Effect of Food Preservatives on Growth and Aflatoxin Production of us Flavus in Liquid Medium

8/6/2019 Effect of Food Preservatives on Growth and Aflatoxin Production of us Flavus in Liquid Medium

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Effects of Food Preservatives on Growth and AflatoxinProduction of A spergillus flavus in Liquid Medium

D usanee Thanaboripat, Thaw atchai Prem sri, N iram ol Punbusayakuland O ratai Sukcharoen

ABSTRACT MATERIALS AND METHODSB e n z o i c acid , s od ium benzoate and pota ss ium me tab is ulphite Organismat 2.0, 6.0 and 10.0 mg/kg were used to control the growthof A sp ergillus flavus a nd a fla to xin p ro du ctio n in liq uid A . fla vu s IM I 102566 obtained from the Internationalm ed iu m. C ultu re s w ere in cu ba te d a t r oo m te mp er atu re fo r 6 MycologlC al Inshtute (1M I), U .K ., w as used thro ughoutdays w ith agitation. The results showed that sodium ben- th Is e xpenmen t. A . fla vu s was grown on potato dex-zoate at 10 m g/kg reduced the grow th of A. flav us by 13% trose ag~r at 30'C for 7 days. Spores were harvested ina nd a fla to xin p ro du ctio n b y 35% at day 6. B en zo ic a cid a t stenle dlshlled water plus 0.1% Tween 80. The spore2.0 m g/kg reduced the grow th a nd aflatoxin p roduction b y suspensIOn was pooled m a stenl~ bottle and the num-72 and 87% after 6 days w hereas benzoic acid at 6.0 and ber of spores counted usmg an Improved Neubrauer10.0 m g/kg com pletely inh ibited both m ycelial g row th and Hemacytometer.aflatoxin production in liquid medium . Potassium .m eta bisu lp hite a t 2 .0 , 6 .0 a nd 1 0.0 m g/kg co mp letely in hib - Food p re se rvahve sited A . flav us growth and aiflatoxin Production. Th t . d b . .d d .t Itpreserva Ives use were enzOlC aCl an 1 s sa ,sodium benzoate and potassium metabisulphite at 2.0,

6.0 and 10.0 mg/kg. Sodium benzoate and potassiumINTRODUCTION metabisulphite were dissolved in water to obtain therequired concentrations w hereas benzoic acid w as dis-Aflatoxins are secondary metabolites of the moulds so lv ed in m eth an ol.A sp erg illu s fla vu s, A . p ara sitic us and A. nomius which

can grow readily on various foods and feeds and pose Mediuma potential health hazard to humans and animals(Diener, Cole, Sanders, Payne, Lee and Klich, 1987; The synthetic medium used for aflatoxin productionKurtzman, Horn and Hesseltine, 1987; Abbas, M irocha, (Venkitasubramaniam, 1977) contained in 1 L distilledKommedahl, Burner, M eronuck and Guntler, 1988). water: sucrose 85 g, L-asparagine 10 g, ammoniumNumerous 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 contam inated CaCI,.2H,O 75 mg, ZnSO,.7H,O 10 mg, MnCl.4H ,Ofoods and feeds. However, preventing fungal growth is 5 mg, FeSO ,.7H,O 10 mg, ammonium molybdate.4H,Othe 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-m L Erlenm eyer flasks.Food preservatives are w idely used to protect foodagainst m icrobial deterioration and their use in reduc- Culture conditionstion of grow th of aflatoxin-producing fungi and afla- . - ..toxin levels have been studied in contam inated food- MedIUm wIth known concentrahons of sodIUm ben-stuffs and culture media (Chipley and Uraih, 1980; zoateand potassium m etabisulphite was autoclaved atUraih and Offonry 1981' Gareis Bauer von M ontgelas 10 pSI for 10-20 mm then moculated wIth 1 mL of sporeand Gedek, 1984;' Than;borip~t, Ramunsri, su~pensio n of A.flavus to obtain a final concentration ofApintanapong and Chusanatasana, 1992; Chou rasia, 10 spores/m L. B enzoIc aC Id dIssolved m m ethanol ,,:as1993). The present study evaluated the effect of sodium added to dry 250-mL Erlenmeyer flasks and the carnerbenzoate, benzoic acid and potassium m etabisulphite solvent was then evaporated m a hot water bath. 100on grow th and aflatoxin production by A . fla vu s in mL of medIUm was added to each flask whIch wasliq uid m ed ium. then autoelaved and inoculated w ith spores of A. f lavus.Flasks w ithout food preservatives served as control.

The inoculated flasks w ere incubated at room tem pera-ture for 7 days on a Gallenkamp orbital shaker at 200

DepartmentofApplied Biology,Facultyof Science,King Mongkut 's rpm. A ll experim ents w ere carried out in triplicate.Insti tute of Technology,LAdkrabang,Bangkok 1OS20,Thailand

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Grow th and aflatoxin an alysisMycelial dry weights were determ ined by filtrationunder reduced pressure using preweighed W hatmanno. 4 filter paper in a Buchner funnel. After washingwith 20 mL distilled water, the mycelial mat was re-moved, dried at 45'C for 48 h., cooled in a desiccatorand w eig hed .The filtrate was extracted for aflatoxin by a modifiedmethod of Shih and Marth (1974). Forty mL of chloro-form were added to 20 mL of filtrate in a separatoryfunnel and shaken vigorously. The chloroform extractwas removed and th e remain in g materia l a gitated tw icew ith 100 m L of chloroform . Pooled extracts w ere evapo-rated to near dryness and transferred to a small vial.A flatoxins w ere separated by thin-layer chrom atogra-phy using toluene: ethyl acetate: chloroform: 90% for-mic acid (70:50:20:20) as the solvent system (Gimeno,1979). Individual aflatoxin bands w ere identified underultraviolet light, eluted with 5 mL methanol and esti-mated spectrophotometrically at 365 nm (Nabney andNesbitt, 1 96 5).S ta tis tic al ana ly sisAn analysis of variance (ANOVA) was calculated toir,dicate the effect of food preservatives on growth andaflatoxin production at significant differences of 0.01and 0.05. The statistical m ethod w as according to M ont-gome ry (1 98 4).

RESULTS AND DISCUSSIONThe production of aflatoxin by A. f l a v u s in liquid me-dium over 7 days (Table 1) shows that aflatoxin yieldson days 4, 5, and 6 were higher than on other days.Thus, therefore further experiments exam ined yieldson days 4 - 6 of incubation.

T he effects of sodium benzoate, benzoic acid and potas-sium metabisulphite on growth and aflatoxin produc-tion of A. fla vu s 1M ! 102566 are shown in Tables 2 and3. Sodium benzoate at 10.0 mg/kg reduced the growthof A. fla vu s and aflatoxin production on day 6 by 13%and 35% respectively whereas 2.0 and 6.0 mg/kg ofsodium benzoate did not have any inhibitory e ffect on


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either growth or aflatoxin production. Sodium ben-zoate (2.0 and 6.0 mg/kg) actually stimulated bothgrow th and aflatoxin production. The statistical analy-sis (Tables 4 and 5) showed significant differences be-tween production of aflatoxin in the cultures w ith andwithout sodium benzoate (2.0 and 6.0 mg/kg). Benzoicacid at 2.0 mg/kg reduced growth and aflatoxin pro-duction on day 6 by 72% and 87% whereas,at 6.0 and10.0 mg/kg both grow th and aflatoxin production werecom pletely inhibited. Potassium m etabisulphite at allconcentrations completely inhibited growth and afla-toxin production of A. f lavlI s.

Statistical analyses showed a correlation between thefood preservative and the concentrations used. It alsoshowed that different food preservatives at differentconcentrations gave different effects on grow th andaflatoxin production of A. f l a v u s .Sodium benzoate at less than 10.0 mg/kg did notinhibit growth or aflatoxin production whereas at 10.0mg/kg a reduction of grow th and aflatoxin was ob-served. Yousef and Marth (1981) found that sorbateconcentration had a major effect on accumulation ofaflatoxins. The levels of aflatoxins seem ed to increase

ASEAN Food JournalVoL 11, No.2, 1996

w ith an in crease in sorb ate con centration to a p oin t andthen decreased at higher levels of sorbate (200 - 300mg/kg). A sim ilar result was reported w ith the appli-cation of sodium chloride at low concentration (20 m g/g) b y C hitaree, K iatsom pob, P anchang and T hanaboripat(1993) which stimulated the production of aflatoxinw hereas high concentrations (40 - 160 m g/ g) inhibitedaflatoxin production. There has been a suggestion thatthe stim ulation of aflatoxin production by low levels ofsalt m ight be a function of sodium ions (Uraih andChip ley , 1 976).The inhibition of growth and aflatoxin production byb enzoic acid at all con cen tration s is in accord an ce w iththe results of Uraih, Cassity and Chipley (1977) andUraih and Offonry (1981). G rowth and aflatoxin pro-duction by a toxigenic strain of A . fla vu s was com-pletely inhibited by benzoic acid and sodium benzoatein synthetic medium at 4 mg/mL (0.4%). Reduction inaflatoxin biosynthesis w as propo rtio nal to the increasedconcentration of either benzoic acid or sodium ben-zoate. The greater effect exhibited by benzoic acid ongrowth and aflatoxin production by A . flavus oversodium benzoate could be explained on the basis of thepH of the medium (Uraih and Chipley, 1976). Theundissociated benzoic acid m olecule m ight be the anti-m icrobial agent affecting grow th and aflatoxin produc-tion by A. f lavus . The organic preservative acids w ouldcease to have any action w hatever or retain only a veryslight action in the neutral pH range (Leuck, 1980).Potassium metabisulphite at all concentrations pre-vented the growth of A . fla vlls . Thus, no aflatoxinproduction w as detected. Sim ilarly, Chourasia (1993)found that sodium metabisulphite at concentrations of0.1 and 0.5% did not perm it mycelial grow th andaflatoxin biosynthesis in liquid medium . Tabata,K am im ura, !be, H ashim oto and Tam ura (1994) reportedthe degradation of aflatoxins by various food preserva-tives including potassium metabisulphite. Sulphite isan unusually multifunctional food preservative andcan exhibit antim icrobial agent properties at both lowand high pH (Gould and Russell, 1991) which is differ-ent from benzoic acid and its salt.

CONCLUSIONThis study indicates that potassium metabisulphitecould be better exploited in the protection of food fromA. f lavus and aflatoxin contam ination than benzoic acidand sodium benzoate because sulphur dioxide (in theform of potassium metabisulphite) is a more activefood preservative than the w eak acid preservative, ben-zoic acid and its salt. In addition, benzoic acid andsodium benzoate (only at high concentration) could beeffective fu ngicides or antim icro bial agen ts against to xi-genic fungi in food at low pH levels.

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ACKNOWLEDGEMENTS Leuck, E. Antim icrobial food preservatives. Berlin:S pring er V erlag, 1980 .We would like to thank Assistant Professor VararatRuangrattanam etee for her assistance in the statistical M ontgom ery, D .C D esign and analysis of experim ents.analyses. N ew Y ork: W iley, 1984.

Nabney, J. and Nesbitt, B .P. A spectrophotometricREFERENCES m ethod for determ ining the aflatoxins. A nalyst 90:155-160 ; 1965 .Abbas, H .K ., M irocha, CJ., Kommedahl, T., Burner,

P.M ., M eronuck, RA and Guntler, R Toxigenicity Samarajeewa, 0., Sen, A .C , Cohen, M .D . and W ei, Cr.of Fusarium prol iferatum an d other Fusarium species Detoxification of aflatoxin in foods and feeds byisolated from com ears in M innesota. Phytopathol. physical and chem ical m ethods. J. Food Prot. 53(6):7 8: 1 25 8-1 26 0; 1 98 8. 4 89 -501 ; 1 990.

Chipley, J.R and Vraih, N . Inhibition of Aspergillus Shih, CN . and M arth, E.H . Aflatoxin formation, lipidgrow th and aflatoxin release by derivatives of ben- synthesis and glucose metabolism by Aspergilluszoic acid. A ppl. Env. M icrobiol. 40: 352-357; 1980. parasiticus during incubation with and without agi-tation. Biochim . Biophys. A cta 338: 286-296; 1974.

Chitaree, K ., Kiatsompob, T., Panchang, W . andThanaboripat, D . Effect of salt concentrations on Tabata, S., Kamimura, H ., Ibe, A , Hashimoto, H. andaflatoxin production in peanut by Aspergil lus flavus. Tam ura, Y . D egradation of aflatoxins by food addi-K asetsart J. N at. Sci. 27: 354-357; 1993. tives. J. Food Prot. 57: 42-47; 1994.

Chourasia, H .K . Growth, sclerotia and aflatoxin pro- Thanaboripat, D ., Ram unsri, W ., A pintanapong, M . andduction by Aspergil lus parasit icus: influence of food Chusanatasana, V . Effects of sodium chloride, pro-preservatives. Lett. Appl. M icrobiol. 17: 204-207; pionic acid and ammonium hydroxide on grow th1993. of Aspergil lus f lavus on com and aflatoxin produc-tion. ASEAN Food J. 7(1): 24-29; 1992.

D iener, V .L., C ole, RJ., Sanders, T.H ., Payne, G .A ., Lee,L.S. and Klich, M .A Epidemiology of aflatoxin Vraih, N . and Chipley, J.R Effect of various acids' andformation by A spergillus flavus. Ann. Rev. salts on growth and aflatoxin production by As-P hy to path ol. 25: 249 -270; 198 7. pergillus flavus NRRL314 5. M icro bio s 1 7: 5 1-5 9; 1 97 6.

Gareis, M ., Bauer, J., von M ontgelas, A and Gedek, B . Vraih, N . and Offonry, S. Inhibition of aflatoxin pro-Stim ulation of aflatoxin B1 and T-2 to xin p ro du c- duction in groundnut w ith benzoic acid derivativestion by so rbic acid . A ppl. E nv . M icrob io l. 47 (2): 41 6- and possible toxic effect of their arom atic residues.4 18 ; 1 98 4. M icrobios 31: 9 3-10 2; 198 1.

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Gould, G .w . and Russell, N .J. Sulphite. Russell, N .J. its control. Rodricks, J.Y ., Hesseltine, cw. andan d Gou ld , G .W . (ed s.) F oo d P reserv ativ es. G lasg ow : M ehlman, M .A M ycotosins in Human and AnimalB lac kie , 1 991. 7 3-8 8. H ealth. Illinois: Pathotox Publishing Inc, 1977. 83-90.

Kurtzman, CP., Horn, B.W . and Hesseltine, CW . As-pergi ll us nom ius , a new aflatoxin producing species Yousef, AE. and Marth, E.H . Growth and synthesis ofrelated to Aspergil lus f lavus an d Asperg il lu s tamarii . a fla to xin by Aspergil lus parasit icus in the presence ofA ntonie van Leeuw . 53: 147-158; 1987. sorbic acid. J. Food Prot. 44: 736-741; 1981.

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Effect of Food Preservatives on Growth and Aflatoxin Production of us Flavus in Liquid Medium