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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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