Effects of Food Preservatives on Growth and Aflatoxin

Production of Aspergillus flavus in Liquid Medium

Dusanee Thanaboripat, Thawatchai Premsri, Niramol Punbusayakul
and Oratai Sukcharoen

ABSTRACT MATERIALS AND METHODS

Benzoic acid, sodium benzoate and potassium metabisulphite Organism

at 2.0, 6.0 and 10.0 mg/kg were used to control the growth
A. flavus IMI 102566 obtained from the International
of Aspergillus flavus and aflatoxin production in liquid
medium. Cultures were incubated at room temperature for 6 MycologlCal Inshtute (1MI), U.K., was used throughout
days with agitation. The results showed that sodium ben- thIs expenment. A. flavus was grown on potato dex-
zoate at 10 mg/kg reduced the growth of A. flavus by 13% trose ag~r at 30'C for 7 days. Spores were harvested in
and aflatoxin production by 35% at day 6. Benzoic acid at stenle dlshlled water plus 0.1% Tween 80. The spore
2.0 mg/kg reduced the growth and aflatoxin production by suspensIOn was pooled m a stenl~ bottle and the num-
72 and 87% after 6 days whereas benzoic acid at 6.0 and ber of spores counted usmg an Improved Neubrauer
10.0 mg/kg completely inhibited both mycelial growth and Hemacytometer.
aflatoxin production in liquid medium. Potassium .
metabisulphite at 2.0, 6.0 and 10.0 mg/kg completely inhib- Food preservahves
ited A. flavus growth and aiflatoxin Production. . . . .
Th e preserva t Ives use d were b enzOlC aCld an d 1t s sa It ,
sodium benzoate and potassium metabisulphite at 2.0,
6.0 and 10.0 mg/kg. Sodium benzoate and potassium
INTRODUCTION metabisulphite were dissolved in water to obtain the
required concentrations whereas benzoic acid was dis-
Aflatoxins are secondary metabolites of the moulds solved in methanol.
Aspergillus flavus, A. parasiticus and A. nomius which
can grow readily on various foods and feeds and pose Medium
a potential health hazard to humans and animals
(Diener, Cole, Sanders, Payne, Lee and Klich, 1987; The synthetic medium used for aflatoxin production
Kurtzman, Horn and Hesseltine, 1987; Abbas, Mirocha, (Venkitasubramaniam, 1977) contained in 1 L distilled
Kommedahl, Burner, Meronuck and Guntler, 1988). water: sucrose 85 g, L-asparagine 10 g, ammonium
Numerous strategies have been proposed for the detoxi- sulphate 3.5 g, KH,PO, 10 g, MgSO,.7H,O 2 g,
fication or inactivation of aflatoxin in contaminated CaCI,.2H,O 75 mg, ZnSO,.7H,O 10 mg, MnCl.4H,O
foods and feeds. However, preventing fungal growth is 5 mg, FeSO,.7H,O 10 mg, ammonium molybdate.4H,O
the best method for controlling toxin production 2 mg and Na,B.o, 2 mg, adjusted to pH 4.5 with HCl
(Samarajeewa, Sen, Cohen and Wei, 1990). or NaOH. Medium (100 mL) was dispensed into 250-
mL Erlenmeyer flasks.
Food preservatives are widely used to protect food
against microbial deterioration and their use in reduc- Culture conditions
tion of growth of aflatoxin-producing fungi and afla- . - known concentrahons ..
MedIUm wIth of sodIUm ben-
toxin levels have been studied in contaminated food-
zoateand potassium metabisulphite was autoclaved at
stuffs and culture media (Chipley and Uraih, 1980;
10 pSI for 10-20 mm then moculated wIth 1 mL of spore
Uraih and Offonry 1981' Gareis Bauer von Montgelas
and Gedek, 1984;' Than;borip~t, Ramunsri, su~pension of A.flavus to obtain a final concentration of
Apintanapong and Chusanatasana, 1992; Chou rasia, 10 spores/mL. BenzoIc aCId dIssolved m methanol ,,:as
1993). The present study evaluated the effect of sodium added to dry 250-mL Erlenmeyer flasks and the carner
solvent was then evaporated m a hot water bath. 100
benzoate, benzoic acid and potassium metabisulphite
mL of medIUm was added to each flask whIch was
on growth and aflatoxin production by A. flavus in
then autoelaved and inoculated with spores of A. flavus.
liquid medium.
Flasks without food preservatives served as control.
The inoculated flasks were incubated at room tempera-
ture for 7 days on a Gallenkamp orbital shaker at 200
Departmentof Applied Biology,Facultyof Science,KingMongkut's rpm. All experiments were carried out in triplicate.
Institute of Technology,LAdkrabang,Bangkok1OS20,Thailand

ASEAN Food Journal Vol. 11, No.2, 1996 6]

Growth and aflatoxin analysis

Mycelial dry weights were determined by filtration

under reduced pressure using preweighed Whatman
no. 4 filter paper in a Buchner funnel. After washing
with 20 mL distilled water, the mycelial mat was re-
moved, dried at 45'C for 48 h., cooled in a desiccator
and weighed.

The filtrate was extracted for aflatoxin by a modified

method of Shih and Marth (1974). Forty mL of chloro-
form were added to 20 mL of filtrate in a separatory
funnel and shaken vigorously. The chloroform extract
was removed and the remaining material agitated twice
with 100 mL of chloroform. Pooled extracts were evapo-
rated to near dryness and transferred to a small vial.
Aflatoxins were separated by thin-layer chromatogra-
phy using toluene: ethyl acetate: chloroform: 90% for-
mic acid (70:50:20:20) as the solvent system (Gimeno,
1979). Individual aflatoxin bands were identified under
ultraviolet light, eluted with 5 mL methanol and esti-
mated spectrophotometrically at 365 nm (Nabney and
Nesbitt, 1965).

Statistical analysis

An analysis of variance (ANOVA) was calculated to

ir,dicate the effect of food preservatives on growth and
aflatoxin production at significant differences of 0.01
and 0.05. The statistical method was according to Mont-
gomery (1984).

RESULTS AND DISCUSSION

The production of aflatoxin by A. flavus in liquid me- The effects of sodium benzoate, benzoic acid and potas-
dium over 7 days (Table 1) shows that aflatoxin yields sium metabisulphite on growth and aflatoxin produc-
on days 4, 5, and 6 were higher than on other days. tion of A. flavus 1M! 102566 are shown in Tables 2 and
Thus, therefore further experiments examined yields 3. Sodium benzoate at 10.0 mg/kg reduced the growth
on days 4 - 6 of incubation. of A. flavus and aflatoxin production on day 6 by 13%
and 35% respectively whereas 2.0 and 6.0 mg/kg of
sodium benzoate did not have any inhibitory effect on
either growth or aflatoxin production. Sodium ben- with an increase in sorbate concentration to a point and
zoate (2.0 and 6.0 mg/kg) actually stimulated both then decreased at higher levels of sorbate (200 - 300
growth and aflatoxin production. The statistical analy- mg/kg). A similar result was reported with the appli-
sis (Tables 4 and 5) showed significant differences be- cation of sodium chloride at low concentration (20 mg/
tween production of aflatoxin in the cultures with and g) by Chitaree, Kiatsompob, Panchang and Thanaboripat
without sodium benzoate (2.0 and 6.0 mg/kg). Benzoic (1993) which stimulated the production of aflatoxin
acid at 2.0 mg/kg reduced growth and aflatoxin pro- whereas high concentrations (40 - 160 mg/ g) inhibited
duction on day 6 by 72% and 87% whereas,at 6.0 and aflatoxin production. There has been a suggestion that
10.0 mg/kg both growth and aflatoxin production were the stimulation of aflatoxin production by low levels of
completely inhibited. Potassium metabisulphite at all salt might be a function of sodium ions (Uraih and
concentrations completely inhibited growth and afla- Chipley, 1976).
toxin production of A. flavlIs.
The inhibition of growth and aflatoxin production by
benzoic acid at all concentrations is in accordance with
the results of Uraih, Cassity and Chipley (1977) and
Uraih and Offonry (1981). Growth and aflatoxin pro-
duction by a toxigenic strain of A. flavus was com-
pletely inhibited by benzoic acid and sodium benzoate
in synthetic medium at 4 mg/mL (0.4%). Reduction in
aflatoxin biosynthesis was proportional to the increased
concentration of either benzoic acid or sodium ben-
zoate. The greater effect exhibited by benzoic acid on
growth and aflatoxin production by A. flavus over
sodium benzoate could be explained on the basis of the
pH of the medium (Uraih and Chipley, 1976). The
undissociated benzoic acid molecule might be the anti-
microbial agent affecting growth and aflatoxin produc-
tion by A. flavus. The organic preservative acids would
cease to have any action whatever or retain only a very
slight action in the neutral pH range (Leuck, 1980).

Potassium metabisulphite at all concentrations pre-

vented the growth of A. flavlls. Thus, no aflatoxin
production was detected. Similarly, Chourasia (1993)
found that sodium metabisulphite at concentrations of
0.1 and 0.5% did not permit mycelial growth and
aflatoxin biosynthesis in liquid medium. Tabata,
Kamimura, !be, Hashimoto and Tamura (1994) reported
the degradation of aflatoxins by various food preserva-
tives including potassium metabisulphite. Sulphite is
an unusually multifunctional food preservative and
can exhibit antimicrobial agent properties at both low
and high pH (Gould and Russell, 1991) which is differ-
ent from benzoic acid and its salt.

Statistical analyses showed a correlation between the CONCLUSION

food preservative and the concentrations used. It also
showed that different food preservatives at different This study indicates that potassium metabisulphite
concentrations gave different effects on growth and could be better exploited in the protection of food from
aflatoxin production of A. flavus. A. flavus and aflatoxin contamination than benzoic acid
and sodium benzoate because sulphur dioxide (in the
Sodium benzoate at less than 10.0 mg/kg did not form of potassium metabisulphite) is a more active
inhibit growth or aflatoxin production whereas at 10.0 food preservative than the weak acid preservative, ben-
mg/kg a reduction of growth and aflatoxin was ob- zoic acid and its salt. In addition, benzoic acid and
served. Yousef and Marth (1981) found that sorbate sodium benzoate (only at high concentration) could be
concentration had a major effect on accumulation of effective fungicides or antimicrobial agents against toxi-
aflatoxins. The levels of aflatoxins seemed to increase genic fungi in food at low pH levels.

ASEAN Food Journal VoL 11, No.2, 1996 63

ACKNOWLEDGEMENTS Leuck, E. Antimicrobial food preservatives. Berlin:
Springer Verlag, 1980.
We would like to thank Assistant Professor Vararat
Ruangrattanametee for her assistance in the statistical Montgomery, D.C Design and analysis of experiments.
analyses. New York: Wiley, 1984.

Nabney, J. and Nesbitt, B.P. A spectrophotometric

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64 ASEAN Food Journal Vol. 11, No.2, 1996