In vitro effect of phenolic antioxidants on germination, growth and aflatoxin B1 accumulation by peanut Aspergillus section Flavi

In vitro effect of phenolic antioxidants on germination, growthand aflatoxin B1 accumulation by peanut Aspergillus sectionFlavi

M.A. Passone1 S.L. Resnik2 and M.G. Etcheverry11Departamento de Microbiologıa e Inmunologıa, Facultad de Ciencias Exactas Fısico Quımicas y Naturales, Universidad Nacional de

Rıo Cuarto, Cordoba, and 2Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Buenos

Aires, Buenos Aires, Argentina

2004/1158: received 5 October 2004, revised and accepted 24 November 2004

ABSTRACT

M.A . PASSONE, S.L . RESNIK AND M.G. ETCHEVERRY. 2005.

Aims: The effectiveness of the food-grade antioxidants butylated hydroxytoluene (BHT), trihydroxybutyro-

phenone (THB), propyl paraben (PP) and butylated hydroxyanisole (BHA) at 1, 10 and 20 mmol l)1

concentrations on germination, growth, and aflatoxin B1 (AFB1) production by Aspergillus section Flavi strains

was determined.

Methods and Results: Assays on the lag phase of germination, germination percentage, germ tube elongation

rate, lag phase, growth rate and AFB1 production by three strains of Aspergillus flavus and three of Aspergillusparasiticus were carried out in vitro on peanut extract meal agar conditioned at different water activities (aw: 0Æ982,0Æ971, 0Æ955, 0Æ937). The antioxidants PP and BHA efficiently inhibited the germination of the two species tested at

the doses 10 and 20 mmol)1. The antioxidants PP and BHA at 1 mmol l)1 and THB at 20 mmol l)1 reduced

the germ tube elongation rate most effectively, regardless of aw levels. An increase in the lag time and a reduction

in the growth rate of 100% of the strains was observed, this was due to the action of BHT at the doses 10 and

20 mmol)1 at 0Æ982, 0Æ971 and 0Æ955 aw, although these treatments stimulated the AFB1 accumulation in most of the

fungi tested. The more effective antioxidants were PP and BHA, which increased the lag phase, reduced the

growth rate and AFB1 production in all of the strains at the four aw assayed. At concentrations 10 and 20 mmol l)1,

these antioxidants totally inhibited fungal development.

Conclusions: The study shows that the antioxidants BHA and PP are effective fungal inhibitors to peanut

Aspergillus section Flavi in wide range of water activity.

Significance and Impact of the Study: The results suggest that phenolic antioxidants, BHA and PP, can be

effective fungitoxicants on aflatoxigenic strains in peanut at industrial level.

Keywords: antioxidants, Aspergillus, chemical control, peanut, water activity.

INTRODUCTION

A variety of oil-rich seeds, particularly peanut (Arachishypogaea L.) offer important substrates for the growth and

subsequent aflatoxin production by different members of

Aspergillus section Flavi: Aspergillus flavus Link, Aspergillusparasiticus Speare, Aspergillus nomius, Aspergillus pseudota-marii and Aspergillus bombycis. These fungi are able to

produce aflatoxins which are potent hepatotoxic carcinogenic

metabolites (IARC 1993). These species can infect cereals

before and after harvest (Cotty 1997; Barros et al. 2003) Theaflatoxin-producing fungi in preharvest peanuts occur under

Correspondence to: Miriam Etcheverry, Departamento de Microbiologıa e

Inmunologıa, Facultad de Ciencias Exactas, Fısico Quımicas y Naturales, Universidad

Nacional de Rıo Cuarto, Ruta Nacional 36, km 601, 5800, Cordoba, Argentina

(e-mail: [email protected]).

ª 2005 The Society for Applied Microbiology

Journal of Applied Microbiology 2005, 99, 682–691 doi:10.1111/j.1365-2672.2005.02661.x

conditions of heat and drought stress during the latter stages

of the growing season (Pettit et al. 1971; Hill and Lacey 1983;

Wilson and Stansell 1983; Blankenship et al. 1984; Cole et al.1989; Dorner and Cole 2002). Preharvest peanut seeds

contain mycelia and spores of aflatoxicogenic fungi, which

can result in a significant decrease in grain quality when they

are stored. If the stored conditions are not good, these seeds

may cause serious damage and aflatoxin accumulation at

higher than internationally accepted levels. Because peanuts

are primarily used for food, strict regulatory limits for the

amount of aflatoxin in peanut products have been established.

The aflatoxin legislation has tolerance levels of 20 ppb of total

aflatoxins (aflatoxins B1, B2, G1, G2) for raw and processed

peanut destined to human consumption (http://www.

micotoxinas.com.br/legisla-in.htm). Thus, peanuts con-

taminated above that level cannot be used for food,

which may result in great economic losses to the peanut

industry. Studies realized by Park and Njapau (1989) in

Argentina showed that the occurrence levels of aflatoxin in

peanuts were between 20 and 200 ppb. Likewise, Barros

et al. (2002), in a study also carried out in Argentina

determined that the media levels of occurrence of aflatoxin B1

(AFB1) in peanut were between 4Æ3 and 134 ppb. Thus

alternative strategies are necessary to inhibit the growth and

aflatoxin production of these important mycotoxicogenic

species.

Many chemical products are used to prevent bacteria and

yeast growth. Antioxidants such as propyl paraben (PP), and

other organic acids such as propionic, benzoic and sorbic

acids are being used to control fungal spoilage of food.

Parabens has been shown to have antimicrobial action,

inhibiting the growth of Clostridium botulinum and its toxin

production (Robach and Piederson 1978). The butylated

hydroxyanisole (BHA) and butylated hydroxytoluene

(BHT) have been associated with an antimicrobial action

on different bacterial and fungi genus (Davidson et al. 1981;Eubanks and Beuchat 1982; Shelef and Liang 1982). Studies

by Thompson (1991, 1992), Etcheverry et al. (2002) and

Nesci et al. (2003) suggested that the phenolic antioxidants

could control conidial germination and growth of some

mycotoxicogenic Aspergillus spp. and Fusarium spp. in

maize.

There is no information available on the effect of

antioxidants on the growth and aflatoxin production by

these toxicogenic fungi in peanut. Thus, the objective of this

study was to determine the effectiveness of a range of food

antioxidants, BHT, trihydroxybutyrophenone (THB), PP

and BHA, in the control on (i) the lag phase of germination,

(ii) the percentage of germination, (iii) the germ tube

elongation rate, (iv) the lag phase of growth, (v) the growth

rate and (vi) AFB1 accumulation by A. flavus and

A. parasiticus on a peanut extract medium under different

water availability conditions.

MATERIALS AND METHODS

Fungal isolates

Six isolates belonging to the genus Aspergillus: A. parasiticus(CHG24), A. parasiticus (CHG44), A. parasiticus (CHG54),

A. flavus (CHG28), A. flavus (CHG40) and A. flavus(CHG46), were used in these experiments. These toxico-

genic isolates were all recovered from peanut seeds (Barros

et al. 2003). The isolates were maintained at 4�C on slants of

malt extract agar (MEA) and in 15% glycerol at )80�C.

Culture medium

The medium used in this study was 3% peanut meal extract

agar (PMEA). The water activity was adjusted at 0Æ982,0Æ971, 0Æ955 and 0Æ937 by the addition of known amounts of

the non-ionic solute glycerol (Dallyl and Fox 1980). The

water activity of all media was determined with a Thermo-

constanter Novasina TH200 (Novasina AG, Zurich, Swit-

zweland). The medium was autoclaved at 121�C for 20 min

before cooling to 50�C and poured into 9-cm sterile Petri

dishes.

Antioxidants

All the chemical antioxidants were obtained from Sigma

Chemical (Dorset, UK). These were benzoic acid, 2(3)-tert-butyl-4 hydroxyanisol (BHA); 2,6-di (tert-butyl)-p-cresol(BHT); 3,4,5-trihydroxybenzoic acid (n-propyl gallate,

THB); n-propyl p-hydroxybenzoato (PP). Stock solutions

of BHA, BHT, THB and PP (1, 10 and 20 mmol l)1) were

prepared in 95% ethyl alcohol, and the appropriate

concentration of each antioxidant was added to PMEA.

Flasks of molten media were shaken prior to pouring, to

ensure that the antioxidant was dispensed in each Petri dish.

Spore germination determinations

Spores of 5-day-old cultures obtained from each test fungus

grown on MEA plate were suspended in 10 ml of sterile

glycerol/water solutions at the required water availability.

This conidial suspension was poured over PMEA (supple-

mented with the appropriate concentration of antioxidants),

to cover the entire surface, and the excess of liquid was

drained off. Control fungi were prepared without antioxi-

dants. Petri dishes of the same aw treatment were sealed in

polythene bags and incubated at 25�C. All experiments were

replicated at least three times per treatment. Periodically,

three agar discs (5 mm diameter) were aseptically removed

from each replicate plate using a cork borer. The discs were

placed on a slide, and examined microscopically. For this

experiment, spores were considered germinated when the

germ tube was longer than the diameter of the spore. A test

CONTROL OF PEANUT AFLATOXIGENIC FUNGI BY ANTIOXIDANTS 683

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 682–691, doi:10.1111/j.1365-2672.2005.02661.x

https://www.researchgate.net/publication/229798387_Influence_of_parahydroxybenzoic_acid_esters_on_the_growth_and_toxin_production_of_Clostridium_botulinum_10755A?el=1_x_8&enrichId=rgreq-01083000-d437-40f7-8c92-b2d7d597d5b6&enrichSource=Y292ZXJQYWdlOzc2NTE1NjQ7QVM6MTUxOTM4MDU4NDI4NDE2QDE0MTMyMzYyNjQ4Nzc=

was considered positive when at least 10% of the spores had

germinated. Germination was reported as a percentage of

the conidial population, determined periodically (hours) by

microscopy and compared with the appropriate control. The

lag phase for germination was the number of hours needed

for 10% of spores to germinate. The elongation germ tube

was measured periodically (hours). The elongation rate

(lm h)1) was calculated (Marın et al. 1995).

Growth studies

Fungi were grown on PMEA for 5 days at 25�C to obtain

heavily sporulated cultures. PMEA plates at different awconditions were amended with the appropriate concentra-

tion of each antioxidant and a spot of spores suspended in

semisolid agar (0Æ2% water agar) were inoculated in the

centre of each plate. Petri dishes of the same aw values were

sealed in polythene bags. The inoculated plates were

incubated at 25�C and the colony radius was measured

daily. For each colony, two radii, measured at right angles,

were averaged to find the mean radius. All colony radii were

determined by using three replicates for each test fungus.

The radial growth rate (cm h)1) was subsequently calculated

by linear regression of the linear phase for growth and the

time at which the line intercepts the x-axis was used to

calculate the lag phase in relation to fungal isolate,

antioxidant concentration and water activity (Marın et al.1995). After evaluating growth, all samples were frozen for

later extraction and AFB1 quantification.

Aflatoxins B1 analysis

A piece of PMEA (1 · 1 cm) from each colony of each

treatment was taken and weighed, transferred to an Epp-

endorf tube. A quantity of 500 ll of chloroform was then

added. The mixture was agitated at 850 g for 20 min. The

piece of agar was removed and the chloroform extract

evaporated to dryness under nitrogen gas. The residue was

redissolved in 10 ll of chloroform for screening by thin

layer chromatography. The samples were applied onto silica

gel plates G60 (0Æ25 mm) without fluorescent indicator (E.

Merck, Darmstadt, Germany). An acetone/chloroform

(10 : 90) solvent system was used to develop the plaques.

The spots were visualized under UV light (360 nm).

Positives samples were quantitatively determined by HPLC,

following the detection methodology proposed by Trucksess

et al. (1994). An aliquot (200 ll) was derivatized with 700 llof trifluoroacetic acid/acetic acid/water (20 : 10 : 70). The

derivatized aflatoxins (50 ll solution) were analysed using a

reversed-phase HPLC/fluorescence detection system. The

HPLC system consisted of a Hewlett Packard 1100 pump

(Palo Alto, CA, USA) connected to a Hewlett Packard 1046

programmable fluorescence detector, interfaced to a Hewlett

Packard Chem Station. Chromatographic separations were

performed on a stainless steel, C18 reverse phase column

(150 mm · 4Æ6 mm i.d., 5 lm particle size; Luna-Phenom-

enex, Torrance, CA, USA). Water/methanol/acetonitrile

(4 : 1 : 1) was used as the mobile phase, at a flow rate of

1 ml min)1. Aflatoxin derivative fluorescence was recorded

at excitation and emission wavelengths of 360 and 440 nm

respectively. Standard curves were constructed with differ-

ent levels of AFB1 and AFG1. These toxins were quantified

by correlating peak height of sample extracts and those of

standard curves. The limit of detection of the analytical

method was 1 ng g)1.

Statistical analyses

Statistical analyses were made using SigmaStat program

Version 2.03 (SPSS Inc, Chicago, IL, USA). Mean

germination rate, growth rate and AFB1 data were per-

formed by analysis of variance (ANOVAANOVA) (P < 0Æ001). F-valuewas found using ANOVAANOVA. The significant differences for

germination rate and growth rate were established by using

Duncan’s New Multiple Range Test at P ¼ 0Æ05 level.

Fisher’s LSD test (P ¼ 0Æ05) was made to compare mean

values of treatments for AFB1 production to determine the

significant differences between the treatments and control.

RESULTS

Antioxidants and water activity, effects ongermination

Table 1 shows the effect of a concentration range of BHT

and THB on germination percentage of three isolates of

A. parasiticus (CHG24, CHG44, CHG54) and three isolates

of A. flavus (CHG28, CHG40, CHG46). The conidial

germination at 25�C was fastest at 0Æ982 aw and slowest

under water stress (0Æ937 aw). At 6 h of incubation and at the

highest water activity (0Æ982 aw), the germination percentage

ranged from 0 to 28% for all species tested, while at 0Æ937aw, the germination percentage ranged from 0 to 17% and 0

to 22% for A. parasiticus and A. flavus respectively. Theseresults suggest that the conidial germination of Aspergillussection Flavi species tested have a very similar lag phase of

germination. Under these water availability conditions

(0Æ982, 0Æ937 aw) the 100% germination of the conidia

ranged from 20 to 27 h and 27 to 35 h respectively. The

antioxidants BHT (1, 10, 20 mmol l)1) and THB (1,

10 mmol l)1) did not reduce the germination percentage

of all isolates when compared with the controls at the

environmental conditions assayed.

The time required for 10% germination of spores of

A. flavus and A. parasiticus was affected by the water activity

conditions (Table 2). At 0Æ982 aw the lag time of germina-

684 M.A. PASSONE ET AL.


tion was shortest and ranged between 2Æ4 and 6 h, whereas

the longest lag time of germination (5Æ7–17Æ4 h) was

observed at the lowest water condition (0Æ937 aw). The lag

time of germination also changed with the antioxidant used.

At 0Æ982 aw and 1 mmol l)1 concentration the fungal isolates

had slightly longer lag times using PP and BHA, which

ranged between 5Æ8 and 21Æ5 h. When THB and BHT were

used at the same water activity (0Æ982 aw) and antioxidant

concentration (1 mmol l)1), the lag time ranged from 2Æ9 to

10Æ1 h, thus results very similar to the control were

obtained. PP at the dose of 1 mmol l)1 showed to be more

effective as the water activity decreased.

The effect of antioxidants on germ tube elongation rate at

different levels of water activity is shown in Figs 1a–d and

2a–d. The germ tube elongation rate of A. flavus isolate wasstimulated by THB at 1 mmol l)1 concentration, regardless

of aw, whereas for A. parasiticus, the germination rate

decreased between 12 and 97% when middle water activities

Table 1 Effect of the antioxidants on ger-

mination percentage of fungal isolates on

peanut extract agar at two water activities

Strains

Incubation hours

Germination (%)

0Æ982 aw 0Æ937 aw

C BHT

(mmol l)1)

THB

(mmol l)1)

C BHT

(mmol l)1)

THB

(mmol l)1)

0 1 10 20 1 10 20 0 1 10 20 1 10 20

CHG24

6 15 14 13 13 19 23 15 – 14 – – 14 17 –

13 34 84 46 56 48 50 33 11 31 13 16 30 36 16

20 100 100 100 100 100 74 80 29 74 71 28 56 64 42

27 100 100 100 100 100 100 100 46 100 100 88 93 94 62

35 100 100 100 100 100 100 100

CHG44

6 11 22 19 20 14 18 – – 16 – – 10 10 –

13 100 100 100 100 100 100 23 – 34 – – 31 39 –

20 100 100 100 100 100 100 85 32 72 45 47 56 64 12

27 100 100 100 100 100 100 100 51 100 97 96 86 95 43

35 79 100 100 100 100 100 79

CHG54

6 16 26 28 22 21 19 – – – – – – – –

13 91 95 79 76 100 83 31 41 14 12 10 – – –

20 100 100 100 100 100 100 91 72 46 43 40 25 26 –

27 100 100 100 100 100 100 100 100 95 90 92 49 58 –

35 100 100 100 100 64 75 13

CHG28

6 25 24 27 28 22 20 19 14 21 16 16 22 17 –

13 86 100 100 78 84 66 42 30 45 34 36 49 44 18

20 100 100 100 100 100 100 94 58 90 94 77 100 100 62

27 100 100 100 100 100 100 100 100 100 100 100 100 100 83

35 100 100 100 100 100 100 100 100

CHG40

6 14 16 10 – 14 13 12 – 15 – – – – –

13 90 76 59 63 80 59 41 – 33 15 12 19 28 25

20 100 100 91 97 100 91 63 22 66 57 39 61 64 48

27 100 100 100 100 100 100 85 59 100 100 96 100 100 100

35 100 100 100 100 100 100 100 76 100 100 100 100 100 100

CHG46

6 10 22 13 12 10 19 14 – 6 – – – 18 –

13 59 62 59 62 64 100 61 17 48 28 26 23 41 29

20 91 96 90 96 99 100 93 57 85 68 74 72 87 74

27 100 100 100 100 100 100 100 95 100 100 100 100 100 100

35 100 100 100 100 100 100 100

BHT, butylated hydroxytoluene; THB, trihydroxybutyrophenone.



Table 2 Lag time before germination of Aspergillus parasiticus and A. flavus isolates on peanuts extract agar supplemented with BHT, THB, PP and

BHA at different water availabilities

Strains Control

Lag phase (h)

BHT (mmol l)1) THB (mmol l)1) PP (mmol l)1) BHA (mmol l)1)

1 10 20 1 10 20 1 10 20 1 10 20

0Æ982 aw CHG24 4 4Æ8 7Æ8 3Æ5 3Æ7 9Æ2 11Æ7 5Æ8 >30 >30 8Æ1 >30 >30

CHG44 5Æ5 3Æ4 11 3Æ5 3Æ1 5 23 14 >30 >30 10 >30 >30

CHG54 3Æ8 10Æ1 5Æ6 10Æ1 4Æ6 10Æ1 14Æ2 14Æ6 >30 >30 11 >30 >30

CHG28 2Æ4 2Æ9 6Æ2 4 5Æ4 7Æ9 8Æ3 16Æ5 >30 >30 10Æ4 >30 >30

CHG40 4Æ3 4Æ9 7Æ3 9Æ1 8Æ6 6Æ3 14Æ5 21Æ5 >30 >30 6Æ5 >30 >30

CHG46 6 3Æ3 6Æ6 4Æ8 8Æ5 4Æ7 6Æ9 16Æ1 >30 >30 6 >30 >30

0Æ971 aw CHG24 6Æ9 6Æ1 19Æ5 5Æ3 4Æ9 3Æ6 7Æ9 8Æ3 >30 >30 11Æ9 >30 >30

CHG44 8Æ7 8Æ7 7Æ4 10Æ1 8Æ7 14Æ7 18Æ9 25 >30 >30 14Æ7 >30 >30

CHG54 5Æ9 4Æ7 4Æ7 4Æ1 4 10Æ1 15 32Æ1 >30 >30 15 >30 >30

CHG28 3 6Æ5 4Æ8 6Æ8 6Æ8 11Æ5 16Æ7 10Æ2 >30 >30 13 >30 >30

CHG40 5Æ5 4Æ9 7Æ3 9Æ1 8Æ6 6Æ3 14Æ5 21Æ5 >30 >30 6Æ5 >30 >30

CHG46 3 3Æ3 6Æ6 4Æ8 8Æ5 4Æ7 6Æ9 16Æ1 >30 >30 6 >30 >30


CHG44 8Æ7 8Æ7 7Æ4 10Æ1 8Æ7 14Æ7 18Æ9 25 >30 >30 14Æ7 >30 >30

CHG54 5Æ9 4Æ7 4Æ7 4Æ1 4 10Æ1 15 32Æ1 >30 >30 15 >30 >30

CHG28 3 6Æ5 4Æ8 6Æ8 6Æ8 11Æ5 16Æ7 10Æ2 >30 >30 13 >30 >30

CHG40 5Æ6 6 7Æ3 9Æ1 8Æ6 6Æ3 14Æ5 21Æ5 >30 >30 6Æ5 >30 >30

CHG46 4Æ1 4Æ9 6Æ6 4Æ8 8Æ5 11Æ2 16Æ5 16Æ1 >30 >30 12Æ9 >30 >30

0Æ937 aw CHG24 14Æ2 4Æ3 10 8Æ5 5 2Æ7 8Æ4 28Æ5 >30 >30 15Æ5 >30 >30

CHG44 14Æ5 3Æ8 14 14 4Æ2 3Æ3 17Æ1 29Æ6 >30 >30 14Æ5 >30 >30

CHG54 8Æ1 9Æ4 10Æ9 12Æ9 14Æ5 14Æ5 27Æ1 27Æ1 >30 >30 9Æ9 >30 >30

CHG28 5Æ7 2Æ9 3Æ9 3Æ8 2Æ7 3 3Æ2 22Æ3 >30 >30 7Æ5 >30 >30

CHG40 17Æ4 3Æ9 9Æ8 10Æ7 9Æ4 4Æ9 5Æ3 27Æ5 >30 >30 19Æ3 >30 >30

CHG46 8Æ4 2Æ8 4Æ7 5Æ1 5Æ8 3Æ2 4Æ5 22Æ3 >30 >30 17Æ3 >30 >30

BHT, butylated hydroxytoluene; THB, trihydroxybutyrophenone; PP, propyl paraben; BHA, butylated hydroxyanisole.

14

(a) (b)

(c) (d)

a

aab

ab

cc c

a

aa

ab

ababc

cdebcd

bcd

dee e

a

a a a

a

a

a

eede

b bcbc cd

a

c d d

ab

121086420

10

8

6

4

2

00 1 10 20

Concentration (mmol l–1)

Ger

min

atio

n ra

te (

µm h

–1)

0 1 10 20

0 1 10 20

0·982aw 0·971aw

0·955aw 0·937aw

0 1 10 20

141210

86420

14121086420

Fig. 1 Effect of antioxidants BHT, THB, PP

and BHA on the germination rate of the iso-

late CHG44 (Aspergillus parasiticus) under

different water activity conditions: (a) 0Æ982;(b) 0Æ971; (c) 0Æ955 and (d) 0Æ937. j, BHT;d,

THB; m, PP; ·, BHA. Data with the same

letter for each antioxidant are not significantly

different according to Duncan’s New Mul-

tiple Range Test (P ¼ 0Æ05)



(0Æ971, 0Æ955 aw) and three dose of THB (1, 10,

20 mmol l)1) were assayed. Under the driest environmental

condition (0Æ937 aw) a germination rate stimulation in the

presence of the two lowest THB and BHT concentration (1,

10 mmol l)1) was observed. The lowest concentration

(1 mmol l)1) of BHA and PP, regardless of the water

conditions, reduced the germ tube elongation rate between

24 and 86% for A. parasiticus and between 23 and 77% for

A. flavus. This assay showed complete inhibition of the

germination conidia for all isolates over a 72-h period by

antioxidants, BHA and PP at the concentrations of 10 and

20 mmol l)1 (data not shown).

Effects of antioxidants and water activity ongrowth and AFB1 production

The lag phase increased from 4Æ6 to 24 h and from 32Æ9 to

47Æ7 h when the water availability varied from optimal to

marginal conditions (0Æ982–0Æ937 aw) for both A. parasiticus(CHG24, CHG44, CHG54) and A. flavus (CHG28,

CHG40, CHG46) (Table 3). The lag phase of all species

tested was increased for all antioxidants treatments at the

two highest water conditions (0Æ982, 0Æ971 aw) assayed.

Aspergillus section Flavi strains showed an increase in their

lag phase between 37 and 90% and 2 and 81% when grown

with 1 mmol l)1 of PP and BHA, respectively, regardless of

the aw.The profiles of growth rates (Figs 3a–d and 4a–d) show

that, in general, the antioxidants BHT and THB, at the

lowest concentration used (1 mmol)1), stimulated the

growth of the fungal isolates. The sensitivity of A. parasiticusisolates to 10 mmol l)1 of BHT was demonstrated. A

growth reduction between 49 and 91% was observed when

compared with control. However, a considerable inhibitory

effect was exerted by 20 mmol l)1 of BHT and THB on all

these aflatoxicogenic fungi, decreasing significantly the

growth rate at the two lowest water activities (0Æ955, 0Æ937aw) assayed. BHA at the dose of 1 mmol l)1 reduced the

growth rate of the fungal isolates between 36 and 74% for

A. parasiticus isolate and between 20 and 50% for A. flavusstrain, regardless of the aw. The antioxidant PP at

1 mmol l)1 reduced significantly the growth rate of both

A. parasiticus CHG24 and A. flavus CHG46, with a

reduction percentage ranging between 49 and 91% and 20

and 50%, respectively, at all water activities conditions.

BHA and PP completely suppressed mycelial growth of all

aflatoxicogenic fungi tested at concentrations greater than

10 mmol l)1, regardless of aw.Aflatoxin B1 production followed the same general

patterns as was observed with the growth rate when the

fungi tested grew under the action of the chemicals PP,

BHA at 1 mmol l)1 and THB at 20 mmol l)1, which

significantly affected this metabolite secondary production

(Table 4). After 11 days of incubation, AFB1 was reduced

between 30 and 99% with PP 1 mmol l)1 concentration at

0Æ982 aw, whereas this metabolite was reduced from 79 to

93% and from 51 to 97% in the presence of BHA

1 mmol l)1 dose and THB 20 mmol l)1 respectively. These

antioxidants were also effective at 0Æ971 aw, which reduced

the AFB1 production for A. parasiticus isolates between 20

and 94%, whereas the AFB1 production by A. flavus isolateswas reduced between 4 and 95%. In general, it was

observed that the levels of AFB1 production by the

Aspergillus isolates tested was stimulated using BHT,

0·6a a

aa

c c

d

eg f

hh

bbc

ed

f

f

h h

g

c

c

a

aa

ab

bd

ef f

bb

d

e

aa

f

0·50·40·30·20·1

0

0·60·50·40·30·20·1

0

0·4

0·3

0·2

0·1

0

0·4

0·5

(a) (b)

(c) (d)

0·3

0·2

0·1

00 1 10 20

0·982aw 0·971aw

0·937aw0·955aw

0


Gro

wth

rat

e (c

m h

–1)

1 10 20

0 1 10 20 0 1 10 20

Fig. 2 Effect of antioxidants BHT, THB, PP

and BHA on the germination rate of the iso-

late CHG46 (Aspergillus flavus) under differ-

ent water activity conditions: (a) 0Æ982; (b)0Æ971; (c) 0Æ955 and (d) 0Æ937. j, BHT; d,




tiple Range Test (P ¼ 0Æ05)



regardless of the aw. At the two lowest levels of water

conditions (0Æ955, 0Æ937 aw), none of the isolates were able

to produce AFB1.

The fungi tested in this investigation differed in their

sensitivity to the different antioxidants, although BHA and

PP were effective in preventing the growth and AFB1

Table 3 Effect of antioxidants on the lag phase means of aflatoxicogenic fungi at different water activities

Strains Control

Lag phase (h)

BHT (mmol l)1) THB (mmol l)1) PP (mmol l)1) BHA (mmol l)1)

1 10 20 1 10 20 1 10 20 1 10 20

0Æ982 aw CHG24 21Æ1 28Æ4 32Æ3 31Æ6 30Æ3 30 31Æ6 33Æ3 >90 >90 30Æ3 >90 >90

CHG44 14 22Æ5 36Æ8 38Æ2 22Æ7 24Æ7 29Æ5 38 >90 >90 30 >90 >90

CHG54 17Æ6 25Æ8 44Æ5 41 25Æ1 26Æ2 30Æ4 35Æ9 >90 >90 18 >90 >90

CHG28 24 27 39Æ1 36 28Æ8 29Æ1 29 50 >90 >90 25 >90 >90

CHG40 4Æ6 26Æ3 39Æ3 34Æ5 21Æ4 24Æ1 29Æ3 45Æ1 >90 >90 24Æ3 >90 >90

CHG46 8Æ9 21Æ1 33 25Æ8 19Æ4 21Æ1 25Æ1 21Æ4 >90 >90 16Æ9 >90 >90

0Æ971 aw CHG24 24 27 39Æ1 36 28Æ8 29Æ1 29 50 >90 >90 25 >90 >90

CHG44 18 33Æ5 46Æ1 48 29Æ5 26 46Æ6 69Æ1 >90 >90 18Æ3 >90 >90

CHG54 25Æ6 33Æ5 36Æ2 32Æ7 25Æ3 28Æ4 39Æ1 61Æ1 >90 >90 30Æ5 >90 >90

CHG28 11Æ6 28Æ8 36Æ1 44Æ5 27Æ6 28Æ9 33Æ3 28Æ3 >90 >90 24Æ3 >90 >90

CHG40 16Æ3 33Æ4 39Æ1 41Æ2 22Æ4 28 29Æ1 53Æ7 >90 >90 21Æ7 >90 >90

CHG46 14Æ4 28Æ3 41Æ8 45 20Æ8 16Æ2 25Æ7 41Æ4 >90 >90 19Æ9 >90 >90


CHG44 25Æ2 32Æ2 44Æ4 41Æ2 22Æ4 26Æ2 35Æ6 54Æ6 >90 >90 37Æ5 >90 >90

CHG54 26Æ9 28Æ7 34Æ3 27Æ6 19Æ6 23Æ7 31Æ8 65Æ9 >90 >90 45Æ9 >90 >90

CHG28 20Æ6 28Æ9 32Æ4 20Æ7 12Æ7 32Æ1 38Æ5 46Æ1 >90 >90 44Æ2 >90 >90

CHG40 20Æ1 15Æ1 31Æ3 36Æ9 17Æ8 26 29Æ3 56Æ7 >90 >90 46 >90 >90

CHG46 17Æ3 24Æ9 41Æ2 25Æ9 12 24Æ8 31Æ4 49Æ1 >90 >90 40 >90 >90

0Æ937 aw CHG24 45Æ7 38Æ4 54Æ1 43Æ8 39 35Æ8 38Æ6 93 >90 >90 46Æ6 >90 >90

CHG44 42Æ4 34Æ3 38Æ5 38Æ9 32 25Æ8 28Æ8 84Æ6 >90 >90 44 >90 >90

CHG54 47Æ7 34Æ1 47Æ4 45 28Æ8 27Æ4 33 87Æ7 >90 >90 48Æ8 >90 >90

CHG28 32Æ9 30Æ6 34 40 28Æ9 27Æ8 38Æ8 63Æ3 >90 >90 47Æ7 >90 >90

CHG40 41Æ7 32Æ6 38Æ8 46Æ8 35 31Æ7 32Æ5 85Æ8 >90 >90 54Æ1 >90 >90

CHG46 39 31Æ2 36Æ7 46Æ8 33Æ9 26Æ4 38Æ2 68Æ4 >90 >90 41Æ7 >90 >90

BHT, butylated hydroxytoluene; THB, trihydroxybutyrophenone; PP, propyl paraben; BHA, butylated hydroxyanisole.

0·6

a

aa

aa

aa

b

c

c

aa

b

e

b

f

d

gh h

c

a

aa a

b

c

cb

ff

b

a

de

a

a

a

cd

0·50·40·30·20·1

0

0·5

0·4

0·3

0·2

0·1

0

0·4

0·3

0·2

0·1

0

0·4

0·5

0·3

0·2

0·1

00 1 10 20 0 1 10 20

0 1 10 20 0 1 10 20

0·982aw 0·971aw

0·937aw0·955aw


Gro

wth

rat

e (c

m h

–1)

(a) (b)

(c) (d)Fig. 3 Effect of antioxidants BHT, THB, PP

and BHA on the growth rate of the isolate

CHG24 (Aspergillus parasiticus) under differ-

ent water activity conditions: (a) 0Æ982; (b)0Æ971; (c) 0Æ955 and (d) 0Æ937. j, BHT; d,




tiple Range Test (P ¼ 0Æ05) (cm h)1 ¼ radial

growth value)



production to the six aflatoxicogenic strains at concentra-

tions of 10 and 20 mmol l)1.

DISCUSSION

Previous experiments have shown that antioxidants as food

additive have a protective action on the organoleptic

characters and against the modification by oxidation of the

food components, preserving their bromatologic quality.

These antioxidants are considered with the new antimicro-

bial substance (Valle Vega and Florentino 2000), neverthe-

less it is well established that resistance to THB by fungal

pathogens is common (Thompson 1992; Etcheverry et al.

2002; Nesci et al. 2003). Our results provide further

evidence, as in many treatments THB stimulated the

germination, growth and AFB1 production by the aflatoxi-

cogenic species tested. The resistance to benzoic acid-related

fungicides with THB may be considered to result from

altered amino acid sites on the tubulin with a reduced

binding affinity for the fungicides (Martin et al. 1992), andmay have been encouraged by the use of fungicides with the

same mode of action for control of foliar disease in the

plantations (Slabaugh and Grove 1982).

Butyl hidroxytoluene and its analogues probably act via a

nonspecific mechanism, involving the perturbation of

membrane function, for example, BHT appears to cause a

Table 4 Effect of antioxidants on aflatoxin B1 production by Aspergillus parasiticus and A. flavus strains

Treatments/

concentration

(mmol l)1)

Aflatoxin B1 (ng g)1)

A. parasiticus A. flavus

CHG24 CHG44 CHG54 CHG28 CHG40 CHG46

0Æ982 aw 0Æ971 aw 0Æ982 aw 0Æ971 aw 0Æ982 aw 0Æ971 aw 0Æ982 aw 0Æ971 aw 0Æ982 aw 0Æ971 aw 0Æ982 aw 0Æ971 aw

Control/0 13072Æ0 a 5233Æ1 abc 78Æ0 a 9161Æ0 bc 11239Æ3 a 9304Æ0 cd 557Æ2 c 597Æ2 e 9078Æ6 ab 8Æ2 cd 123Æ4 b 108Æ3 b

BHT/1 4115Æ1 bcd 5312Æ6 abc 25Æ9 bcd 12269Æ8 a 7029Æ9 abc 16918Æ1 ab 456Æ5 c 1939Æ2 b 3288Æ0 c 46Æ0 b 576Æ3 b 821Æ5 b

BHT/10 6524Æ1 b 6534Æ9 ab 58Æ2 ab 11124Æ5 ab 8652Æ9 ab 15152Æ3 abc 1731Æ3 b 1330Æ2 c 13148Æ1 a 108Æ1 a 4225Æ2 a 5270Æ4 a

BHT/20 7106Æ4 b 7558Æ1 a 39Æ9 bcd 7532Æ4 cd 6445Æ8 abcd 9913Æ8 cd 2940Æ7 a 2616Æ5 a 7280Æ4 bcd 37Æ2 bc 4790Æ9 a 5634Æ6 a

THB/1 6686Æ8 b 4625Æ9 abc 10Æ9 cd 9959Æ9 abc 7298Æ8 abc 20397Æ3 a 128Æ7 d 1011.9 d 8863Æ1 ab n.d. d 52Æ2 b 251Æ6 b

THB/10 5592Æ3 bc 2377Æ8 cde 41Æ2 abc 9040Æ3 bc 3470Æ3 bcd 13367Æ4 bc 96Æ5 d 90Æ4 f 7708Æ1 abc n.d. d 203Æ0 b 26Æ5 b

THB/20 1472Æ8 d 4205Æ6 bcd 2Æ6 d 5579Æ9 d 1913Æ3 cd 3752Æ2 de 20Æ3 d 50Æ9 f 4468Æ5 bcd 5Æ1 cd 15Æ1 b 30Æ3 b

PP/1 1299Æ6 d 1173Æ3 de 15Æ6 cd 1854Æ6 e 2298Æ1 cd 1372Æ8 e 6Æ8 d 45Æ2 f 6366Æ6 bcd 7Æ9. cd 49Æ3 b 14Æ5 b

BHA/1 2797Æ5 cd 316Æ2 e 10Æ2 cd 2211Æ9 e 892Æ2 d 4818Æ4 de 37Æ0 d 27Æ3 f 1844Æ9 d 3Æ9 d 10Æ9 b 19Æ3 b

Mean values based on triplicate data. Mean in a column with a letter in common are not significantly different according to LSD test (P < 0Æ05).Detection limit: 1 ng g)1 (ppb).

BHT, butylated hydroxytoluene; THB, trihydroxybutyrophenone; PP, propyl paraben; BHA, butylated hydroxyanisole; n.d., not detected.

141210

86420

141210

86420

14121086420

20

aad

aa

a

aba

cd e e

eed

aca

aab

b b b

e e

c

a

aaab

c

ab

abc

abce a ace

ffeabd

15

10

5

00 1 10 20 0


1 10 20

0 1 10 20 0 1 10 20

Ger

min

atio

n ra

te (

µm

h–1

)

0·982aw 0·971aw

0·955aw 0·937aw

(a) (b)

(c) (d)Fig. 4 Effect of antioxidants BHT, THB, PP

and BHA on the growth rate of the isolate

CHG46 (Aspergillus flavus) under different

water activity conditions: (a) 0Æ982; (b) 0Æ971;(c) 0Æ955 and (d) 0Æ937. j, BHT; d, THB;

m, PP; ·, BHA. Data with the same letter for

each antioxidant are not significantly different

according to Duncan’s New Multiple Range

Test (P ¼ 0Æ05) (cm h)1 ¼ radial growth

value)



reduction in the permeability of vesicles of phospholipid

bilayer membranes (Singer and Wan 1977). The conidia of

the fungi tested in this investigation differed in their

sensitivity to BHT levels. Some strains were quite resistant

to BHT and could even produce higher amounts of AFB1 in

the presence of antioxidants, whereas other strains were very

sensitive to inhibition by BHT. This antioxidant was

effective in preventing germination, growth and aflatoxin

production by aflatoxicogenic strains at the concentration of

20 mmol l)1.

The most effective antioxidants assayed were BHA and

PP. Thompson (1994) showed that the minimal inhibition

concentration (MIC) of BHA was 500 lg ml)1 for species of

Aspergillus, whereas PP was the next most effective phenolic

antioxidant. For A. flavus and A. parasiticus the MIC was

500 and 1000 lg ml)1 respectively in PDA. The microbial

action of BHA showed an increase in sugars, amino acids

and proteins loss in Fusarium spp. (Thompson 1996). Also a

direct effect on the mitochondrial electron chain of trypan-

osomes has been shown, which inhibit respiration (Aldunate

et al. 1992). However, PPs appear to act mainly on the cell

membrane, eliminating the DpH component of the proto-

motive force and affecting energy transduction and substrate

transport by alteration of cell membrane permeability

(Liewen 1991; Adams and Moss 1995). In this work, the

growth and AFB1 production by the strains tested decreased

significantly at the lowest concentration of antioxidants PP

and BHA used (1 mmol l)1), as esters of p-hydroxybenzoicacid (parabens) on mycotoxicogenic fungi in the genera

Penicillium, Aspergillus and Fusarium have been shown to be

effective against these fungi but tend to be fungistatic rather

than fungicide (Aalto and Firman 1953; Chang and Branen

1975; Thompson 1992, 1994). Overall, ‡10 mmol l)1 of

BHA and PP completely inhibited growth at all aw levels

tested in this study, and, more importantly, they inhibited

AFB1 accumulation on a 3% peanut extract agar. Similar

results were obtained by Etcheverry et al. (2002). In their

research showed the impact of antioxidants on growth and

fumonisin production by Fusarium verticillioides and Fusa-rium proliferatum on maize meal agar. It was observed the

antioxidants PP and BHA completely inhibited the growth

of both species, regardless of the aw levels. Nesci et al.(2003), showed that the effect of BHA and PP at 1 mmol l)1

on lag phase and growth rate of Aspergillus section Flavi

from maize was maintained in the pH range between 6 and

8. At all pH values the inhibitory effect of BHA was higher

than PP. No AFB1 was detected in treatments of pH and

water activity on a maize meal agar.

Our study showed that it is possible to control the conidial

germination, fungal growth and AFB1 accumulation on a

PMEA by BHA and in a similar way. There is, therefore, a

posibility to develop the use of BHA and PP as food additive

to control aflatoxicogenic fungi. The effects of BHA and PP

on Aspergillus section Flavi strains on peanut grain is being

studied to establish their abilities as fungitoxicants.

ACKNOWLEDGEMENTS

This work was supported by grants from Agencia Cordoba

Ciencia and Secretarıa de Ciencia y Tecnica de la Univer-

sidad Nacional de Rıo Cuarto (Res. N� 077/03), 2003–04.

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Documents

In vitro effect of phenolic antioxidants on germination, growth and aflatoxin B1 accumulation by peanut Aspergillus section Flavi