binding property of aflatoxin by wild-thesis

1

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

2

Dr. ANU APPAIAH. K.A, M.Sc., Ph.D. Scientist, Food Microbiology Department CFTRI, Mysore – 570 020 E-mail:- [email protected]

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 Microbiology, CFTRI, Mysore -570020.

3

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

4

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

5

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

6

Dedicated To

My Beloved Parents

7

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

8

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

9

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

10

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= 4.8×2.18 µm

magnification Χ10 K

Plate 5: Scanning electron micrograph of Yeast culture code 16 (size= 2.24×1.8

µm magnification Χ20 K)

Plate 6: Scanning electron micrograph of culture code 18 (size= 3.06×1.25 µm

magnification Χ10 K)

Plate 7: Scanning electron micrograph of culture code 18 (size=3.06 µm× 1.25

µm magnification Χ 15 K)

Plate 8: Scanning electron micrograph of Yeast culture code 29(size=6×1.8 µm

magnification Χ 10 K)

Plate 9: Scanning electron micrograph of Yeast culture code 29(size= 5.2× 1.9µm

magnification × 10 K)

Plate 10: Scanning electron micrograph of Yeast culture code 30 (size= 3.12 × 0.3 µm magnification Χ 10 K)

Plate 11: Scanning electron micrograph of Yeast culture code 30 (size= 2.92 × 0.78 µm magnification × 20 K)

Plate 12: Scanning electron micrograph of Yeast culture code 37 (size= 3.87 × 2.25µm magnification × 10 K)

Plate 13: Scanning electron micrograph of Yeast culture code 40 (size= 5 × 2.8µm 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

14

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.

15

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

16

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 C17H12O6 312 268-269 21,800 425

B2 C17H14O6 314 286-289 23,400 425

G1 C17H12O7 328 244-246 16,100 450

G2 C17H14O7 330 247-240 21,000 450

M1 C17H12O7 328 299 19,000 425

M2 C17H14O7 330 293 - -

B2a C17H14O7 330 240 20,400 -

G2a C17H14O8 346 190 18,000 -

R0 C17H16O6 314 230-234 14,100 425

B3 C16H14O6 302 233-234 9,700 -

GM1 C14H12O8 344 276 12000 -

P1 C16H10O6 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

microorganism’s yielded aflatoxicol(AFL). Animals also metabolize AFB1 with

ammonia produced AFD1 and AFD2.

17

Fig. 1: Structures of aflatoxins B1, B2, G1 and G2

Fig. 2: Structures of aflatoxins M1, M2, B2A and G2a

.

18

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 249th 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.

19

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 3-4µm in diameter,

although some yeast can reach over 40µm (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.

20

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 24th 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).

21

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.

22

MATERIALS AND METHODS

23

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 Cº 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 30º 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 100µl of Chloroform

24

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)

25

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 100µL

1xPBS buffer 100µl

Culture suspension 100µl

Distilled water 100µl

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 1000µl 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

26

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 (300µm thick). The plates were allowed to dry

for 1-2 hours at room temperature. Plates were activated for 1 hour at 100 ºC 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 Na2HPO4, 2Mm KH2PO4. 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 108 cells

(OD <2)

27

• 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

• 5µl 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 50µl of methanol

• 20µl 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 80º

C evaporation temperature 180ºC

• 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 400µl

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 30ºC

• 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, 200µl yeast lysis buffer and 20µl of proteinase k were

added

• The above mixture was kept in water bath at 55 ºC 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 (250µl)

• To the above solution , approximately 750µl of ice cold isopropyl alcohol in a

ratio 1:3 was added

• 20µl 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 500µl of 70% ice cold ethanol

• The pellet was air dried and resuspended in 20µl 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 (1µl/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 2µl of marker dye (bromophenol blue)

(5µl of each sample in a well)

• Then the samples were electrophoresis at 50v for 1 to 1 ½ hour

• The gel was stained with ethydium bromide (1µl/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(5¹to 3¹ direction) Expected size of the amplicon

BSF 5 ◌ٰ GCATATCAATAAGCGGAGGAAAAG 3 ◌ٰ

BSR 5¹ GGTCCGTGTTTCAAGACGG 3¹

Taq DNA polymerase (Bangalore Genei , India)

10x reaction buffer

Ingredients Amount

Tris buffer 1 00mM

KCl 500mM

MgCl2 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(µl) 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 5µl aliquote of the amplified PCR product was taken , mixed with 2µl 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 ¾ 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 1µl

Alu I

Hae

Buffer 2µl (10x assay buffer)

Template (DNA or PCR product) 7 or 5µl

MQ water 12 µl

Agarose gel 2%

Table 5. Restriction digestion reaction mixture

Amount Volume (µl)

AluI 1

Hae 1

Buffer 2

PCR product 5

Water 12

Total volume 25

34

Methodology: • For restriction digestion, 1µl of enzyme either Alu I or Hae III was used

• To that 2 µl of 10x assay buffer was added with 5µl of template

• For mixture of AluI and Hae III , 0.5 µl of each enzyme with 2 µl of buffer

and template were used and 15 µl of sterile water is added

• The mixture was kept in water bath at 37˚C 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 50µl

Mini preparation spin column

wash solution 700µl

Elution buffer 30µl

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 µl of PCR binding solution to 100µl 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 5µl

of elution buffer to the center of the spin column and centrifuge for 1 minute

at 13000 rpm

Alternatively for increased DNA concentration , 30 µl 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 -20ºC 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 32°C 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)



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

Minutes0 2 4 6 8 10

Vol

ts

0.00

0.01

0.02

0.03

0.62

51.

100

1.55

81.

967

2.47

52.

775

7.52

5

9.04

2

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

Minutes0 2 4 6 8 10

Vol

ts

0.000

0.005

0.010

0.60

00.

958

1.51

71.

950

2.42

52.

642

2.78

3

7.46

7

8.96

7

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

Minutes0 2 4 6 8 10

Vol

ts

0.00

0.02

0.04

0.70

0 1.15

01.

692

2.02

5

3.76

7

6.22

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-200814:44:01STD

m/z305 310 315 320 325 330 335 340 345

%

0

10029070809 4 (0.217) Cm (2:12) TOF MS ES+

586311.36

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-200815:51:40117 P4

m/z300 305 310 315 320 325 330 335 340 345 350

%

0

10030070807 6 (0.321) Cm (6:10) TOF MS ES+

515316.26311.35

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-200816:41:19117 S 4

m/z300 305 310 315 320 325 330 335 340 345 350

%

0

10030070827 8 (0.423) Cm (7:10) TOF MS ES+

248316.31

311.33

303.19 309.28313.32

319.15

340.38

335.31325.35

321.32327.26 333.32

329.29

337.31349.28

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

30-Jul-200816:13:19CCS

m/z300 305 310 315 320 325 330 335 340 345 350

%

0

10030070816 91 (4.773) Cm (91:95) TOF MS ES+

460319.13

311.35

303.16

309.36307.25

315.21

343.24324.40 335.30 339.31 346.82

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 µm and 3.1×0.3 µm 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 6×1.8 µm. Other cells

with an approximate size of 4.8×2.18 µm, 3.06×1.25 µm, 3.87×2.25 µm

and 5×2.8 µm respectively in case of culture number 5, 18, 37 and 40.

Plate 4. Scanning electron micrograph of culture code 5 (size= 4.8×2.18 µm

magnification Χ10 K )

Plate 5. Scanning electron micrograph of Yeast culture code 16 (size= 2.24×1.8

µm magnification Χ20 K)

50

Plate 6. Scanning electron micrograph of culture code 18 (size= 3.06×1.25 µm

magnification Χ10 K)

Plate 7. Scanning electron micrograph of culture code 18 (size=3.06 µm× 1.25

µm magnification Χ 15 K)

51

Plate 8. Scanning electron micrograph of Yeast culture code 29(size=6×1.8 µm

magnification Χ 10 K)

Plate 9. Scanning electron micrograph of Yeast culture code 29(size= 5.2× 1.9µm

magnification × 10 K)

52

Plate 10. Scanning electron micrograph of Yeast culture code 30

(size= 3.12 × 0.3 µm magnification Χ 10 K)


(size= 2.92 × 0.78 µm magnification × 20 K)

53


(size= 3.87 × 2.25µm magnification × 10 K)


(size= 5 × 2.8µm 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


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


29, Lane 5: 30, Lane 6: 37

Restriction pattern of Alu 1 of 18s rRNA


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

CACTCAGGGCATTAGATCATTACGCCAGCATCCTAGTCAAAAGACGCAGCCCTCGATCCAGACAGGCAATATCAGCAGAAGCTATAACACTCCACCGAAGTGAAGCCACATTCAACTGCCATTATCTTGCCATCCGAATCGATGCTGGCCCAGTGAAATACGAGTGCACAACTCAAGAAGAGAAGATAATCGTAAAACACCAAGTCTGATCTAATGCCCTTCCCTTTCAACAATTTCACGTACTTTTTCACTCTCTTTTCAAAGTTCTTTTCATCTTTCCATCACTGTACTTGTTCGCTATCGGTCTCTCGCCAATATTTAGCTTTAGATGGAATTTACCACCCACTTAGAGCTGCATTCCCAAACAACTCGACTCTTCGATAGCACCTTACATAGGAATGGGCATCTCATCAGACGGGATTCTCACCCTCTATGACGTCCTGTTCCAAGGAACATAGACAAGAGCCAAACCCAAGGTTACCATCTTCAAATTACAACTCAAACACCGAAGGTGCTAGATTTCAAATTTGAGCTTTTGCCGCTTCACTCGCCGTTACTGAGGCAATCCCTGTTGGTTTCTTTTCTCGTTAATATGTATATAGCAAA

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.

TAATTTTCACCAGGCTTGCACCATTACGCCAGCGTCCTAGAATCGCAGGCCTCGAAAGGGATGGAGGCGTCAACACGAGCTATAACACGCGCGCCCGAAGGTGCGCGCCACATTCTCGAGTTCTTGTTCCTCCCCCCTTTTCGACGCTGGCCCGGTAAAACCGTGTCTGCTTGCAAGCCCTTCCCTTTCAACAATTTCACGTGCTGTTTCACTCTCTTTTCAAAGTGCTTTTCATCTTTCCATCACTGTACTTGTTCGCTATCGGTCTCTCGCCAATATTTAGCTTTAGATGGAATTTACCACCCACTTAGAGCTGCATTCCCAAACAACTCGACTCGTCGGAGCCGCGGTGCAAAGAGTCGGCGTGCGCCATACGGGGCTCTCACCCTCCCAGGCGCCATGTTCCAATGGACTTGGGCGCGGCCGACTCAGACCACGAAACCTTCAAATTACAATTCCCGCAGGATTTCAAATTTGAGCTTTTGCCGCTTCACTCGCCGTTACTGGGGCAATCCCTGTTGGTTTCTTTTCCTCCGTTTAAATGGATATGCA

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.

TCACCAGTCTTGGATCATTATGCCAGCATCCTAGGTATCACCGGAGGCATCAGTCGGGCGGGTTGGTTCAGACGAGGGCTAGGCTACACCACCGGGACCGTGCCACTTCCCAACGCCCTTCTCCTGCCGCCCAAACTGATGCTGGCCCGATAAACTGTGTAGGCCACCCCCGAAGAAGTAACATACAAAATACCCCCTCTGATCTCAAGCCCTTCCTTTTCTTCAATTTCTCGTACTTTTTCTCTCTCTTTTCAAAGTTCTTTTCATCTTTCCATCACTGTACTTGTTCGCTATCGGTCTCTCGCCAATATTTAGCTTTAGATGGAATTTACCACCCACTTAGAGCTGCATTCCCAAACAACTCGACTCTTCGAAGGAACTTTACATAGGCCTGGATCATCTCATCGCACGGGATTCTCACCCTCTGTGACGTTCTGTTCCAAGAAACATAGACAAGAGCCAGACCCAAAGATACCTTCTTCAAATTACAACTCGGACTCTGAAAGAGCCAGATTTCAAATTTGAGCTTTTGCCGCTTCACTCGCCGCTACTAAGGCAAT 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

REFERENCES

65

REFERENCES

• A.O A.C. (1975 ). Natural poisons in official methods of analysis. 12th ed. 209-

269.

• Bandyopadhyay, U., Biswas, K., Chatterjee, R., Ganguly, C.K., Chakraborty, T.,

Bhattacharya, K., Banerjee, Rok. (2002). Gastroprotective effect of neem bark

extract . Life Sciences. 71, 2845-2865.

• Barnett, J.A. (1992). Yeast. 8, 1.

• Bass, D., Howe, A., Brown, N., Barton, H., Demidova, M., Michelle, H., Li.,

Sanders., Watkinson, S.C., Willcock, S., Richards, T.A. (2007). Yeast forms

dominate fungal diversity in the deep oceans. Proc Biol Sci. 962, 10-16.

• Bassappa, S.C., Shantha, T. (1996). Methods of detoxification of Aflatoxin in

foods and feeds, A critical appraisal. J. food Sci Technol. 33, 95-107.

• Bejaoui, H., Mathieu, F., Taillandier, P. Lebrihi, A.( 2004). Ochratoxin A removal

in synthetic and natural grape juices by selected ocnological Saccharomyces

strains.Journal of Appl. Microbiol. 97, 1038-1044.

• Bhunia, A.K., Jhonson, M.C., Ray, B. (1987). Direct detection of an antimicrobial

peptide of pediococcus in sodium dodecyl sulphate poly acrelamide gel

electrophoresis,. Indian J Microbiol. 2, 319-322.

• Cryer, D.R., Eccleshall, R., Marmuri, J.(1975) . Isolation of yeast DNA. Methods

in Cell Biology. 12, 39- 44.

• Davis, N.D., Dickens, J.W., Freie, R.L., Hamilton, P.B., Shotwell, O.L, Wyllie,

T.D., Fulkerson, J.F. (1980). Mycotoxins : protocols for surveys, sampling, Post

collection handling and analysis of grain samples involved in mycotoxin

problems. J. A. O . A. C. 6189, 95-102.

• Davis, N.D., Diener, U.L., Eldridge, D.W. (1966). Production of aflatoxins B1

and G1 by Aspergillus flavus in a semisynthetic medium. Appl. Microbiol. 14,

378-380.

66

• Ehrlich, K.C, Montalbano, B.G., Cotty, P.J. (2003). Sequence comparision of

Aflatoxin from different Aspergillus species provides evidence for variability in

regulation of aflatoxin production. Fungal Genetics and Biology. 38, 63-74.

• Environmental health criteria 11. Mycotoxins WHO. (1979).

• Haskard, C.A., Elnezami, H.S., Kankaanpaa, P.E., Salminen, S., Ahokas, J.T.

(2001). Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria. Appl.

Environmental Microbiol. 7, 3086-3091.

• Hoffman., Winston. (1987). Gene. 57, 267-272.

• Jiang, Y., Jolly, P.E ., Ellis, W.O., Wang, I.S., Phillips, T.D., Whilhams, J.H.

(2005). Aflatoxin B, Albumin adduct levels and cellular immunostatus in Ghana ,

Ians. Int. Immunol. 17, 807-814.

• Klis, F.M,. (1994). Review: Cell wall assembly in yeast. Yeast 10, 851-869.

• Kulwant Singh., Jens, C. Frisvad,. Ulf Throm., Mathur, S.B. (1991). An illustrated

manual on Identification of some seed borne Aspergilli, Fusaria, Pencilia and

their mycotoxins. 1st ed. P-38-39.

• Kurtzman, C.P., Fell, J.W. (2006). Yeast systematic and phylogeny, Implications

of molecular identification methods for studies in ecology. Springer Verlag Berlin

Herdelberg. 11-30.

• Kurtzman, C.P., Piskur, J. (2006). Taxonomy and Phylogenetic diversity among

the yeasts. Comparative genomics. Springer Verlag Berlin Herdelberg. 15, 29-46.

• Lewis, L. Onsongo M., Niapau, H. Schurz Rogers H., Luber, G., Kieszak, S.,

Nyanongo, J., Backer, L, Dahiye, A.M., Misore, A. (2005). Aflatoxin

contamination of commercial maize products during an outbreak of accute

aflatoxicosis in eastern and central Kenya . Environmental Perspectives . 113,

1763-1767.

• Lewis, S.J,. (2004). Binding of aflatoxin B1 to cell wall components of

Lactobacillus rhamnosus strain GG . Food Additives and Contaminants. 21, 158-

164

67

• Line, J.E., Brackett, R.E. (1995). Role of toxin concentration and second carbon

source in microbial transformation of Aflatoxin B1 by Flavobacterium

aurantiacum. Alimantary Pharmacology and Theraputics.12, 807-822.

• Mann , R. (1977). Degradation of aflatoxin B1 by yeasts , molds and Bacteria .

Zeitschrift for Lebensmittel Forschung.163, 39-43.

• Nout, M.J.R., Motarjemi ,Y. (1997). Assesment of fermentation as a household

technology for improving food safety : A joint FAO/WHO workshop . Food

Control. 8, 221-226.

• Odhav, B., Naicker,V. (2002). Mycotoxins in south African traditionally brewed

beers. Food Additives and Contaminants. 19, 55-61.

• Orrihage, K.E., Sillerstrome, E. Gustafsson., J.A. Nord, C.E., Rafter, J. (1994).

Binding of mutagenic heterocyclic amines by intestinal lactic acid bacteria.

Mutation Research. 311, 239-248.

• Pons, A., Walter., Louise, S., Lee., Leonard stoloff. (1980). Revised method for

Aflatoxins in Cottonseed Products and Comparision of Thin Layer and High

performance Liquid Chromatography Determinative Steps : Collaborative Study .,

J. chemistry. 63, 4.

• Sambrook, J., Russel, D.W. (2001). Molecular cloning, A laboratory manual. 3rd

ed. Cold spring Harbour NY. 1125-1130.

• Shetty, H P., Jespersen, L.( 2006). Saccharomyces cerevisiae and lactic acid

bacteria as potential mycotoxin decontaminating agents ., Trends in food Sci

technol. 17, 48-55.

• Shetty, H.P., Hald Benedicte., Jespersen Lene. (2007). Surface binding of

aflatoxin B1 by Saccharomyces cerevsiae strains with potential decontaminating

abilities in indigenous fermented foods. J. food microbiol. 113, 41-46.

• Shih, C.N., Marth, E.H. (1973). Some cultural conditions that control Biosynthesis

of lipid and Aflatoxin by Aspergillus parasiticus .Appl. microbiol. 27 , 452-456.

• Shih, C.N., Marth, E.H. (1973). Some cyltural conditions that control Biosynthesis

of Lipid and flatoxin by Aspergillus parasiticus. Appl. Microbiol. 27, 452-456.

68

• Stanly, V.G., Ojo, R.,Woldensenbet, S., Hutchinson, D.H. (1993). The use of

Saccharomyces cerevisiae to suppress the effects of aflatoxins in broiler chicks.

Poultry Sci. 72, 1867-1872.

• Ventura, M., Gomez, A., Anaya, I., Diaz, J., Broto, F., Agut, M., Comellas, L.

(2004). Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by

liquid chromatography- tandem mass spectrometry. J. of Chromatography. 1048,

25-29.

• Walker, K., Skelton H., Smith,K. (2002). Cutaneous lesions showing giant yeast

forms of Blastomyces dermatitidis. J. Cutan Pathol. 29, 616-618.

• Walker, R. (2003). Ochratoxin A in coffee, A Risk assessment. J. Coffee Res. 20,

150-153.

• Wei, Xiao. (1984). Methods in molecular biology , Yeast protocols. 2nd ed.

Human press publishers. 4-19.

• Whitaker, T.B., Dickens, J.W. (1980). Water slurry method of extracting aflatoxin

from peanuts. JAOCS. 6189, 54.

• Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V. (1990).

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.

Nucleic Acids Res. 18, 6531-6535.

• Williams, J.H., Phillips, T.D., Jolly, C.M., Agarwal, D.(2004). Human

Aflatoxicosis in developing countries: A review of toxicology, exposure, potential

health consequences and interventions. American J. Clinical Nutrition. 80, 1106-

1122.

• Williams, N. (1996). Genome Projects: Yeast genome sequence . Science. 481,

272-262.

• Wilson, J.P., Junevic, Z., Hanna, W.W., Wilson, D.M., Potter, T.L., Coy, A.I .

(2006). Host specific variation in infection by mycotoxins in pearl millet and corn

Mycopathologia. 161, 101-107.

binding property of aflatoxin by wild-thesis

Documents