxin.
The supernatent and pellet samples were subjected to thin layer chromatography and
observed for fluorescence under U V. Results of TLC are recorded in table no 7.


Plate 3. TLC plate showing fluorescent spots

Table 7: Potential for aflatoxin binding by yeast culture s:

SL NO
SUPERNATENT (S) OR
PELLET (P) FRACTIONS
CONCENTRATION
1 S -
P -
2 S -
P LC
3 S -
P LC
4 S +
P LC
5 S LC
P +
6 S +
P LC
7 S +
P +
8 S +
P LC
9 S +


39

P LC
10 S LC
P LC
11 S +
P LC
12 S -
P LC
13 S +
P LC
14 S +
P LC
15 S -
P -
16 S LC
P +
17 S +
P LC
18 S LC
P +
19 S LC
P LC
20 S +
P LC
21 S LC
P +
22 S -
P -
23 S -
P -
24 S +
P +
25 S LC
P +
26 S LC


40

P -
27 S -
P -
28 S +
P LC
29 S LC
P +
30 S LC
P +
31 S LC
P -
32 S +
P LC
33 S +
P LC
34 S LC
P LC
35 S LC
P LC
36 S +
P LC
37 S LC
38 S LC
P +
39 S LC
P LC
40 S LC
P +

LC= Lower concentration


41

Based on the efficiency of binding, five cultures were selected for further
studies. They were culture number 5, 16, 18, 29, 30, 37, and 40. The selected cultures
were subjected for further studies on their Aflatoxin binding efficiency.

HPLC RESULTS :
Screened cultures were treated with aflatoxin and incubated for different
intervals of time. The supernatant and cell pellets were subjected to HPLC
fluorescence detection. Concentration of the toxin were calculated based on the
standards.The retention times of the various toxins were compared with the standards
(fig 3c). It is observed that the elution was over within a short period of 3 minutes.
But for all samples, the chromatogram was not for 10 minutes to observe for any
further degraded/ transformed product (fig 3a). Aflatoxin B1 was eluted at 0.7
minutes. Where as B2, G1, and G2 were eluted at 1.15, 1.69 and 2.02 minutes after
injection. Pellet and supernatant of all the treated samples at time intervals of 0, 2 and
4 hours were separately analyzed as recorded in table 8.

Table 8: Aflatoxin binding of selected yeast isolates at various time intervals

0 hour incubation
Retention time(min)
2 hour incubation
Retention time(min)
4 hour incubation
Retention time(min)
5 pellet 1.37 2.00 2.50 0.642 1.058 1.683 0.542 0.933 1.467 1.875
5supernatent 1.658 2.050 2.800 1.067 1.56 2.oo 0.64 1.508
16 pellet 1.408 1.992 0.650 1.200 1.700 0.742 1.150 1.683 2.083
16supernatent 1.408 2.142 3.967 1.467 2.842 0.625 1.100 1.558 1.967
18 pellet 1.267 1.833 0.633 1.158 1.692 0.600 0.958 1.517 1.950
18supernatent 1.408 1.992 0.675 1.117 1.725 1.542 2.00 2.792
29 pellet 1.275 1.542 0.633 1.017 1.667 1.158 1.625
29 supernatent 1.183 1.517 1.950 1.608 1.992 0.667 1.242 1.642
30 pellet 1.025 1.500 5.175 0.658 1.092 1.700 0.675 1.275 1.617
30 supernatent 1.058 1.458 1.858 0.692 1.725 1.592
37 pellet 1.258 1.583 2.042 0.658 1.058 1.700 0.658 1.108 1.608 2.042
37 supernatent 1.400 1.900 0.592 0.983 1.650 1.392 2.033 2.825
standard 0.7 1.150 1.692 2.025


42

On further analysis, the concentration of B1 at 0 hour of incubation was high
in the pellet for samples 5,16, 18 and 29. However, in the supernatants of 16 and 18,
the concentrations of B1 was quite high (table 9). After 2 hours of incubation, there
was change in the binding capacities of G2 was observed in all the cultures tested,
which was absent in culture 16 and 29 at 0 hour. By 4 hours, all the toxins were drawn
to culture5, 16, 18, 30 and 37. Where as in 29, G1 and G2 were absent. G1 was absent
in both culture and supernatant at 2 hours of incubation, which needs further
investigation. High binding immediately to the cell wall was observed earlier by
Shetty et al (2007). Four hours of incubation time was recorded for binding test using
lactic acid bacteria by Haskard et al (2001).

Table 9: Aflatoxin binding capacity of various yeast isolates

AFLATOXIN
SAMPLE NO PELLET SUPERNATENT
0 HOUR
5
6
18
29
37
30
B1,B2,G2
B1,B2
B1,B2, G2
B1, B2
B1, B2, G2
B1, B2, G2
B1,B2,G2
B1, B2, G2
B1, B2
B1, B2, G1, G2
B1, B2, G2
B1, B2, G1, G2
2 HOUR
5
6
18
29
37
30
B1, B2, G2
B1, B2, G2
B1, B2, G2
B1, B2, G2
B1, B2, G2
B1, B2, G2
B1, G2
B1, G2
B1, B2, G2
B2, G2
B1, B2, G2
B1, B2
4 HOUR
5
6
18
29
37
30
B1, B2, G1, G2
B1, B2, G1, G2
B1, B2, G1, G2
B1, B2
B1, B2, G1, G2
B1, B2, G1, G2
B1, B2, G1, G2
B1, B2, G1, G2
B1, B2, G2
B1, B2, G2
B1, B2, G1, G2
B2
STD B1, B2, G1, G2

With the help of following graphs we can study the aflatoxin
concentration.The different aflatoxins elute in reverse face c18 column in the order of
Aflatoxin B1, B2, G1 and G2.


43

Minutes
0 2 4 6 8 10
V
o
l
t
s
0.00
0.01
0.02
0.03
0
.
6
2
5
1
.
1
0
0
1
.
5
5
8
1
.
9
6
7
2
.
4
7
5
2
.
7
7
5
7
.
5
2
5
9
.
0
4
2

Fig 3 (a). CHROMATOGRAM OF SAMPLE NO 18 P4

Minutes
0 2 4 6 8 10
V
o
l
t
s
0.000
0.005
0.010
0
.
6
0
0
0
.
9
5
8
1
.
5
1
7
1
.
9
5
0
2
.
4
2
5
2
.
6
4
2
2
.
7
8
3
7
.
4
6
7
8
.
9
6
7


Fig 3 (b). CHROMATOGRAM OF SAMPLE NO 18 S4

Minutes
0 2 4 6 8 10
V
o
l
t
s
0.00
0.02
0.04
0
.
7
0
0
1
.
1
5
0
1
.
6
9
2
2
.
0
2
5
3
.
7
6
7
6
.
2
2
5

Fig 3 (c). CHROMATOGRAM OF STANDARD SAMPLE




Type of toxin Retention time
B1
B2
G1
G2
0.700
1.150
1.690
2.025


44

Concentrations of B2 absorbed by Yeast cultures was calculated based on
HPLC analysis (Table 10). It was observed that in culture number 16, initial adhesion
was high. After 2 hours of incubation, all the B2 had adhered to culture number 5 and
16. At the end of 4 houres of incubation, the concentration of B2 adhered to the cell
was high for all the yeast cultures except culture number 18, where equal
concentrations in cell pellet and supernatant was obsereved. And in culture number
16, supernatant has higher concentration than cell pellet.
Table 10. Concentration of Aflatoxin B2 (mg/100ml) adsorbed by yeast cultures























Further the results were analyzed using mass spectra.The method is useful for
the simultaneous determination of aflatoxin B1, B2 G1 and G2 .Chromatographic
SAMPLE NO. PELLET SUPERNATENT
0 hour incubation
16 238 121
18 119 238
29 057 119
30 042 075
37 076 238
2 hour incubation
5 108 -
16 015 -
18 091 266
29 042 333
30 033 05
37 091 025
4 hour incubation
5 041 -
16 075 133
18 041 041
29 108 043
30 025 -
37 108 075


45

separation was performed using a short column that allows rapid determination
obtaining sharp chromatographic peaks and minimizing consumption of mobile
phase.The molecular indicated that these are derivations are basically B1 (table 11).
Such derivations were easily observed by Ventura et al(2004). The distribution of B1
and B2 were more in pellet, indicating the efficiency of adhesion by the yeast cells
(fig 4).

MS DATA OF AFLATOXIN BINDING TO YEAST CELLS:
29-Jul-200814:44:01 STD
m/z
305 310 315 320 325 330 335 340 345
%
0
100
29070809 4 (0.217) Cm (2:12) TOF MS ES+
586 311.36
309.39
307.35
316.32
331.28
325.38
321.31
327.36
335.31
342.31


Fig 4(a). MS data of standard sample

30-Jul-200815:51:40 117 P4
m/z
300 305 310 315 320 325 330 335 340 345 350
%
0
100
30070807 6 (0.321) Cm (6:10) TOF MS ES+
515 316.26
311.35
309.34
307.36
302.27
340.37
324.36
335.33

Fig 4(b). MS data of sample No 37 P4



46




30-Jul-200816:41:19 117 S 4
m/z
300 305 310 315 320 325 330 335 340 345 350
%
0
100
30070827 8 (0.423) Cm (7:10) TOF MS ES+
248 316.31
311.33
303.19
309.28
313.32
319.15
340.38
335.31
325.35
321.32
327.26
333.32
329.29
337.31
349.28


Fig 4(c). MS data of sample No 37 S4




30-Jul-200816:13:19 CCS
m/z
300 305 310 315 320 325 330 335 340 345 350
%
0
100
30070816 91 (4.773) Cm (91:95) TOF MS ES+
460 319.13
311.35
303.16
309.36
307.25
315.21
343.24 324.40
335.30
339.31
346.82


Fig 4(d). MS data of negative control sample






47

Table 11: Lc/ms results at different incubation time:

S= supernatant
P=pellet
A few unknown compounds were present which are having molecular weight
of about 319.13, 340.39, 327.23. These may be either the degraded products or cell
extracts, has molecule with the molecular weight of 319.13 was observed in both
negative control (cells grown without aflatoxin) and experimental cell pellet data.
On comparison of the binding capacity, B1 was found to adhere more than B2
in the first two hours of experiment (table 7). But at 4 hours of incubation B2 was
found to adhere more than B1.



Sample
No.
0 hour 4hour 4 hour
5p B1 B2 B1+Na B1 B2 B1 B2 B1+Na
5s B2 340.39 342.26 B2 324.37 340.36 B1 B2 B1+Na
16p B1 B2 B1+Na B2 B1+Na B2 324.38 B1+Na
16s B1 B2 B1+Na B2 G2 B1+Na
18p B1 B2 B1+Na B2 G2 B2 G2 340.38
18s B2 B1+Na 340.37 B2 340.40
29P B1 B2 B1+Na B2 B1+Na 340.39 B2 B1+Na 340.41
29S B2 G1 340.39 B2 G2 B1+Na
30p B1 B2 B1+Na B2 324.39 327.23 B2 327.28 340.35
30s B2 G1 B1+Na B2 B1+Na 342.2 6
37p B2 B1+Na B2 340.37 B2 327.23 340.36
37s B2 G1 B1+Na B2 B1+Na 340.38


48

Table 12: Binding capacity of aflatoxin to yeast cells as indicated by
MS data

SCANNING ELECTRON MICROGRAPH OF ISOLATES:
The isolated cultures were subjected to scanning electron
microscopy to study the morphology. It was observed that, culture
number 16 and 30 ware rod shaped with an approximate size of 2.24
1.8 m and 3.10.3 m respectively. Rest of the isolates were oval and
showed budding. Culture number 29 were slightly rod shaped with
Sample No. Pellet Supernatant
0 hour incubation
5 B1 B2
16 B2 B2
18 B2 B1
29 B1, B2 B2
30 B2 B2
37 B2 B2
2 hour incubation
5 B1
16 B1, B2
18 B2
29 B1, B2
30 B1
37 B1, B2
4 hour incubation
5 B2 B1, B2
16 B2 B2
18 B2 B2
29 B2 B2
30 B1, B2 B2
37 B1 B2


49

noticeable with clamp connections with a size of 61.8 m. Other cells
with an approximate size of 4.82.18 m, 3.061.25 m, 3.872.25 m
and 52.8 m respectively in case of culture number 5, 18, 37 and 40.


Plate 4. Scanning electron micrograph of culture code 5 (size= 4.82.18 m
magnification 10 K )



Plate 5. Scanning electron micrograph of Yeast culture code 16 (size= 2.241.8
m magnification 20 K)




50



Plate 6. Scanning electron micrograph of culture code 18 (size= 3.061.25 m
magnification 10 K)


Plate 7. Scanning electron micrograph of culture code 18 (size=3.06 m 1.25
m magnification 15 K)





51


Plate 8. Scanning electron micrograph of Yeast culture code 29(size=61.8 m
magnification 10 K)



Plate 9. Scanning electron micrograph of Yeast culture code 29(size= 5.2 1.9m
magnification 10 K)





52


Plate 10. Scanning electron micrograph of Yeast culture code 30
(size= 3.12 0.3 m magnification 10 K)



Plate 11. Scanning electron micrograph of Yeast culture code 30
(size= 2.92 0.78 m magnification 20 K)





53


Plate 12. Scanning electron micrograph of Yeast culture code 37
(size= 3.87 2.25m magnification 10 K)




Plate 13. Scanning electron micrograph of Yeast culture code 40
(size= 5 2.8m magnification 10 K)




54

DNA ISOLATION FROM SELECTED CULTURES:
The yeast DNA was isolated from selected cultures , which showed potential
for aflatoxin binding . The DNA was isolated from the cultures namely 5, 16,
18,29,30 and 37. The samples were run in agarose gel electrophoresis , which was
separated according to its size. Isolated DNA was confirmed by gel electrophoresis
(0.8% ) on agarose gel . Presence of clear bands indicated the presence of clear bands
indicated the presence of yeast DNA (plate ).

Band separation for isolated yeast DNA in agarose gel



Plate 14. Agarose gel electrophoresis , Lane1:5, Lane2: 16, Lane 3: 18, Lane 4:
29 , Lane5:30, Lane 6:37.

PCR ANALYSIS OF 18s rRNA GENE OF YEAST:
The 18s rRNA gene analysis was carried out for the amplification of the
isolates specific to the primers. This was carried out using the BSF and BSR primers .
The approximate size of amplified rDNA of isolates varied considerably. Isolated
DNA of yeast ran for PCR under condition, as explained in materials and methods.
Completion of PCR was confirmed by running the product in gel electrophoresis
(1.2% agarose). Appearance of clear band indicate the completion of reaction (plate ).
Bands obtained were compared with marker with approximate size and
generated PCR product of about 1.4 kb. PCR product was purified and sent for
sequence analysis.


1 2 3 4 5 6


55

PCR analysis of 18s rRNA gene of yeast



Plate 15. Agarose gel electrophoresis, Lane 1: 5, Lane 2: 16, Lane 3: 18, Lane 4:
29, Lane 5: 30, Lane 6: 37

RESTRICTION DIGETION:

Mixed restriction digestion:
Restriction fragmentation was performed using the enzymes Alu 1and Hae
111 (plate ). They are used both as separately and simultaneously. Results indicate
that the Yeast of culture code 16 and 30 had similar restriction pattern, hence
concluded to be the same species. Further sequencing of culture 30 was performed.

Restriction pattern of Hae 111 and Alu 1 from the yeast cultures



Plate 16. Agarose gel electrophoresis, Lane 1: 5, Lane 2: 16, Lane 3: 18, Lane 4:
29, Lane 5: 30, Lane 6: 37
M 1 2 3 5 6
1 2 3 4 5 6


56

Restriction pattern of Hae 111 of 18s rRNA



Plate 17. Agarose gel electrophoresis, Lane 1: 5, Lane 2: 16, Lane 3: 18, Lane 4:
29, Lane 5: 30, Lane 6: 37


Restriction pattern of Alu 1 of 18s rRNA



Plate 18. Agarose gel electrophoresis, Lane 1: 85, Lane 2: 96, Lane 3: 98, Lane 4:
109, Lane 5: 110, Lane 6: 117

SEQUENCING RESULTS:
The nucleotide sequence analyzed by BLAST searches performed with the
nucleotide option at the National Center for Biotechnology Information (NCBI) Gene
Bank Data Library . the gene sequence reported are deposited in gene bank.

M 1 2 3 4 5 6
1 2 3 4 5 M 6


57

PHYLOGENETIC TREE
Phylogenetic trees for all the cultures were constructed and shown below.
The sequence had a total 606 nucleotides. Which, when analyzed with the
nucleotide sequence analyzer. Sequence data of culture number 5 indicated a 97%
similarity with Pichia anomala. The sequence got is as shown below

CACTCAGGGCATTAGATCATTACGCCAGCATCCTAGTCAAAAGACGCAGCCCTCGATCC
AGACAGGCAATATCAGCAGAAGCTATAACACTCCACCGAAGTGAAGCCACATTCAACTG
CCATTATCTTGCCATCCGAATCGATGCTGGCCCAGTGAAATACGAGTGCACAACTCAAG
AAGAGAAGATAATCGTAAAACACCAAGTCTGATCTAATGCCCTTCCCTTTCAACAATTTCA
CGTACTTTTTCACTCTCTTTTCAAAGTTCTTTTCATCTTTCCATCACTGTACTTGTTCGCTA
TCGGTCTCTCGCCAATATTTAGCTTTAGATGGAATTTACCACCCACTTAGAGCTGCATTC
CCAAACAACTCGACTCTTCGATAGCACCTTACATAGGAATGGGCATCTCATCAGACGGG
ATTCTCACCCTCTATGACGTCCTGTTCCAAGGAACATAGACAAGAGCCAAACCCAAGGTT
ACCATCTTCAAATTACAACTCAAACACCGAAGGTGCTAGATTTCAAATTTGAGCTTTTGCC
GCTTCACTCGCCGTTACTGAGGCAATCCCTGTTGGTTTCTTTTCTCGTTAATATGTATATA
GCAAA

BG_85b-CFTRI.AA.9.RP_2008-10-07_003.seq Pichia anomala

Fig 5 (a). phylogenetic tree of culture number 5



58

Similar analysis for culture number 18 indicated as 97 % similarity with
Clavispora lusitaniae and the sequence is as shown below. The sequence had a total
of 547 nucleotides.

TAATTTTCACCAGGCTTGCACCATTACGCCAGCGTCCTAGAATCGCAGGCCTCGAAAGG
GATGGAGGCGTCAACACGAGCTATAACACGCGCGCCCGAAGGTGCGCGCCACATTCTC
GAGTTCTTGTTCCTCCCCCCTTTTCGACGCTGGCCCGGTAAAACCGTGTCTGCTTGCAA
GCCCTTCCCTTTCAACAATTTCACGTGCTGTTTCACTCTCTTTTCAAAGTGCTTTTCATCT
TTCCATCACTGTACTTGTTCGCTATCGGTCTCTCGCCAATATTTAGCTTTAGATGGAATTT
ACCACCCACTTAGAGCTGCATTCCCAAACAACTCGACTCGTCGGAGCCGCGGTGCAAAG
AGTCGGCGTGCGCCATACGGGGCTCTCACCCTCCCAGGCGCCATGTTCCAATGGACTT
GGGCGCGGCCGACTCAGACCACGAAACCTTCAAATTACAATTCCCGCAGGATTTCAAAT
TTGAGCTTTTGCCGCTTCACTCGCCGTTACTGGGGCAATCCCTGTTGGTTTCTTTTCCTC
CGTTTAAATGGATATGCA

BG_83b-CFTRI.AA.7.RP_2008-10-07_016.seq Clavispora lusitaniae




Fig 5 (b). Phylogenetic tree of culture number 18





59

Culture number 37 was identified as Candida tropicalis with a sequence of 586
nucleotides. It indicated 96% similarity.

TCACCAGTCTTGGATCATTATGCCAGCATCCTAGGTATCACCGGAGGCATCAGTCGGGC
GGGTTGGTTCAGACGAGGGCTAGGCTACACCACCGGGACCGTGCCACTTCCCAACGCC
CTTCTCCTGCCGCCCAAACTGATGCTGGCCCGATAAACTGTGTAGGCCACCCCCGAAGA
AGTAACATACAAAATACCCCCTCTGATCTCAAGCCCTTCCTTTTCTTCAATTTCTCGTACT
TTTTCTCTCTCTTTTCAAAGTTCTTTTCATCTTTCCATCACTGTACTTGTTCGCTATCGGTC
TCTCGCCAATATTTAGCTTTAGATGGAATTTACCACCCACTTAGAGCTGCATTCCCAAACA
ACTCGACTCTTCGAAGGAACTTTACATAGGCCTGGATCATCTCATCGCACGGGATTCTCA
CCCTCTGTGACGTTCTGTTCCAAGAAACATAGACAAGAGCCAGACCCAAAGATACCTTCT
TCAAATTACAACTCGGACTCTGAAAGAGCCAGATTTCAAATTTGAGCTTTTGCCGCTTCA
CTCGCCGCTACTAAGGCAAT
CCCTGTTGGTTTCTTTTCCTCAGTTTATTTGAAAAGCCAAAAAA

BG_81b-CFTRI.AA.5.RP_2008-10-07_012.seq Candida tropicalis



Fig 5 (c). Phylogenetic tree of culture number 37







60













SUMMARY AND CONCLUSION



















61

SUMMERY
The work was undertaken to study the aflatoxin binding capacity of yeast.
Aspergillus flavus culture from laboratory culture collection was used to infect
groundnuts to produce aflatoxin. The extracted toxin was used for aflatoxin binding
test.
Cultures drawn from the yeast culture collection of the laboratory were treated
with aflatoxin. The binding property was confirmed by subjecting the pellet and
supernatant samples to Thin Layer Chromatographic analysis. By observing the
intensity of fluorescent spots under UV at 360nm, the yeast isolates were screened
which were having potential for aflatoxin binding. Based on the efficiency of binding,
five cultures were selected for further studies. Culture number 5, 16, 18, 29, 30, 37
and 40 were the cultures selected for further study. Of the cultures selected, culture
number 47 did not posses considerable binding ability, hence was delimited from
further evaluations. Cultures were treated with aflatoxin and incubated at different
intervals of time and subjected to HPLC, compared with standards. Pellet and
supernatant of all the treated samples at time intervals of 0, 2 and 4 hours were
separately analyzed and recorded. The concentration of B1 at 0 hour of incubation
was high in the pellet for samples 5, 16 and 29. However, in the supernatents of 16
and 18, the concentration of B1 was quite high. After 2 hours of incubation, there was
change in the binding capacities of G2 was observed in all the cultures tested, which
was absent in culture number 16 and 29 at 0 hour. By 4 hours, all the toxins were
drawn to culture 5, 16, 18, 29,30 and 37. Where as in 29, G1 and G2 were absent. G1
was absent in both culture and supernatant at 2 hours of incubation, which needs
further investigation.
Further the results were analyzed using mass spectra, where simultaneous
determination of aflatoxin B1, B2, G1 and G2 was carried out. The distribution of B1
and B2 were more in pellet, indicating the efficiency of adhesion by the yeast cells.
The isolated cultures were subjected to Scanning Electron Microscopy to
study the morphology and their approximate size was also determined.
The yeast DNA was isolated from selected cultures which showed potential
for aflatoxin binding.The DNA was isolated from the culture number 5, 16, 18, 29,
30 and 37. Further the 18s rRNA gene analysis was carried for the amplification of
the isolates specific to the primers by performing polymerase chain reaction.


62

Restriction fragmentation was performed using the enzymes Alu 1 and Hae 111.
Results indicate that the Yeast of culture code 16 and 30 had similar restriction
pattern, hence concluded to be the same species. By sequence analysis, culture
number 5 indicated 97% similarity with Pichia anomala, culture number 18 showed
97% similarity with Clavispora lusitaniae and culture number 37 indicated 96%
similarity with Candida tropicalis. Phylogenetic trees for all the cultures were
constructed. Analysis of other cultures were awaited.





























63


CONCLUSION

Of the total 40 yeast isolates, 5 cultures had the potential for aflatoxin binding.
Isolates like Pichia anomala, Clavispora lusitaniae and Candida tropicalis are
common resident organisms of most of fermented foods. Their ability to bind
aflatoxin indicates its utility in decontamination of cereals and pulses during
fermentation. Earlier such work was done only with Saccharomyces cerevisiae and
this is the first report of its kind where other yeasts have been observed for their
aflatoxin binding capacity. This we have shown will open up a new field in food
fermentation and aflatoxin management.









64














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