Indian Journal of Experimental Bi ology Vol. 39 , July 2001 , pp . 673 -677 Mechanism of removal of aflatoxin B 1 by a resistant bacterium S Vaithiyanathan * & D P Mishra Department of Biochemistry and G K Garg Department of Molecular Biology & Genetic Engineering, Coll ege of Basic Scienc es & Hum aniti es , G. B.P.U.A.T., Pantnaga r 263 145. India Received 20 September 2000; revised 28 March 2001 A bacterium resistant to aflatoxin B I iso lated from the so il was s hown to remove a maximum 26 % of toxin at 3 IJ.g mr 1 • A compariso n of spectro photometric and radioisotope meth ods s howed that a max imum of 48 hr was sufficient to remove the toxin. Radioiso to pe analysis showed that the radioactiv it y d ecreased in the chl oro for m ph ase w hil e it increased in the aqueous phase during the time course of the experiment. An ana ly sis of the s upe rn atant of culture medium showed that the bacterium had converted aflatoxin B I to wa ter soluble compounds with A 111 ,. of 338 and 374. 'J atural toxicant s occurring in hum an foods and mim al feeds prese nt a potential health hazard. Most )Otent of these are th e aflatoxins produced by 'ilamentous fungi . They are hi ghly stable and cannot )e completel y destroyed by any method s. )etoxifi ca tion of aflatoxins by biological means is eceiving much att ention in recent times 1 . The work m the degradation of atlatoxins by Flavobacterium rurantiacunl received much attention . It wa s uggested th at aflatoxin B 1 (AFB 1) first adsorb on to he cell walls and su bsequently modified by a process ::ading to non-extrac table forms of toxins 3 . Later it v as demonstrated that F. aurantiacum metabolized 1 C-AFB 1 into water soluble products and C02 4 . ' hey suggested th at the mecha ni sm of removal of might be through minera li zation phenomenon. :urther it has been reported that a few bacteria were !so capable of transforming AFB 1 to other ompounds which may be le ss ac tive than the parent om pound 1 . Since scanty information is availab le on 1e bacterial degradation of AFB 1, we thought of :udying the removal of AFB I by a resistant acterium. In the present study, experiments have een carried out to st udy the removal of AFB 1 by eg radation or transformation by a resistant bacterium alated from the soils. 'resent address: Animal Nutriti on Division, Central Sheep & 'ool Research In stitute, Avikanagar 304 50 I, In dia ; nail: svaith @cswri .ra j.nic . in and Fax No. +91-01437-2 8!63 Materials and Methods Aspergillus parasiticus SRRC 148 was obtained as a gift from Commodity Safety Re search, USDA , New Orl eans, LA , USA for the isolation of AFB 1 to be use d in the following experiments. All the cultures work we re performed in the autoclaved medium (121 °C and 15 psi for 15 min) . AFB 1 re sistant bacterium Bacillus spp was reported earliers had a high re sistance le ve l. It was routinely str eaked with heat activated spores on nutrient agar once in every month. AFB 1 was also purchased from Sigma Chemical Co. and a ll other chemicals were of analytical grade. Unlabeled AFB 1 and 14 C-labeled AFB 1 were isolated 6 · 7 and purified 8 and its quantity determin ed 9 . Purified AFB 1 was routinely checked on TLC. Isolation and purification unlabeled AFBJI- A. parasiticus was routinely streaked on Potato dextrose agar slant s at monthly intervals to hav e fresh stock of cu lture. A spore suspension was prepared by adding 5 ml sterile water containing a drop of (0.1 %) Tween 80 to 1 -3 week old slant and shaking it vigorously. This spo re suspension was used for inoculation. Isolation of AFB 1 wa s as described in the previous papers. Isolation and purification of 14 C-labeled AFBJ- Acetate-I-1 4 C: Sodium acetate- 1- 14 C (Specific activity 46.15 mCi/mmole) was purchased from BARC, Bombay. For the production of 14 C-AFB I, the activity was diluted with unlabeled sodium acetate and used in the replacement medium.
Transcript
Indian Journal of Experimental Biology Vol. 39, July 2001 , pp. 673-677
Mechanism of removal of aflatoxin B 1 by a resistant bacterium
S Vaithiyanathan* & D P Mishra Department of Biochemistry
and
G K Garg Departme nt of Molecu lar Biology & Genetic Engineering, College of Bas ic Sciences & Humanities, G.B.P.U.A.T.,
Pantnagar 263 145. Indi a
Received 20 September 2000; revised 28 March 2001
A bacterium resistant to aflatoxin B I isolated from the soil was shown to remove a maximum 26% o f toxin at 3 IJ.g mr 1
• A comparison of spectrophotometric and radioisotope methods showed that a maximum of 48 hr was suffic ient to remove the toxin . Radioisotope ana lys is showed that the radioact ivity decreased in the chloroform phase whil e it increased in the aqueous phase during the time course of the experiment. An analysis of the supernatant of culture medium showed that the bacterium had converted aflatoxin B I to water soluble compounds with A111,. of 338 and 374.
'Jatural toxicants occurring in human foods and mimal feeds present a potential health hazard. Most )Otent of these are the aflatoxins produced by 'ilamentous fungi . They are highly stable and cannot )e completely destroyed by any methods. )etoxification of aflatoxins by biological means is eceiving much attention in recent times 1. The work m the degradation of atlatoxins by Flavobacterium rurantiacunl received much attention . It was uggested that aflatoxin B 1 (AFB 1) first adsorb on to he cell walls and subsequently modified by a process ::ading to non-extractable forms of toxins3
. Later it vas demonstrated that F. aurantiacum metabolized 1C-AFB 1 into water soluble products and C024 . 'hey suggested that the mechani sm of removal of ~FBI might be through mineralization phenomenon. :urther it has been reported that a few bacteria were !so capable of transforming AFB 1 to other ompounds which may be less active than the parent ompound 1. Since scanty information is avai lable on 1e bacterial degradation of AFB 1, we thought of :udying the removal of AFB I by a resistant acterium. In the present study, experiments have een carried out to study the removal of AFB 1 by egradation or transformation by a resistant bacterium alated from the soils.
'resent address: Animal Nutrition Di vision, Central Sheep & 'ool Research Inst itute, Avikanagar 304 50 I , India; nail: svaith @cswri .ra j.nic .in and Fax No. +91-01437-28!63
Materials and Methods Aspergillus parasiticus SRRC 148 was obtained as
a gift from Commodity Safety Research, USDA, New Orleans, LA, USA for the isolation of AFB 1 to be used in the following experiments. All the cultures work were performed in the autoclaved medium (121 °C and 15 psi for 15 min) .
AFB 1 resistant bacterium Bacillus spp was reported earliers had a high resistance leve l. It was routinely streaked with heat activated spores on nutrient agar once in every month.
AFB 1 was also purchased from Sigma Chemical Co. and all other chemicals were of analytical grade. Unlabeled AFB 1 and 14C-labeled AFB 1 were isolated6
·7 and purified8 and its quantity determined9
.
Purified AFB 1 was routinely checked on TLC. Isolation and purification unlabeled AFBJI
A. parasiticus was routinely streaked on Potato dextrose agar slants at monthly intervals to have fresh stock of cu lture. A spore suspension was prepared by adding 5 ml sterile water containing a drop of (0.1 %) Tween 80 to 1-3 week old slant and shaking it vigorously. This spore suspension was used for inoculation. Isolation of AFB 1 was as described in the previous papers.
Isolation and purification of 14C-labeled AFBJAcetate-I-14C: Sodium acetate- 1-14C (Specific activity 46.15 mCi/mmole) was purchased from BARC, Bombay. For the production of 14C-AFB I, the activity was diluted with unlabeled sodium acetate and used in the replacement medium.
674 INDIAN J EXP BIOL, JULY 200 1
For isolation of 14C-Iabeled AFB 1, approximately 103 spores of A. parasiticus SRRC 148 were inoculated in 100 ml basal medium (contained per litre: glucose 50 g; (NH4hS04 4 g; KH2P04 l 0 g; MgS04.7H20 2g; and the tracemetals Na2B40 7.l 0 H20 0.7 mg; (NH4)6M07h4AH20 0.5 mg; FeS04 10 mg; CuS04.5H20 0.3 mg; MnS04.H20 0.11 mg; ZnS04.7H20 17.6 mg) kept at 28°C for 4 days with shaking at 100 rpm. On 4' 11 day, mycelia were collected, washed and resuspended (approximately 1 g) in 100 ml of replacement medium (contained per litre: KH2P04 5 g; MgS04.7H20 0.5 g; KCI 0.5 g and the trace metals as used in the above basal medium). The desired precursors were then added to resting cells. The respiring cells were prevented from growing by the absence of a nitrogen source. This resting culture was kept at 28°C for 40 hr wi th shaking at l 00 rpm. Isolation of AFB 1 was as described above. Radioactivity was measured with a Rack Beta Counter (Beckman, USA) using dioxane based scintillation fluid .
Aflatoxin removal by the bacterium (a) Spectrophotometric method= To determine the
removal of AFB I by the bacterium, cells (108
activated spores) were grown on 50 ml M9 medium 11
pH 7 (ingredients per litre: Na2HP04 6.0 g; KH2P04 3.0g; NaCI 0.5 g; NH4CI 1.0 g; FeS04 0.1 g containing 0.1 ml of 0.1 M CaC[z and 1.0 ml of 1 M MgS04.7H20) supplemented with 0 .06% tryptone, 0.03% yeast extract and 0.03% NaCl containing 0 to 60 p,g mr1 AFB 1 at 37°C with shaking at 100 rpm. One ml culture was centrifuged at 4000 g for 10 min after 0, 24 and 48 hr and the supernatant was analyzed spectrophotometrically for AFB 1 in UV-Vis spectrophotometer (DU-70 Beckman Inc, USA) 12.
(b) HPLC method: Cells (lOs activated spores) were grown on 25 ml M9 medium prepared as mentioned above containing 0 to 10 p,g mr1
AFB 1 (Sigma) at 37°C with shaking at 100 rpm. One ml culture was centrifuged at 4000 g for 10 min after 0, 24 and 48 hr and the supernatant was analyzed for AFB 1 by reverse phase HPLC with mic;o-Bonda Pak C-18 column (Waters, USA) 13
. The mobi le phase used was acetonitrile + water {(50 + 50 (v/v)}, flow rate was 1.5 mllmin and detection at 362 nm with AUFS 0.002. Standard curve was prepared using peak areas.
(c) Radioisotope method: Cells (lOs activated spores) were exposed to 14C-AFB1 (10 p,g mr1)
(uniformly labeled) in 10 ml M9 medium prepared as above in a specially fabricated Erlenmeyer fl ask of l 00 ml capacity with ground joint. This fl ask had a small glass well with (20 mm height and 7 mm di a.) at the center of the base and was inserted with Whatman No. 1 paper strip (5 mmx30mm) and filled with 0.5 ml 1 M KOH to absorb 14C02 released by the bacterium. Incubation was continued for 72 hr at 37°C shaking at l 00 rpm with appropriate posi ti ve and negative control s (triplicate flasks for each time interval of 12 hr) . The medium was centrifuged at 4000 g for 10 min . The cell pellet was suspended in 1 ml medium and to this 10 ml of dioxane based scintillation fluid 14 with cabosil was added to make it as a gel so that the cells were distributed evenly. Supernatant was extracted thrice with chloroform. The aqueous layer (l ml) was added to 9 ml scinti llation fluid. The chloroform fractions were combined and evaporated to dryness in a rotary film evaporator and then dissolved in 10ml scintillation fluid . Paper strip and KOH solution from the well were added into scintillation fluid . After adding the scintillation fluid , they were kept in dark for 2 hr before counting. Radioactivity was measured tn a Rackbeta counter (Beckman Inc., USA).
Transformation product of AFBJ-Cells (lOs act ivated spores) were grown in 10 ml M9 medium prepared as mentioned above containing AFB I 10 p, g mr1 at 37°C with shaking at 100 rpm for 24 hr. Medium was centrifuged as mentioned above and the supernatant was extracted thrice with chloroform. The aqueous phase was extracted thrice with methylene chloride. They were then evaporated to dryness in ·a rotary film evaporator and redissolved in methanol. They were run on TLC on chloroform + acetone (99: 1 v/v) until the solvent front reached 14 em. The total area was divided into equal upper and lower portions, scrapped off separately and eluted with methanol. They were then scanned in UV-Vis spectrophotometer. A control was run without cells.
Comparison of microorganisms-To determine the removal of AFB 1 by other bacterial species, Escherichia coli, Pseudomonas fluorescence, Bacillus sphaericus and B. thuringiensis were grown in 10 ml M9 medium prepared as above containing AFB 1 10 p,g mr1 (Sigma) at 37°C for 48 hr. Culture was centrifuged and the supernatant was analyzed for AFB 1 by spectrophotometric method as mentioned above.
The data obtained were analyzed by Student's t
test 15•
VAITHIYANATHAN eta/. : MECHANISM OF REMOVAL OF AFLATOX IN B I 675
Results The resistant bacterium was exposed to different
concentrations of AFB I and the removal of toxin by bacterium was determined by three different methods .
AFB 1 (lab purified) concentrations in the supernatants are presented in Table I. The results show that the bacterium removed 2 to 21 % of toxin and the amount of toxin present at 24 hr and 48 hr were significantly (P < 0.0 I) lower than that at 0 hr. However, between 24 and 48 hr there was no significant difference. The total amount of toxin removed ranged from 53 to 171 Jl g in 50 ml medium (Table I). Similarly the bacterium exposed to AFB I (Sigma) showed that the concentrations at 24 and 48 hr were significantly (P < 0.01) lower than that at 0 hr (Table 2) . The per cent removal ranged from 7 to 26 (Table 2) and the amount removed was 15 to 20 Jlg in 25 ml medium.
Jn order to find out the change in toxin concentration between time intervals as well as the movement of tox in to various fractions, radiotracer technique was used by employing 14C-AFB I. The amount of 14C-AFB I isolated was 4 mg and the radioactivity of the labeled toxin was 0.16 J.LCi. The bacterium was grow n in the medium containing AFB I 10 Jl g mr ' (0.08 J.LCi ) and the results are presented in Fig. I. At 0 hr more than 95% radio activity was observed in chloroform phase while the aqueous phase, cell pellets and KOH solution and Whatman
No.I paper had nearly I% of radioactivity. However, with increase in the growth of bacteria there was a significant (P < 0.01 ) decrease in radioactivity in the chloroform phase. But in other fractions there was an increase in the radioactivity. The increase in the radioactivity in KOH solution was not signi ficant throughout the time course while in aqueous phase of the medium and cell pellets the increase in activity was significant (P < 0.05) at 36 hr.
A compari son of the radioisotope method and spectrophotometric method using lab purified AFB I revealed that magnitude of removal in AFB I was maximum at 48 hr of growth (Table 3).
Transformation of AFBJ-Results presented in Table 4 showed that there was a Amax at 338 in the upper and at 378 in the lower half portion of methylene chloride fraction of the supernatant of culture grown in presence of AFB I wh ile it was absent in medium containing AFB I but without culture. The presence of these A111ax were important because of its polarity and its solubility in aqueous phase.
Comparison of microorganisms-AFB I removal by the resistant bacterium and its comparison with that of E. coli, P. fluo rescence, B. sphaericus and B. thuringiensis (Table 5) showed that after 48hr of incubation, the resistant bacterium removed significantly (P < 0.01) higher amount of toxin than the other bacterial spec ies. E. coli showed a removal
Table !- Results on changes in concentration of AFB I (lab purified) ex posed to the bacterium as measured by spectrophotometric method
Cone. Incubati on time (hr) Amount (Jl g) removed Removal (Jlg mr') Aflatoxin B I (Jl g/ml ) from 50 ml medium after 48 hr (%)
0 24 48
10 10.008±0.064" 8. 125±0.1!1 b 7. 894±0.034b 106 21 20 20.005±0.050" !6.72 ! ±0.088b !6.603±0.058b 170 17 30 29 .978±0.024" 26. 1 04±0.191 b 26.563±0.1 0 I b 171 II 40 40.007±0. 192" 36.708±0.141 b 36.7 56±0.055b 163 8 50 50.034±0.375" 47. !9 !±0. !46b 47.24 !±0. 1! 6b 140 6 60 59.986±0.070" 59.725±0.059b 58.919±0.081 b 53 2
In a row figures with d iffe rent superscripts differ significant ly P<O.O I
Table 2- Rcsu lts on changes in concent ra tion of AFB I (Sigma) ex posed to the bac terium as measured by HPLC method
In a row figures with d ifferen t superscripts d iffer significantly a, b P<O.Ol; c, d P<0.05
676 INDIAN J EXP BIOL . .JULY 2001
Table 3-Results on comparison of Radioisotope method and spectrophotometric method for changes in the concentration of
14C-anatoxi n B I and AFB I respectively exposed to the bac teri um
Time (hr)
0
12
24
36
48
60
72
Radioisotope method Cpm %
removal
9064±25 0
8077±98 10.89
7866±98 13.22
7317±188 19.27
7360±189 18.80
7440±192 17.92
7309±72 19.36
Spectrophotometric method 11glml %
removal
I 0. 156±0.063 0
8.475±0. 11 1 16.55
8.042±0.034 20.82
Table 4-Results on UV- Vis spectrum of aqueous fractions
Area \nax in Medium+ Medium+ MeOH AFBI+ AFBI
Upper Half Portion
Lower Half Portion
558 338 274
224/222 2 10/212
558 510 378 274 232 208
+ = Present, - = Absent
culture
+ + + + + + + + +
+ + +
+ + +
+
Table 5-Results on comparison of microorganisms in removi ng AFB I (Sigma) as measured by spectrophotometric method
Incubat ion time (hr) % Anatox in B I (gg/ml) remo-
Microorganisms 0 hr 48 hr ved
Isolated bacterium 9.949±0.0 18 9.292±0.083 7
Escherichia coli 9.998±0.034 9.900±0.046
Pseudomonas I 0.000±0.096 9.992±0.072 0 fluorescence
Bacillus 10.000±0.096 I 0.625±0. 154 0 thuringiensis
Bacillus 10.000±0.096 10.91 1±0.114 0 se.haericus
of 1% while the other bacteria did not show any removal.
Discussion The resistant bacterium showed the resistance
level up to 70 p,g mr' 5 and it was tested in this study for its property of either to remove or to degrade the toxin. As it was seen from three methods and the toxin purified at two different methods, it showed (Tables 1, 2 and Fig. 1) that there was removal of AFB 1 from liquid medium (21% at 10 p,g mr' and 26% at 3 p, g mr1
). The difference in the AFBl removal at 10 p,g mr' between toxin purified in the lab and the toxin from Sigma, may probably be due to some contaminants in the AFB 1 purified in the lab. The experiments were carried out at pH 7 and hence the decrease in the concentrat ion was not due to acid ity and alkalinity . However, this removal of toxin
I · · F' . ? i ? 16 was ess 111 companson to .aurant/Qcunc -· . Further the removal of toxin was not proportionate to concentration indicating this was not enzymatic process of degradation but a si mple removal phenomenon either through binding or transformation .
Radioisotope studies showed that there was significant (?<0.05) reduction in AFB 1 (Fig. 1) caused by the growth of bacterium. This reduction may be through metabolism of AFB 1 or through conversion of AFB 1 into other fractions. The significant (?<0.05) increase in the radioactivity in the aqueous phase of supernatant and in the cell pellet may suggest that toxins were transformed to water soluble products and subsequently released into medium by the cells. Moreover, the radioactivity in KOH + Whatman paper NO. 1 (representing C02
released by the cells) was not changed significantly. However, it was reported that F. aurantiacum metabolized 14C-AFB 1 and released 14C02
4• Thus it
appears from the resul ts that mechanism involved in the removal of AFB 1 by the bacteria may be through transformation of AFB l .
Fig. !-Changes in CPM of 14C-AFB I ex posed to bacterium
Y AITHIY ANA THAN et al.: MECHANISM OF REMOY AL OF AFLATOXIN B I 677
Biotransformation plays an important role in the biological activity and di sposition of aflatoxin s by living organisms. Experiments carried out to study the
transformation of AFB I showed the presence of Amax at 338 in the upper and A111ax 378 in the lower half portion (Table 4) indicating its proximity to Amax of AFB 1 which is at 362. But the appearance of these
Amax in aqueous phase showed its transformation (Amax 338 in the upper portion showed the greater polarity
than the Amax 378 in the lower portion). Similarly in the radioisotope study, there was a significant increase in the rad ioactivity in aq ueous phase (Fig. 1 ). It has been reported that microorganisms including bacteria were capable of transforming AFB 1 to aflatoxin (R0 )
1, which may be conjugated before
elimination from the system 17• The enzyme
responsible for the reduction of B 1 to Ro was identified as a soluble NADPH-dependent 17-hydroxy steroid dehydrogenase 1
• Thi s transformation of AFB 1 into a more polar compound may help in conjugation and subsequent elimination from the biological system. Thus in the present study the evidences indirectly suggest that the removal of toxin may have been effected through transformation by the bacterium. Further research is necessary to identify the compounds.
Compari son of different microorgani sms in removal of toxin showed that the resistant bacterium was more effective than the other bacteria (Table 5). It was reported that E. coli Wl485 was relatively insensitive at 37°C to AFB 1, but at 45°C AFB 1 had inhibitory effect 18
• So the removal of 1% of AFB 1 by E. coli may be an apparent one. Further, the resistant bacterium grew well in the presence of AFB I at 45°C. Moreover it was reported that 12 strains of 19 species of Bacillus were sensiti ve to 10 fl g mr 1 of AFBI 19
•
It may be concluded that the bacterium removed a maximum 26% of AFB 1 at 3 fl g mr1 and the removal may be effected through biotransformation of AFB I.
Acknowledgement Authors are very much thankful to the Dean,
CBSH for providing the necessary facilities and to Dr D. Bhatnagar for arranging A. parasiticus SRRC148 from Commodity Safety Research, OSbA. The senior author (SY) is thankful to the Director, CSWRI (JCAR) for giving the opportunity to pursue Ph.D and to Dr Pankaj Mishra for providing other bacterial cultures.
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