JOURNAL OF BACTERIOLOGY, Jan., 1967, p. 59-64
American Society for
Vol. 93, No.
Printed in U.S.A.
TADASHI ARAI, TATSUYA ITO, AND YASUMASA KOYAMA
Department of Antibiotics, Institute ofFood Microbiology, Chiba University, Narashino, Chiba, and
Pharmaceutical Faculty, Chiba University, Chiba, Japan
Received for publication 18 July 1966
Antimicrobial activity of a crude aflatoxin preparation and of aflatoxin B1
studied. They were found inactive against common gram-positive and gram-nega-
tive bacteria at a concentration of 100 jAg/rn1. Both samples of aflatoxin did, how-
ever, exhibit antimicrobial activity, though narrow and limited, against various
strains of Streptomyces and Nocardia. The antibiotic action of aflatoxin
confirmed by bioautogram after thin-layer chromatography. Among seven strains
ofnmicroorganisms, including aflatoxin-sensitive and -resistant strains, N. asteroides
IFM 8 was found to reduce aflatoxin B1, in addition
components in the crude preparation.
to other minor fluorescent
Although numerous data concerning biological
activity of aflatoxins (12, 13), the toxic secondary
metabolites of Aspergillus flavus, have been thus
into the growth
strate inhibited 12 species of the genus Bacillus,
one species of Clostridium, and one of Strepto-
myces among the 329 microorganisms tested at a
concentration of 30lAg/ml.The crude preparation
on an alumina column
TABLE 1. Antimicrobial activity ofcrude aflatoxin and aiflatoxin B1"
Minimal inhibitory concn (pug/mi)
Bacillus subtilis PCI 219...............75b
B. cereus ATCC1072.OO
Streptomyces vinaceus IFM 1017...........25
S. olivoreticuli IFM 1018...............10
S. lavendulae IFM 1025................25
S. roseochromogenes IFM10950
S. virginiae IFM 1028................25
S. halstedii IFM 1032................50
S. netropslis IFM 1035................25
S. aureofaciens IFM 1042...............100
S. antibioticus IFM10925
S. griseoluteus IFM 1055...............25
Nocardia leishmanii IFM 27.............100
N. asteroides IFM150
N. asteroides IFM225
N. asteroides IFM325
N. asteroides IFM8>100
N. coeliaca IFM 30.................25
N. rangoonensis IFM 23...............
N. brasiliensis lFm 65................50
Medium: glucose-nutrient agar for Bacillus and Sabouraud agar(1%0glucose) for Streptomyces and
Nocardia. Incubation: 27 C for 72 hr for Streptomyces, 37 C for 24 hr for Bacillus, and 37 C for 72 hr for
bGrowth was retarded down by concentrations as low as 50 JAg/mi.
ARAI, ITO, AND KOYAMA
was also found by Miyaki and Aibara (personal
communication) to exhibit antimicrobial activity
against Bacillus species. The active prinaciple in
the preparation, however, was removed as the
purification of the toxin proceeded.
Most of carcinogenic four- or five-membered
lactones are known to possess some antimicro-
bial activity. Aflatoxins are the first unsaturated,
six-membered cyclic lactones which have been
found to be carcinogenic, and studies on the
antimicrobial activity of aflatoxins are of some
This paper deals with the antimicrobial activity
of aflatoxin and the reduction of the toxins by a
strain of Nocardia asteroides.
MATERIALS AND METHODS
Crude aflatoxin material was prepared as described
by Asao et al. (2). A.flavus MOOI (ATCC 15517), a
gift from G. N. Wogan, Massachusetts Institute of
Technology, was grown in submerged culture in
Adye's medium (1), and the active principle was ex-
tracted with chloroform and precipitated with petro-
leum ether. It was further purified by chromatography
on an alumina column (activity grade 1, M. Woelm
Eschwege, Germany). By developing the column with
chloroform, aflatoxin B1 was selectively eluted. The
preparation thus obtained was 55% pure by calcula-
tion with the extinction coefficient, and no fluorescent
contaminant was revealed by thin-layer chromatog-
All test strains were obtained from the culture col-
lection of our laboratories. They were maintained in
appropriate culture media sealed with mineral oil.
Prior to use, test bacteria were grown on nutrient agar
for 24 hr at 37 C; Streptomyces species were grown on
Nocardia species were grown on potato extract-agar
(Kelner-Morton; 7) for 1 week at 37 C. The usual agar
streak method was employed for the determination of
antimicrobial activity. Methanol solutions of aflatoxin
were added to the agar plates to give a solvent dilu-
tion of 1:20, which was not inhibitory to any of the
test organisms. Growth from Streptomyces or Nocardia
cultures was homogenized, when necessary, with glass
beads to make a suspension. Media used and incuba-
tion periods are indicated in Table
chromatography of aflatoxins was run with a solvent
system of chloroform-methanol-formic acid (95: 5: 1)
on a 250 u thick layer of Kieselgel G (E. Merck AG,
Darmstadt, Germany). For detection of antimicrobial
activity (bioautogram), the layer was dried to remove
formic acid, laid upside down on a potato extract-
agar plate seeded with a suspension of S. virginiae
spores, and covered with sterile filter paper. After
20 min at room temperature, the plate was removed
with the filter paper. The treated agar plate was incu-
bated at 27 C for 18 hr. Results had to be read before
heavy growth with sporulation of the organism ob-
scured the inhibition zone. To prepare the cell sus-
pensions for reduction studies, the organisms were
cultured on a rotary shaker at 27 C for 3 days for
1 week at 27 C, and
Streptomyces and 1 week for Nocardia. Escherichia coli
was cultured without shaking at 37 C for 48 hr. The
mediumcontained: glucose, 5 g; starch, 5 g; meat ex-
tract, 5 g; Polypeptone (BBL), 10 g; NaCl, 3 g; and
distilled water, 1 liter (pH 7.2). The cells were col-
lected by centrifugation. They werewashed three times
with salineand resuspended in 0.067M phosphate buffer
ofpH 6.0 and 7.0. To 35 ml ofsuspensions containing
0.87 ml of packed cells was added 2.0 ml of 50%
methanol solution of aflatoxin crude preparation or
aflatoxin B1 to give final concentrations of 9 and 12
,ug/ml, respectively. The suspensions were then in-
cubated on a shaker in a water bath at 37 C. For
spectrophotometric determination, a 5-ml sample was
withdrawn, extracted with 10 ml of chloroform, and
applied to a Hitachi spectrophotofluorometer (model
EPU-2). Fluorescence at 425 mpA was determined by
using an excitation wavelength of 365 m,u.
One strain each of Staphylococcus aureus, S.
citreus, S. albus, Streptococcus faecalis, E. coli,
Salmonella paratyphi A and B, Shigella dysen-
teriae Shiga, Serratia marcescens, Sarcina lutea,
1. Comparison of chromatograms of crude
aflatoxin as determined under visible and ultraviolet
light, and by antimicrobial activity. Fluorescence:
violet; FV, faint violet; GR, green; Y, yellow.
ANTIMICROBIAL ACI1VITY OF AFLATOXINS
and Pseudomonas aeruginosa was not inhibited
with a concentration of up to 100 jig/ml of
aflatoxins. As shown in Table 1, the crude prepa-
ration exhibited very limited antibiotic activity
against Bacillus subtilis and B. cereus. Complete
inhibition of B. subtilis was not obtained even
at a concentration of 100 pg/ml of aflatoxin B1,
although growth was retarded at a concentration
of 75 and 50 ,ug/ml. A similar effect on B. cereus
was observed. Most test strains of Streptomyces
and Nocardia were inhibited at a concentration
of 25 to 50 ,ug/ml of the crude preparation.
being inhibited at a concentration of 10 ,ug/ml.
A comparable antimicrobial spectrum was ob-
tained with purified aflatoxin B1. S. aureofaciens,
N. asteroides IFM 8, and N. rangoonensis were
found to be resistant to both the crude prepara-
tion and B1. The results of thin-layer chromatog-
light, and by bioautogram (antimicrobial activ-
ity), are presented in Fig. 1. Under visible light,
the crude preparation had three yellow spots
as revealed by
visible and ultraviolet
0 0 0 0 0 0 0
unrelated to those detected by other means.
spots in addition to two
major spots, a violet fluorescent spot at the same
height as B1 and a green fluorescent one which is
presumably G1. By antimicrobial detection, both
the crude preparation and B1 had single inhibi-
tory spots of an RF value corresponding to that
The fate of aflatoxins in the washed-cell sus-
pensions of four resistant and three sensitive or-
ganisms was examined by thin-layer chromatog-
raphy (Fig. 2). Complete disappearance of char-
acteristic fluorescent spots was observed only
with N. asteroides IFM 8; no change of pattern
was noted with other organisms after 38 hr of
incubation. The reduction of aflatoxin by N.
asteroides IFM 8 was further followed quantita-
tively by fluorometric measurement and qualita-
tively by thin-layer chromatography and ultra-
violet-absorption spectra. The results are shown
in Fig. 3-7. Immediate quenching of fluorescence
occurred both in pH 6.0 and in 7.0 buffers with
the preparation had
0 0 0 0 0
FiG. 2. Reducing effect ofNocardia asteroides IFM 8 cells on aflatoxin shown by thin-layer chromatography.
Fluorescence: V, violet; GR, green.
VOL. 93, 1967
ARAI, ITO, AND KOYAMA
the crude preparation. The relative intensity of
fluorescence dropped to 20 and 14% of the
original, respectively. It was also revealed by
thin-layer chromatography that the spots cor-
responding to aflatoxin G, and minor fluorescent
compounds disappeared first, whereas that of
aflatoxin B1 persisted for 24 hr at pH 6.0 and 6
hr atpH 7.0. This was also the case with aflatoxin
B1. On the other hand, the reduction of fluores-
cence was slow and not so significant with B1
as with the crude preparation. Changes in the
spectral patterns of the aflatoxin crude prepa-
ration and B1 in cell suspensions at both pH
values are shown in Fig. 6 and 7. Absorbance
at 362 m,u was reduced with time, whereas the
absorbance at 265 m,u increased at first and then
gradually decreased. Characteristic absorption
maxima at 265 and 362 mu were lost after 12 hr.
Consequently, the relative intensity of these two
absorption maxima was reversed after the contact
of Nocardia cells.
The difficulty of determining the antimicrobial
activity of a
aflatoxins is evident when one considers that
some antibiotics inhibit the growth of bacteria
at the concentration of 6.0 x 10-8 Ag/ml (xan-
purified material like
in pH 6.0 buffer
aspergilli of the flavus group are known to pro-
duce several antimicrobial agents, such as kojic
acid (10), aspergillic acid (11) and other pyrazine
compounds (5, 8), and flavacidin (9), making
it necessary to use caution in attributing the
antibiotic action to aflatoxin in the crude prepa-
ration. In our experiments, the antimicrobial
activity of the toxin against various strains of
actinomycetes increased as the preparation was
Time in hours
FIG. 3. Rate ofreduction ofaflatoxin crude prepara-
tion by Nocardia asteroides IFM 8 as determined
GR 0 0.46
V 0 0.13
N.asteroides IFM 8
N.asferoides IFM 8
Time in hours
FIG. 4. Reduction of aflatoxin crude preparation by Nocardia asteroides IFM 8 as revealed by thin-layer
chromatography. Fluorescence: V, violet; GR, green.
ANTIMICROBLAL ACrIVrIY OF AFLATOXINS
FIG. 5. Rate ofreduction ofaflatoxin BA by Nocardia
asteroides IFM 8 as determinedfluorometrically.
Wave length Inmp.
FIG. 6. Changes in shapes of spectral patterns of
aflatoxin crudepreparation by Nocardia asteroidesIFM
8 (pH 7.0).
Wave length In m.>
FIG. 7. Changes in shapes of spectral patterns of
aflatoxinB, by Nocardiaasteroides IFM 8 (pH 7.0).
further purified, patterns of antimicrobial spectra
of crude preparation and purified B1 were almost
identical, and the location of the active principle
thin-layer chromatogram corresponded
exactly to that of aflatoxin B1. This evidence
supports the assumption that aflatoxin B1 is the
major antimnicrobial principle in the preparation.
However, other aflatoxins may have antimicro-
bial activity, but their amounts in the crudeprepa-
ration are not large enough to be manifested by
the bioautogram. The antimicrobial spectrum of
aflatoxin is narrow and limited, inhibiting only
various strains belonging to the family Actino-
Streptomyces and Nocardia which are resistant
to the agent. Specific reistance of these strains,
however, is mostly irrelevant to the production of
The reduction of aflatoxin by N. asteroides
IFM 8 was more rapid and complete at neutral
pH than at pH 6.0, and the disappearance of
sooner with the crude
preparation than with B1. It is not yet clear
whether this is due to the fact that fluorescent
components other than B1 in the crude prepara-
tion are much more sensitive to the microbial
action or to the presence of some additional
quenching agent in the crude preparation.
Since Geiger and Conn (6) reported the mecha-
nism of antibiotic action of clavacin and penicillic
acid, the antimicrobial activity of unsaturated
lactones has been correlated to their reaction with
sulfhydryl compounds. Dickens and Jones (4)
studied the carcinogenic activity of a series of
reactive lactones and formulated basic types of
chemical structure in relation to carcinogenicity.
Although selection of compounds for carcino-
genic test was made on the basis of their anti-
the relationship between
cinogenicity and antibiotic action of these
saturated lactones is not yet clear. As the com-
pounds studied in their experiments were four-
and five-membered ring lactones, the current
data on aflatoxin at least provide some informa-
tion on these two types of biological activity.
We thank G. N. Wogan for his kindness in supply-
ing a culture of a toxic strain ofA. flavus as well as an
authentic sample of aflatoxin B1. Thanks are also due
to Haruo Kaji for his excellent technical assistance.
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