Journal of Applied Bacteriology 1988,64,293-297 2621/07/87

Antimicrobial activity and inhibition of aflatoxin Bi

formation by olive plant tissue constituents

N. PASTER*?, B.J. JUVEN$ & H. HARSHEMESH~ ?Department of Stored Products,

$Department of Food Science and §Department of Oleiculture and Viticulture, Agricultural
Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel

Received 31 July 1987, revised 5 November 1987 and accepted 12 November 1987

PASTER, N., JUVEN, B.J. & HARSHEMESH, H . 1988. Antimicrobial activity and
inhibition of aflatoxin B, formation by olive plant tissue constituents. Journal of
Applied Bacteriology 64,293-297.
Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which
Aspergillus Jlauus was grown, inhibited aflatoxin production by 90% without inhi-
biting the fungal growth. The extract was found to contain mainly calTeic acid and,
to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity
of caffeic acid, catechin, coumarin and p-, 0- or m-coumaric acid were tested and
only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory
effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/
I totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested,
catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudo-
monas aeruginosa and Staphylococcus aureus.

Mycotoxins are secondary metabolites produc-
ed by many moulds, mainly those belonging to
the genera Aspergillus, Penicillum and Fusarium.
The toxic effects of mycotoxins on animals have
been widely described and circumstantial evi-
dence indicates that mycotoxins are also
involved in human intoxications (Bullerman
1978).
Formation of mycotoxins is closely linked to
fungal growth and controlling the factors affect-
ing growth (especially relative humidity and
temperature) may prevent toxin production.
Fungal growth and the subsequent mycotoxin
formation in stored grains can be inhibited by
physical methods (aeration, cooling, modified
atmospheres, etc.) or by fungistats, of which the
propionic, acetic and sorbic acids are the most
commonly used. Herbs, spices and essential oils
have been found to have antibacterial and anti-
fungal activity and some of them also inhibit
mycotoxin formation (Morozumi 1978; Azzouz
& Bullerman 1982; Bahk & Marth 1983).
Aqueous extracts of some plants have also
been found to inhibit aflatoxin production to
* Corresponding author.
varying degrees (Bilgrami et al. 1979). Phenolic
compounds are known for their antimicrobial
and antifungal activity (Friend 197% and their
inhibitory effect on aflatoxin production has
also been reported (Sinha & Singh 1981; Bil-
grami et al. 1982). Phenolic compounds are
widely distributed in olive plant tissues and
the following have been found in significant
amounts in the pulp of olive fruits: oleuropein,
free p-coumaric acid, derivatives of caffeic acid
and mainly an ester of caffeic acid and glucose
(Vazquez Roncero et al. 1974).
The aim of the present study was to deter-
mine whether extracts of olive plant tissue pro-
duced under controlled conditions can inhibit
the growth of Aspergillusflavus cf aflatoxin pro-
duction. In addition, some substances identified
as constituents of the extracts were also studied
for their antifungal and antibacterial activity.
Materials and Methods
OLIVE C A L L U S G R O W T H A N D E X T R A C T I O N
Olive callus cv. Manzanillo was grown on
double inorganic modified White’s media
294 N. Paster
(21-MW) with indolacetic acid and YY-
dimethyl-allylaminepurine (Lavee & Messer
1969). Fresh tissues (15 g) taken from the first
subculture, were used for the extraction of phe-
nolic acids. The final volume of the extract was
10 ml. The phenolic compounds were extracted
by a method based on ethylacetate (JendeStrid
1985) with some modification (Harshemesh
1987). We used 80% hot ethyl alcohol instead of
acetone for the first step. All the samples were
tested after acidic hydrolysis for 30 min.
The 80% ethyl alcohol extracts were separat-
ed by paper chromatography and analysed by
HPLC using external standards (Harshemesh
1987).
TEST O R G A N I S M S A N D I N O C U L U M
Aspergillus jlauus NRRL 3251 was grown on
Potato Dextrose agar (PDA; Oxoid) at
26" 1°C. Agar discs (0.3 cm in diameter)
covered with mycelium served as the source of
the inoculum. The discs were cut from the edge
of 7 d colonies and a single disc was placed in
the centre of a petri dish containing the test sub-
stances. The antibacterial activity of the various
phenolic compounds was tested against Staphy-
lococcus aureus (enterotoxin A-producing strain)
and Pseudomonas aeruginosa. The test organ-
isms were cultured for 24 h in tryptic soy (TS)
broth at 30°C.
ASSAY
The assays used to demonstrate inhibitory
properties were performed in either TS broth
(for the bacteria) or PDA (for the fungus). The
phenolic compounds were dissolved in 5 ml of
ethanol followed by the addition of 3 ml of dis-
tilled H,O at 80°C. The solutions (100 mmol/l)
were then filter sterilized and decimal dilutions
were made in the PDA for A.flavus, to obtain
the desired final concentrations.
For the bacterial assays, the filter-sterilized
solutions were added to TS broth. Tryptic soy
broth (10 ml), either unsupplemented or suppie-
mented with a phenolic compound, was inocu-
lated with each of the test bacteria to yield
approximately lo4 cfu/ml. The inoculated
samples, in 25 ml Erlenmeyer flasks, were incu-
bated for 3 d at 30°C in a gyratory incubator
shaker at 100 rev/min.
After inoculation and incubation, samples
were taken, diluted, and the number of
et al.
organisms extracted by (a) plate counts on TS
agar and @) a 3-tube most probable number
(MPN) method with TS broth; the tables of de
Man (1975) were used to calculate the counts.
The results are typical of those obtained from
replicate experiments.
D E T E R M I N A T I O N OF A F L A T O X I N A N D
MYCELIAL D R Y W E I G H T
After 12 d the entire contents of four petri
dishes were taken for aflatoxin B, analysis. The
toxin was determined according to the AOAC
method (Anon. 1980) using TLC. Standard &a-
toxin B, was purchased from Makor Chemicals
(Jerusalem, Israel).
The mycelial weights were determined after
melting the media in each of two separate plates
and filtering them through a cotton plug. The
mycelia were then oven-dried at 80°C for 24 h
(Lisker et al. 1983).
ReSdtS
The main component of the callus tissue
extracts identified by chromatographic methods
was caffeic acid (ca 1 mg/g fresh weight of
tissue). The other components identified were
catechin and chlorogenic acid, 0.5 and 0-3 mg/g
fresh weight of tissue respectively. A group of
coumarins was also identified.
ANTIBACTERIAL ACTIVITY
The antibacterial effect of individual phenolic
compounds added to TS broth at a concentra-
tion of 10 mmol/I is shown in Table 1. One day
after inoculation, a reduction in viable counts
was only observed in samples containing caffeic
acid and inoculated with Ps. aeruginosa. After
3 d, reductions in viable counts of Ps. aerugin-
osa and Staph. aureus to non-detectable values
(< 1 viable cell/ml) were observed in samples
containing caffeic acid or catechin. Growth was
inhibited 1 d after inoculation with Ps. aerugin-
osa in samples containing catechin, and with
Staph. aureus in samples containing m-,0- or p-
coumaric acid, catechin and caffeic acid.
EFFECT O N F U N G A L G R O W T H A N D
AFLATOXIN B FORMATION
An inhibitory effect on the formation of aflatox-
in B, was recorded for ocoumaric acid at all
 Inhibition of ajlatoxin B, formation 295
Table 1. Antibacterial activity of phenolic compounds
Viable bacterial counts/ml*
Ps. aeruginosa Staph. aureus
Addition Initial
to TSBt
~~
DH Id~
3d ~
Id
~~
3 ___
d
None 7.4 2 x 108 4 x 108 lo8 109
m-coumaric 6.8 2 x lo8 2 x 10' 3 x lo5 2 x lo8
o-coumaric 6.4 108 2 x 108 2 x 105 108
pcoumaric 6.8 108 3 x 108 2 x 105 2x 108
Catechin 7.3 2 x 104 <0.4 104 <0.4
Caffeic 7.0 4 <0.4 lo4 (0.4
* Initial bacterial counts were 5 x 104/ml for Pseudomonas aeruginosa
and lO'/ml for Staphylococcus aweus.
The various phenolic compounds were added to trypic soy broth
(TSB) to yield a final concentration of 10 mmol/l.

the concentrations tested, for caffeic acid at
10 mmol/l and for the callus extract at 0.5 and
1.0% (Table 2).
Callus extract and the o-coumaric acid
reduced the amount of the toxin that accumu-
lated to 1&20% of the amount in the control,
while the reduction by caffeic acid was only
50%. The other two isomers of the coumaric
acid, as well as catechin, coumarin and ethanol,
did not inhibit aflatoxin formation and its pro-
duction was even slightly better (20-25% over
the control) when the fungus was grown on
media supplemented with p-coumaric acid (at
10 and 1 mmol/l) or catechin (at 10 mmol/l).
None of the substances tested affected mycel-
ial growth except for coumarin, which totally
inhibited the growth of the fungus when applied
at a concentration of 10 mmol/l. In all the treat-
ments which supported growth, the values of
the mycelial dry weight were almost identical
and did not exceed a level of 10% over the
control. The pH values of the media containing
the test materials (separately) were also quite
uniform, with a range of 5.5-6.5 recorded for
the different treatments.
Discussion
The kinds of damage caused to edible commod-
ities by contaminating micro-organisms have
been widely reported and, of these, the presence
of toxin metabolites was recognized as a
problem. Since the risk of residues in the food
limits the use of antimycotic agents only a
restricted number of chemicals have been
approved now as widely accepted safe materials.
Consequently, efforts have been made to evalu-
ate the preservative action of natural sub-
stances, e.g. plant constituents or plant extracts,
as well as that of spices or herbs, which are
widely used in the food industry. Phenolic com-
pounds, which comprise a major group of plant
constituents, are well known as antimicrobial
agents and their important role in the resistance
of plants to diseases has been described. The
ability of phenolics to inhibit aflatoxin pro-
duction was also reported. Sinha & Singh (1981)
reported that pyrocatechol, chloroglucinol,
ferulic acid and vanillin were inhibitory to afla-
toxin production in varying degrees, even at a
concentration of 100 n@g. Bilgrami et al.
(1982) also noted that o-vanillin did not support
aflatoxin production. Olives contain large quan-
tities of phenols and the antibacterial activity of
green olive fruit extracts, oleuropein and some
of its hydrolysis products has also been
described (Juven et al. 1968; Juven & Henis
1970; Fleming et al. 1973). Recently, Bullerman
et al. (1986) reported that aflatoxin production
on both whole and damaged fresh olives and
olive paste was very low or non-existent. The
effect of specific olive compounds was not
studied, however, except for that of oleuropein,
which was found to reduce aflatoxin production
by more than 80%. Hence, the inhibitory activ-
ity of olives may be attributed to phenolic com-
pounds. As the amount of the phenolics in the
different olive plant tissues may vary within the
growth period, thus affecting their inhibitory
effect, we decided to use callus tissue as a model
in our studies. Callus tissues are grown under
controlled conditions, thus enabling us to
achieve similar compositions of extracts taken
296 N . Paster et al.
Table 2. The effect of ethanolic callus tissue extract .and some of its com-
_
uonents on growth of AsDerailius fluuus and aflatoxin B, formation
_ _ _ ~

Supplements pH of Mycelial dry weight Aflatoxin

and concns.' medium (g/15 ml of medium) (% of control)t
m-coumaric acid
0.5 - 0.130 100
1 6.0 0.133 85
5 - 0.106 100
10 5.5 0.131 100
o-coumaric acid
0.5 - 0.128 20
1 6.0 0.130 15
5 - 0.125 15
10 5.5 0.119 20
p-coumaric acid
0.5 - 0.121 100
1 6.0 0.105 125
5 - 0.115 100
10 6.0 0.125 120
Catechin
0.5 - 0.131 90
1 6.0 0.128 100
5 - 0.130 100
10 6.5 0.110 125
Coumarin
0.5 - 0.115 85
1 6.0 0.128 105
5 - 0.120 85
10 6.0 No growth
Caffeic acid
0.5 - 0.130 100
1 6.5 0.128 100
5 - 0.120 100
10 6.5 0.125 50
Callus extract
0.5% 6.5 0.135 10
1.0"/" 6.5 0.131 10
Ethanol
0.5'Yo 6.5 0.135 100
1.O"h 6.5 0.128 100
Control 6.5 0.135 100
* Concentrations of coumaric acids, catechin, coumarin and caffeic acid
are in mmol/l.
t Control = 20 pgikg

after the same growth period. The extracts
which were used in our study after growth for
30 d contained mainly caffeic acid and, to a
lesser cxterit, catechin and coumarins. As cou-
marin may lead to coumaric acid the latter was
also studied for its inhibitory effect. Of the sub-
stances tested, however, only catechin and
caffeic acid had a bacterial effect against Ps.
~ J C T U ~ ~ ~ and
O S L Staph.
I aureus.
These findings corroborated data appearing
in the literature about the antibacterial activity
of caffcic acid, as reviewed by Davidson &
Branden I 198 1).
Only o-coumaric acid and caffeic acid at a
concentration of 10 mmol/l were found to
inhibit the aflatoxin production in addition to
the callus extract itself which also exhibited a
strong inhibitory effect.
Nevertheless, the activity of caffeic acid could
be explained by its activity as a n antioxidant,
since it has been reported that the activity of
this substance was almost the same as that of
the well known antioxidant butylated hydroxy-
toluene (Hare1 & Kanner 1985).
Fanelli et al. (1985) have also reported that
several antioxidants strongly inhibited aftatoxin
 Inhibition of ujlutoxin B, formation
production by Aspergillus parasiticus. It there-
fore seems likely that aflatoxin production by A.
jlauus, studied in our trials, was also influenced
by the antioxidative activity of caffeic acid. The
strong inhibitory effect of the callus extract as
compared with isolated phenolic compounds
can be explained in part by the presence of o-
coumaric acid which may be formed via
coumarins-a group found among the callus
constituents. However, inhibition due t o a syn-
ergistic action of various constituents of the
callus cannot be ruled out. This assumption
should be clarified in further studies.
W e are grateful t o H. Weisslowicz, M. Mena-
sherov a n d H. Cohen for their technical asis-
tance and also acknowledge the contribution
from the Agricultural Research Organization,
The Volcani Center, Bet Dagan, Israel. No.
2125-E, 1987 series.
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