Directory UMM :Journals:Journal of Stored Products Research:Vol 36.Issue1.Jan2000:
Journal of Stored Products Research 36 (2000) 75±87
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Myco¯ora and occurrence of fumonisins in freshly
harvested and stored hybrid maize
Reinaldo B. Orsi a, Benedito CorreÃa a,*, Claudia R. Possi b, Eliana
A. Schammass b, Jose R. Nogueira b, Silvia M.C. Dias c, Marisa A.B. Malozzi c
a
Departamento de Microbiologia, Instituto de CieÃncias BiomeÂdicas, Universidade de SaÄo Paulo, Brazil
b
Instituto de Zootecnia, Universidade de SaÄo Paulo, Brazil
c
Instituto Biologico, Universidade de SaÄo Paulo, Brazil
Accepted 9 June 1999
Abstract
The study of the myco¯ora in stored grain permits an evaluation of cereal storage conditions that
aect grain deterioration and the risk of mycotoxin contamination. Abiotic factors can directly aect
the relative frequency of fungal populations in stored grain. The aim of the present work was to study
the in¯uence of abiotic factors on variations of myco¯ora of freshly harvested and stored maize in
Brazil and the occurrence of fumonisins. Samples (195) of three hybrids of maize were analyzed monthly
during one year. Microbiological analysis revealed a predominance of Fusarium spp, which presented the
greatest total number of colony forming units per gram in the three hybrids, namely: Br 201 (11 104
to 5340 104 CFU/g), C 125 (18 104 to 2790 104 CFU/g) and Cx 322 (25 104 to 2940 104
CFU/g), followed by Penicillium spp, Aspergillus spp and 10 other fungal genera. Fusarium moniliforme
Sheldon was the most prevalent species (59.2% of Fusarium isolates in Br 201, 55.4% in C 125 and
69.2% in Cx 322). Fusarium spp showed signi®cant negative correlations with mean temperature and
relative humidity of the air. Higher temperatures and relative humidity at the end of the study and high
moisture content at the beginning of the study were observed. The CFU/g values recorded for the three
predominant genera exceeded the internationally accepted tolerance limits. The mycotoxicological
evaluation indicated contamination of 176 samples (90.2%) with fumonisin B1 and of 190 samples
(97.4%) with fumonisin B2. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Myco¯ora; Fumonisins; Aspergillus ¯avus; Fusarium moniliforme; Stored maize; Fumonisins in maize
* Corresponding author. Tel.: +55-11-818-7295; fax: +55-11-818-7354.
E-mail address: correabe@usp.br (B. CorreÃa)
0022-474X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 4 7 4 X ( 9 9 ) 0 0 0 2 9 - 6
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R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
1. Introduction
In Brazil, maize has historically had an important role in both human and animal nutrition.
Even before the arrival of European colonizers, the Brazilian natives already cultivated this
cereal. At present, Brazil is ranked as the third largest producer of maize in the world, coming
after the USA and China. Of the Brazilian annual yield of about 32 million tonnes in recent
years (Tsuneshiro and Okawa, 1996), 41% is used by the poultry and swine industries. A
substantial part of this crop comes from small producers and approximately 24.7% of the
harvest is stored and used on the producing farms (Pedrosa and Dezen, 1991).
In the state of SaÄo Paulo, maize plantations make up 7% of the cultivated land (Francisco
et al., 1997). The mean productivity of 2747 kg/ha recorded for this region during the 1990±
94 period re¯ects a poor local technology (Tsuneshiro and Okawa, 1996). The maizeproducing farms in this state have a mean area of 13.3 ha; 61.9% of the farmers receive
technical assistance, 44.1% use soil analysis, and 46.2% use hybrid seed (Francisco et al.,
1997).
The overall world loss of grains is considered to be 5% of the total production (FAO/WHO/
UNEP, 1977). In Brazil, losses of 10±25% (Pedrosa and Dezen, 1991) have been estimated to
occur throughout the trading process, partly due to contamination with toxigenic fungi and
mycotoxins. Improved storage conditions would allow a 10±20% increase in the supply of food
available to people (Christensen and Kaufmann, 1969).
The fumonisins are secondary metabolites produced by Fusarium moniliforme Sheldon and F.
proliferatum (Matsushima) Niremberg (Bacon and Nelson, 1994). They show a worldwide
distribution and can be isolated from maize and maize-based food and feedstus naturally
contaminated with Fusarium. Of the seven fumonisins currently identi®ed (Bezuidenhout et al.,
1988; Cawood et al., 1991; Musser, 1996), fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) are the
most frequently detected in fungal cultures or in naturally contaminated maize, a component
of human and animal feed in many countries (Sydenham et al., 1991; Doko et al., 1995;
Visconti, 1996; Thiel et al., 1996).
The fumonisins have been associated with leukoencephalomalacia (ELEM) in equines
(Ross et al., 1991; Sydenham et al., 1992), porcine pulmonary edema (PPE; Colvin and
Harrison 1992; Osweiler et al., 1992), diarrhea and reduced body weight in broiler chicks
(Brown et al., 1992), carcinogenicity in rats (Gelderblom et al., 1991) and
leukoencephalomalacia and hemorrhage in the brain of rabbits (Bucci et al., 1996). In
addition, epidemiological evidence suggests a correlation between the consumption of F.
moniliforme contaminated maize and a high incidence of human esophageal carcinoma (Thiel
et al., 1992, Rice and Ross, 1994).
Based on the above considerations, the present investigation was undertaken in order to: (a)
identify the myco¯ora of local maize hybrids freshly harvested and after storage in normal
facilities in SaÄo Paulo, Brazil; (b) determine the occurrence of fumonisins B1 and B2 in the
cereal grains; and (c) correlate the results with data on abiotic factors (temperature, moisture
content, water activity, and relative humidity).
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
77
2. Material and methods
2.1. Samples
A total of 195 samples of three maize hybrids: Br 201 (Embrapa Milho e Sorgo, Rod. MG
424, Km 65, Sete Lagoas/MG, Brazil, CP 151, Cep 35701-970), C125 (Cargill, R. Jacarezinho,
AndiraÂ/PR, Brazil, Cep 86380-000) and Cx 322 (ICI Sementes, Rod. Anhanguera, Km 296,
Cravinhos/SP, Brazil, Cep 14140-000), which are commonly planted in the State of SaÄo Paulo,
were used. The samples (15 of freshly harvested grains and 180 of stored grains) were collected
in RibeiraÄo Preto, State of SaÄo Paulo, Brazil, a region characterized by humid tropical weather
with hot and rainy summers and dry winters. After harvest, the maize grains were put in 60 kg
jute sacks (5 sacks per hybrid), stacked over wooden boards and stored for 12 months in a
well-ventilated warehouse, located near the production area. Monthly samples were collected
from various points of each sack (Fonseca, 1991), until a total of 1 kg grains/sack was
obtained (one sample per month per sack).
2.2. Moisture content and water activity
The moisture content of the maize grains was determined in the grain-storage areas
immediately after sampling, using a ``Brow Duvel'' moisture meter (Model CA 2511
manufactured by Gehaka Co., SaÄo Paulo, Brazil). Water activity was determined by automated
analysis with the equipment AQUALAB CX-2 (Decagon Devices Inc.). Each sample was
measured ®ve times.
2.3. Recovery, identi®cation and enumeration of the myco¯ora
A 10 g sample of maize from each of the 195 samples was ground and mixed with 90 ml of
sterile distilled water to obtain a 10ÿ1 stock dilution, from which 10-fold serial dilutions up to
10ÿ6 were made using the same diluent. Duplicate 1 ml volumes of each dilution were added to
Petri dishes containing 10±15 ml of Potato Dextrose Agar (Swanson et al., 1992). The plates
were then incubated at 258C for 5 days and observed daily. Plates that contained 15 to 150
CFU were used for counting and the results were expressed as CFU per gram of sample
(Mislivec et al., 1992). The fungal colonies recovered were identi®ed according to the methods
for each genus (Raper and Fennell, 1965; Von Arx, 1974; Nelson et al., 1983; Nelson, 1992).
2.4. Determination of fumonisins
The maize samples were analyzed for fumonisins B1 (FB1) and B2 (FB2) according to the
method of Ross et al. (1991), with some modi®cations. Brie¯y, 10 g of maize kernels were
added to 50 ml of a solution of acetonitrile/water (1:1) and stirred for 30 min. The extract was
then ®ltered through ®lter paper. Next, 2 ml of the ®ltrate were added to 5 ml of water and the
mixture was applied to a preconditioned Sep-Pak cartridge (Waters, Division of Millipore
Corp., Milford, MA). The cartridge was washed with 2 ml of a 20:80 mixture of acetonitrile/
78
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
water and the toxin eluted with 2 ml of a 70:30 mixture of acetonitrile/water. The ®nal extract
was collected in Eppendorf tubes and kept at ÿ208C until use.
Two hundred microliters of the ®nal extract were derivatized with 50 ml of a solution of ophthaldialdehyde (OPA) (40 mg of OPA dissolved in 1 ml of methanol and diluted in 5 ml of
0.1 M sodium tetraborate containing 50 ml of mercaptoethanol). The product of this reaction
was analyzed by a reverse-phase isocratic HPLC system consisting of a Shimadzu SCL-6B
pump, an RF55 ¯uorescent detector (Shimadzu; excitation and emission wavelength of 355 and
440 nm, respectively) and a 150 4.6 mm C18 column (5ODS-20, Phenomenex). The eluent
was methanol/sodium acetate buer (77:23), pH 3.6. Calibration of the apparatus was
undertaken using stock solutions of standard fumonisins (Sigma) at the concentrations 0.0125,
0.025 and 0.05 mg/ml for FB1 and 0.005, 0.01 and 0.02 mg/ml for FB2. A recovery experiment
was carried out in quadruplicate with toxin levels that varied from 4±24 ng of FB1 and 8±56
ng of FB2 per gram of ground maize. The recoveries of FB1 and FB2 were 88% and 94%,
respectively. The detection limit was 50 ng/g for both FB1 and FB2.
2.5. Climatic data
During storage, the climatic conditions relating to temperature (8C) and relative humidity
(%) were recorded as monthly averages.
2.6. Statistical analysis
The experimental design used for each hybrid was totally randomized, with 13 storage time
periods analyzed as orthogonal polynomials to obtain the regression equations. The dependent
variables (CFU/g of Aspergillus spp, Fusarium spp, Penicillium spp and fumonisins B1, and B2)
were transformed into loge (x + 1) (Steel and Torrie, 1960). Stepwise multiple regression and
studentized residuals were used to obtain the regression models.
Table 1
Absolute and relative (%) frequencies of fungi recovered from 195 samples of maize grains (hybrids Br 201, C 125
and Cx 322) collected over a one-year period in Brazila
Aspergillus
Fusarium
Penicillium
Trichosporon
Cladosporium
N. S. F.b
a
b
Br 201
C 125
Cx 322
22.5
55.0
50.5
16.0
29.0
2.5
20.0
60.0
47.0
13.5
20.0
3.0
13.0
56.0
33.5
7.5
31.5
4.0
(34.6)
(84.6)
(77.7)
(24.6)
(44.6)
(3.8)
For each hybrid, 65 samples were analyzed.
Non-sporulated fungi.
(30.8)
(92.3)
(72.3)
(20.8)
(30.8)
(4.6)
(20.0)
(86.1)
(51.5)
(11.5)
(48.5)
(6.1)
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
79
3. Results and discussion
The fungi identi®ed in the 195 samples of maize analyzed (hybrids Br 201, C 125 and Cx
322) showed that Fusarium spp were the most frequent molds, followed by Penicillium spp,
Aspergillus spp, Trichosporon spp, Cladosporium spp, and non-sporulated fungi (Table 1).
Another 8 genera of fungi were isolated at very low frequencies: Epicoccum spp, Mucor spp,
Rhizopus spp, Neurospora spp, Rhizoctonia spp, Curvularia spp, Trichoderma spp, and
Nigrospora spp. Fusarium moniliforme was the predominant Fusarium species and, within the
genus Aspergillus, A. alutaceus Berkeley and Curtis (A. ochraceus ) had the greatest frequency
of recovery. Penicillium isolates were not identi®ed to species.
The majority of the fungal isolates were recovered at aw values of 0.7±0.79 (Table 2). The
speci®c frequencies of the total species identi®ed were: F. moniliforme 59.2%, F. proliferatum
36.1%, F. anthophilum A. Braun 4.6%, A. alutaceus 10.8%, A. chrysellus Kwon and Fennell
3.8%, A. tamani Kita 3.8%, A. niger Van Tieghem 3.1%, A. ornatus Raper, Fennell and
Tresner 2.3%, A. cremeus Kwon and Fennell 2.3%, A. nidulans Eidam 1.5%, A. candidus Link
1.5% and A. ¯avus Link 0.8% for hybrid Br 201; F. moniliforme 55.4%, F. proliferatum 42.3%,
F. anthophilum 9.2%, A. alutaceus 10.8%, A. tamarii 5.4%, A. chrysellus 4.6%, A. cremeus
3.1%, A. niger 2.3%, A. ¯avus 2.3%, A. glaucus Link 2.3%, and A. versicolor Tiraboschi 0.8%
for hybrid C 125; and F. moniliforme 69.2%, F. proliferatum 38.5%, F. anthophilum 15.4%, F.
clamydosporum Wollenw 0.8%, F. nygamai Burgess 0.8%, A. alutaceus 8.5%, A. ornatus 3.1 %,
A. ¯avus 1.5%, A. chrysellus 0.8%, A. cremeus 0.8%, A. glaucus 0.8%, A. niger 0.8% and A.
raperi Stolk 0.8%. for hybrid Cx 322. The greater prevalence of Fusarium spp found in the
present investigation agrees with the data reported by other authors (Pozzi et al., 1995; Lillehoj
and Zuber, 1988; Asevedo et al., 1994; Castro et al., 1995) who also describe this genus as that
Table 2
Absolute and relative (%) frequencies of the genera Fusarium, Aspergillus and Penicillium in 195 samples of maize
grains (hybrids Br 201, C 125 and Cx 322) and range of water activity (aw) values recorded over a one-year period
in Brazil. For each hybrid, 65 samples were analyzed; AF=Absolute frequency; RF=Relative frequency; nd=not
detected at dilution 10ÿ3
Hybrids
Br 201a
C 125b
Cx 322a
a
Range of aw
Samples in each
aw group
0.90±0.80
0.79±0.70
0.69±0.67
0.90±0.80
0.79±0.70
0.69±0.66
0.90±0.80
0.79±0.70
0.69±0.67
3
7
3
3
7
3
1
11
1
Aspergillus
AF (RF%)
Fusarium
AF (RF%)
Penicillium
AF (RF%)
9(6.9)
26(20.0)
10(7.7)
nd a A
23 (17.7) b A
17 (13.0) b A
nd
23(17.7)
3(2.3)
30(23.0)
52(40.0)
28(21.5)
20(15.4) a B
66(50.8) b B
34(26.2) b B
10(7.7)
93(71.5)
9(6.9)
27(20.8)
51 (39.2)
23(17.7)
18 (13.8) a B
50 (38.5) b B
26 (20.0) b B
8(6.2)
56(43.0)
3(2.3)
(P > 0.05).
Frequencies followed by dierent lower case letters denote dierences in the rows. Frequencies followed by dierent capital letters denote dierences in the columns. Analysis among hybrids by w-square.
b
80
Table 3
Fumonisin levels and number of colony forming units (CFU/g) of the fungi isolated from 195 freshly harvested and stored maize grains (hybrids
Br 201, C 125 and Cx 322) collected over a one-year period in Brazila
Fumonisin levels
(mg/g)b
Br 201
c
FH
S1e
S2
S3
S4
S5
S6
S7
S8
S9
Cx 322
Genera
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
ndd
594.6
6.9
1
1096
276
280
680
1480
60
2616
866
90
5340
1430
187
872
2096
50
460
1790
60
360
600
20
36
163
10
15
nd
460.3
18.7
nd
62.5
104
3
1216
329
72
1523
444
55
286
196
21
2790
121
70
550
620
140
290
120
14
22
34
14
24
nd
669.8
14.9
1
1415
41
nd
868
8
2
677
102
31
170
75
30
2940
260
50
490
50
120
160
100
3
36
13
2
89
Br 201
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
24.0
15.0
13.1
9.8
12.1
7.2
17.1
11.7
17.2
8.4
7.5
6.5
4.5
7.4
9.4
8.0
7.4
5.3
8.0
7.2
11.0
7.1
9.1
6.0
14.1
9.8
6.8
10.1
3.3
7.9
13.0
7.3
26.0
9.2
18.0
8.7
3.6
6.5
4.1
7.5
6.0
7.2
11.0
7.1
10.1
7.5
11.2
6.8
3.0
7.3
3.6
5.8
3.6
7.7
10.5
8.3
11.6
9.0
6.6
5.3
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
Samples
C 125
Table 3 (continued )
Fumonisin levels
(mg/g)b
Br 201
Samples
S11
S12
Meanf/SDg
a
Genera
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
6
2
11
2
6
11
54
5
107
94
59.3/ 2 81.3
938.3/ 2 1442.3
681.8/ 2 734.5
34
9
18
10
2
69
13
5
148
45
31.1/ 2 40.2
573.7/ 2 785.1
160.6/ 2 183.4
For each hybrid, 65 samples were analyzed.
Mean of 5 samples.
c
Freshly harvested.
d
Not detected at dilution 10ÿ3.
e
Storage month.
f
Mean of 65 samples.
g
Standard deviation.
b
Cx 322
11
3
25
11
1
37
2
10
82
37
19.4/ 232.7
589.1/ 2789.6
55.7/ 267.6
Br 201
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
8.9
6.9
15.4
8.0
10.3
7.4
15.3
7.5
5.2
5.3
3.7
6.6
4.4
4.2
4.1
5.7
5.9
6.3
10.5/2 5.9
8.1/ 2 2.5
10.5/2 6.1
7.8/2 1.4
8.0/ 2 3.9
6.8/2 0.9
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
S10
C 125
81
82
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
most frequently associated with Brazilian maize grains. The predominance of F. moniliforme
had already been observed in Brazil, as well as the occurrence of F. proliferatum as the second
most frequent species in maize (Pozzi et al., 1995). However, our results show higher
frequencies of these fungi in the maize hybrids studied.
The three most important fungal genera in terms of toxigenicity produced the greatest
numbers of colony forming units (CFU/g) in hybrid Br 201, varying from 11 104 to
5340 104 with Fusarium spp, from 0 to 280 104 with Aspergillus spp and from 2 104 to
2096 104 with Penicillium spp. The values recorded for these fungal genera in the other two
hybrids were 18 104 to 2790 104 Fusarium spp, 0 to 140 104 Aspergillus spp and 10 104
to 620 104 Penicillium spp for hybrid C 125 and 25 104 to 2940 104 Fusarium spp, 0 to
120 104 Aspergillus spp and 2 104 to 260 104 Penicillium spp for hybrid Cx 322 (Table
3). These numbers exceed the tolerance limits set by the International Commission on
Microbiological Speci®cation for Foods (Elliott, 1980), which are of the order of 102 to 104
CFU/g.
The genus Aspergillus was absent from the myco¯ora of the 15 samples of freshly harvested
grains for the three hybrids studied (Table 3). In the remaining samples analyzed, species of
Aspergillus were found in grains with moisture content of 10.2±16.4%, the greater number of
recoveries occurring between 12.4±16.4% and at water activities of 0.7±0.79 (Tables 2 and 4).
The linear correlation analysis showed a signi®cant positive correlation between the
Table 4
Moisture content (MC) and water activity (aw) values of 195 samples of maize grains (hybrids Br 201, C 125 and Cx
322) listed with the climatic data recorded over a one-year period in Brazil
Mean of 5 samples per month
Samples
FHa
S1b
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
Mean
SIDc
a
Br 201 MC % ± aw
C 125 MC % ± aw
Cx 322 MC % ± aw
Relative humidity
(%)
Mean temperature
(8C)
17.0±0.91
16.4±0.82
16.2±0.81
16.2±0.74
14.2±0.75
12.3±0.68
12.0±0.69
12.6±0.73
11.7±0.70
11.8±0.71
12.6±0.68
10.3±0.72
10.8±0.71
13.3±0.74
2 2.2ÿ 2 0.06
16.8±0.87
16.8±0.82
16.6±0.79
16.4±0.74
14.0±0.80
12.6±0.70
12.0±0.66
12.4±0.74
11.8±0.67
11.9±0.71
12.6±0.69
10.2±0.72
10.9±0.71
13.4±0.74
2 2.2ÿ2 0.06
16.0±0.83
15.8±0.78
15.6±0.78
15.6±0.74
14.2±0.76
12.8±0.74
12.2±0.67
12.0±0.75
12.6±0.71
12.7±0.72
12.7±0.72
10.2±0.74
11.1±0.72
13.3±0.74
2 1.8ÿ 2 0.03
86.9
82.0
82.3
80.6
82.3
83.2
83.0
83.7
85.5
92.7
93.0
92.9
91.6
86.1
2 4.5
20.2
19.8
18.8
19.4
20.0
21.4
22.7
22.2
24.4
24.1
23.5
24.3
23.3
21.8
2 1.9
Freshly harvested.
Storage month.
c
Standard deviation.
b
Average per month
83
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
populations of Aspergillus spp and those of Penicillium spp (P < 0.05; 0.34) and signi®cant
negative correlations between Aspergillus spp and both relative humidity (P < 0.05; ÿ0.35) and
aw (P < 0.05; ÿ0.34). Signi®cant negative correlations were also found between the genus
Penicillium and both mean temperature (P < 0.01; ÿ0.41) and relative humidity (P < 0.01;
ÿ0.49). The regression analysis of number of CFU/g versus storage time showed a statistically
signi®cant correlation in the three hybrids analyzed for the genera Aspergillus and Penicillium
(P < 0.01).
The stepwise multiple regression analysis for number of CFU/g, storage time, aw, moisture
content, mean temperature and relative humidity revealed the most important variables in each
model: for the genus Aspergillus they were storage time in Br 201 (P < 0.05) and storage time,
relative humidity, mean temperature and mean temperature2 in C 125 (P < 0.05); for the genus
Penicillium they were time of storage and aw in Br 201 (P < 0.01) and mean temperature
(P < 0.05) in Cx 322.
The highest numbers of CFU/g of the genus Fusarium were recovered during the ®rst ®ve
storage months (Table 3) at mean temperature values of 18.8±21.48C, grain moisture contents
of 12.6±16.8%, relative humidity of 80.6±83.2% (Table 4). Fusarium spp showed signi®cant
negative correlations with mean temperature (P < 0.01; ÿ0.67) and relative humidity
(P < 0.01; ÿ0.57) and positive correlations with grain moisture content (P < 0.01; 0.52). These
results corroborate the ®nding that this mold, despite being able to survive on desiccated
grains, thrives in more humid conditions. The regression analysis of number of CFU/g versus
storage time showed a statistically signi®cant correlation (P < 0.01) for Fusarium spp. The
stepwise multiple regression analysis for number of CFU/g, storage time, aw, moisture content,
mean temperature, and relative humidity revealed the most important variables for each model
of the genus Fusarium: mean temperature and mean temperature2 for Br 201 (P < 0.01); aw
and relative humidity for C 125 (P < 0.05); storage time for Cx 322 (P < 0.01).
The lowest frequencies of occurrence were observed in hybrid Br 201 for Fusarium spp and
in hybrid Cx 322 for Aspergillus spp and Penicillium spp (Table 1). Hybrids Br 201 and CX
322 did not show statistically signi®cant dierences of Aspergillus, Fusarium and Penicillium
contamination at the aw values detected. However, in hybrid C 125, the higher numbers of
CFU/g for Aspergillus spp were statistically signi®cant when compared to contamination levels
for the other two genera (P < 0.05) at aw values of 0.90±0.80 (Table 2).
Tables 5 and 6 show the occurrence of fumonisins in grains of the Brazilian maize hybrids
analyzed. Out of a total of 195 samples (65 belonging to each of the three hybrids), 176
Table 5
Fumonisin levels in 195 samples of freshly harvested and stored maize grains (hybrids Br 201, C 125 and Cx 322)
from Brazil
BR 201
Positive samples
Mean concentration
Concentration range (mg/g)
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
62(93.38%)
10.54
0.90±46.39
64(98.46%)
8.17
2.51±29.16
56(86.15%)
10.57
0.98±49.31
63(96.92%)
7.80
3.1±20.65
58 (89.2%)
8.05
0.87±37.14
63(96.92%)
6.83
1.96±11.74
84
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
Table 6
Distribution of FB1 and FB2 levels in 195 samples of freshly harvested and stored maize grains (hybrids BR 201, C
125 and Cx 322) in Brazil
BR 201
C 125
CX 322
Total
% of samples
Fumonisins levels (mg/g)
FB1
FB2
FB1
FB2
FB1
FB2
FB1
FB2
FB1
FB2
< 1.0
1.0±3.0
3.1±5.0
5.1±8.0
8.1±10.0
10.1±13.0
13.1±15.0
15.1±18.0
18.1±20.0
20.1±23.0
23.1±25.0
25.1±28.0
28.1±30.0
30.1±50.0
nda
4
20
15
3
5
2
nd
3
4
1
1
nd
5
nd
2
nd
43
5
8
2
nd
1
2
nd
nd
1
nd
1
6
12
7
5
6
2
2
3
4
1
nd
2
5
nd
nd
3
33
16
7
3
1
nd
nd
nd
nd
nd
nd
1
6
14
16
3
5
3
2
2
3
1
nd
nd
1
nd
2
1
45
10
4
1
nd
nd
nd
nd
nd
nd
nd
2
16
46
38
11
16
7
4
8
11
3
1
2
11
±
1.0
8.2
23.6
19.5
5.6
8.2
3.6
2.0
4.1
5.6
1.5
0.5
1.0
5.6
±
2
2
62
15.9
9.7
3.1
0.5
0.5
1.0
±
±
0.5
±
a
4
4
121
31
19
6
1
1
2
±
±
1
±
nd=not detected.
(90.2%) were contaminated with FB1 at concentrations that ranged from 0.87±49.31 mg/g and
190 (97.4%) were contaminated with FB2 at concentrations that ranged from 1.96±29.16 mg/g.
Studies on fumonisin contaminants in maize and maize-based products from several countries
(Shephard et al., 1996; Bullerman, 1996) revealed levels of contamination lower than those
presently reported. A similar ®nding was observed when comparing our results with data on
FB1 and FB2 contamination in maize hybrids or inbred lines grown in countries from three
continents. Of these, Argentina showed the highest concentration of fumonisins (37 mg/g) in 51
samples analyzed (Visconti, 1996). In Brazil, Hirooka et al. (1996) reported maximum levels of
18.5 mg/g for FB1 and 19.3 mg/g for FB2 in 48 samples of maize grains.
With respect to possible dierences in fumonisin contamination between the maize hybrids
studied, their pro®les are compared in Tables 5 and 6. The lowest contamination occurred in
hybrid CX 322. This applied to both FB1 and FB2 mean levels (8.05 and 6.83 mg/g,
respectively) and range of concentrations (0.87±37.14 mg/g for FB1 and 1.96±11.74 mg/g for
FB2). The highest FB1 levels were found in 140 day-storage samples of the C 125 hybrid
(49.31 mg/g) and in freshly harvested samples of the BR 201 and CX 322 hybrids (46.59 and
37.14 mg/g, respectively). A more uniform distribution of mean concentrations of FB2 as
compared to FB1 occurred in all the hybrids.
After 140 days of storage, an overall decrease in the number of CFU/g for Fusarium spp
together with a tendency towards decreased FB1 mean concentrations was observed in the
three hybrids analyzed, although FB1 levels showed some variability. The diversi®ed myco¯ora
of these hybrids, demonstrated by the recovery of 13 distinct fungal genera, presupposes a
competition for nutrients. According to Horn and Wicklow (1983), each fungus carries a factor
that determines its growth and ability to compete with other fungal species. In this respect, it
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
85
should be mentioned that studies carried out by Marin et al. (1998) indicated that fungal
interactions could act as an additional factor in the control of Fusarium spp contaminants in
maize and lead to decreased contamination by this genus, mainly at lower aw. On the other
hand, sample size should also be considered as an in¯uencing factor when trying to account for
the decrease and variability of fumonisin levels recorded during our investigation (Whitaker et
al., 1998).
The statistical analysis showed a negative correlation between fumonisins B1 and relative
humidity (P < 0.001; ÿ0.68769). Negative correlations were also found between the fumonisin
B2 and relative humidity (P < 0.001; ÿ0.64384) and mean temperature (P < 0.001; ÿ0.48662).
FB2 correlated positively with grain moisture content (P < 0.001; 0.56913).
To conclude, the signi®cant levels of contamination with Fusarium spp reported herein, both
in terms of prevalence and number of CFU/g, and the detection of fumonisins in freshly
harvested and stored maize grains highlights the relevance of studies on the occurrence of
fusariotoxins in maize and its by-products in the tropics.
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www.elsevier.com/locate/jspr
Myco¯ora and occurrence of fumonisins in freshly
harvested and stored hybrid maize
Reinaldo B. Orsi a, Benedito CorreÃa a,*, Claudia R. Possi b, Eliana
A. Schammass b, Jose R. Nogueira b, Silvia M.C. Dias c, Marisa A.B. Malozzi c
a
Departamento de Microbiologia, Instituto de CieÃncias BiomeÂdicas, Universidade de SaÄo Paulo, Brazil
b
Instituto de Zootecnia, Universidade de SaÄo Paulo, Brazil
c
Instituto Biologico, Universidade de SaÄo Paulo, Brazil
Accepted 9 June 1999
Abstract
The study of the myco¯ora in stored grain permits an evaluation of cereal storage conditions that
aect grain deterioration and the risk of mycotoxin contamination. Abiotic factors can directly aect
the relative frequency of fungal populations in stored grain. The aim of the present work was to study
the in¯uence of abiotic factors on variations of myco¯ora of freshly harvested and stored maize in
Brazil and the occurrence of fumonisins. Samples (195) of three hybrids of maize were analyzed monthly
during one year. Microbiological analysis revealed a predominance of Fusarium spp, which presented the
greatest total number of colony forming units per gram in the three hybrids, namely: Br 201 (11 104
to 5340 104 CFU/g), C 125 (18 104 to 2790 104 CFU/g) and Cx 322 (25 104 to 2940 104
CFU/g), followed by Penicillium spp, Aspergillus spp and 10 other fungal genera. Fusarium moniliforme
Sheldon was the most prevalent species (59.2% of Fusarium isolates in Br 201, 55.4% in C 125 and
69.2% in Cx 322). Fusarium spp showed signi®cant negative correlations with mean temperature and
relative humidity of the air. Higher temperatures and relative humidity at the end of the study and high
moisture content at the beginning of the study were observed. The CFU/g values recorded for the three
predominant genera exceeded the internationally accepted tolerance limits. The mycotoxicological
evaluation indicated contamination of 176 samples (90.2%) with fumonisin B1 and of 190 samples
(97.4%) with fumonisin B2. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Myco¯ora; Fumonisins; Aspergillus ¯avus; Fusarium moniliforme; Stored maize; Fumonisins in maize
* Corresponding author. Tel.: +55-11-818-7295; fax: +55-11-818-7354.
E-mail address: correabe@usp.br (B. CorreÃa)
0022-474X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 4 7 4 X ( 9 9 ) 0 0 0 2 9 - 6
76
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
1. Introduction
In Brazil, maize has historically had an important role in both human and animal nutrition.
Even before the arrival of European colonizers, the Brazilian natives already cultivated this
cereal. At present, Brazil is ranked as the third largest producer of maize in the world, coming
after the USA and China. Of the Brazilian annual yield of about 32 million tonnes in recent
years (Tsuneshiro and Okawa, 1996), 41% is used by the poultry and swine industries. A
substantial part of this crop comes from small producers and approximately 24.7% of the
harvest is stored and used on the producing farms (Pedrosa and Dezen, 1991).
In the state of SaÄo Paulo, maize plantations make up 7% of the cultivated land (Francisco
et al., 1997). The mean productivity of 2747 kg/ha recorded for this region during the 1990±
94 period re¯ects a poor local technology (Tsuneshiro and Okawa, 1996). The maizeproducing farms in this state have a mean area of 13.3 ha; 61.9% of the farmers receive
technical assistance, 44.1% use soil analysis, and 46.2% use hybrid seed (Francisco et al.,
1997).
The overall world loss of grains is considered to be 5% of the total production (FAO/WHO/
UNEP, 1977). In Brazil, losses of 10±25% (Pedrosa and Dezen, 1991) have been estimated to
occur throughout the trading process, partly due to contamination with toxigenic fungi and
mycotoxins. Improved storage conditions would allow a 10±20% increase in the supply of food
available to people (Christensen and Kaufmann, 1969).
The fumonisins are secondary metabolites produced by Fusarium moniliforme Sheldon and F.
proliferatum (Matsushima) Niremberg (Bacon and Nelson, 1994). They show a worldwide
distribution and can be isolated from maize and maize-based food and feedstus naturally
contaminated with Fusarium. Of the seven fumonisins currently identi®ed (Bezuidenhout et al.,
1988; Cawood et al., 1991; Musser, 1996), fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) are the
most frequently detected in fungal cultures or in naturally contaminated maize, a component
of human and animal feed in many countries (Sydenham et al., 1991; Doko et al., 1995;
Visconti, 1996; Thiel et al., 1996).
The fumonisins have been associated with leukoencephalomalacia (ELEM) in equines
(Ross et al., 1991; Sydenham et al., 1992), porcine pulmonary edema (PPE; Colvin and
Harrison 1992; Osweiler et al., 1992), diarrhea and reduced body weight in broiler chicks
(Brown et al., 1992), carcinogenicity in rats (Gelderblom et al., 1991) and
leukoencephalomalacia and hemorrhage in the brain of rabbits (Bucci et al., 1996). In
addition, epidemiological evidence suggests a correlation between the consumption of F.
moniliforme contaminated maize and a high incidence of human esophageal carcinoma (Thiel
et al., 1992, Rice and Ross, 1994).
Based on the above considerations, the present investigation was undertaken in order to: (a)
identify the myco¯ora of local maize hybrids freshly harvested and after storage in normal
facilities in SaÄo Paulo, Brazil; (b) determine the occurrence of fumonisins B1 and B2 in the
cereal grains; and (c) correlate the results with data on abiotic factors (temperature, moisture
content, water activity, and relative humidity).
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
77
2. Material and methods
2.1. Samples
A total of 195 samples of three maize hybrids: Br 201 (Embrapa Milho e Sorgo, Rod. MG
424, Km 65, Sete Lagoas/MG, Brazil, CP 151, Cep 35701-970), C125 (Cargill, R. Jacarezinho,
AndiraÂ/PR, Brazil, Cep 86380-000) and Cx 322 (ICI Sementes, Rod. Anhanguera, Km 296,
Cravinhos/SP, Brazil, Cep 14140-000), which are commonly planted in the State of SaÄo Paulo,
were used. The samples (15 of freshly harvested grains and 180 of stored grains) were collected
in RibeiraÄo Preto, State of SaÄo Paulo, Brazil, a region characterized by humid tropical weather
with hot and rainy summers and dry winters. After harvest, the maize grains were put in 60 kg
jute sacks (5 sacks per hybrid), stacked over wooden boards and stored for 12 months in a
well-ventilated warehouse, located near the production area. Monthly samples were collected
from various points of each sack (Fonseca, 1991), until a total of 1 kg grains/sack was
obtained (one sample per month per sack).
2.2. Moisture content and water activity
The moisture content of the maize grains was determined in the grain-storage areas
immediately after sampling, using a ``Brow Duvel'' moisture meter (Model CA 2511
manufactured by Gehaka Co., SaÄo Paulo, Brazil). Water activity was determined by automated
analysis with the equipment AQUALAB CX-2 (Decagon Devices Inc.). Each sample was
measured ®ve times.
2.3. Recovery, identi®cation and enumeration of the myco¯ora
A 10 g sample of maize from each of the 195 samples was ground and mixed with 90 ml of
sterile distilled water to obtain a 10ÿ1 stock dilution, from which 10-fold serial dilutions up to
10ÿ6 were made using the same diluent. Duplicate 1 ml volumes of each dilution were added to
Petri dishes containing 10±15 ml of Potato Dextrose Agar (Swanson et al., 1992). The plates
were then incubated at 258C for 5 days and observed daily. Plates that contained 15 to 150
CFU were used for counting and the results were expressed as CFU per gram of sample
(Mislivec et al., 1992). The fungal colonies recovered were identi®ed according to the methods
for each genus (Raper and Fennell, 1965; Von Arx, 1974; Nelson et al., 1983; Nelson, 1992).
2.4. Determination of fumonisins
The maize samples were analyzed for fumonisins B1 (FB1) and B2 (FB2) according to the
method of Ross et al. (1991), with some modi®cations. Brie¯y, 10 g of maize kernels were
added to 50 ml of a solution of acetonitrile/water (1:1) and stirred for 30 min. The extract was
then ®ltered through ®lter paper. Next, 2 ml of the ®ltrate were added to 5 ml of water and the
mixture was applied to a preconditioned Sep-Pak cartridge (Waters, Division of Millipore
Corp., Milford, MA). The cartridge was washed with 2 ml of a 20:80 mixture of acetonitrile/
78
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
water and the toxin eluted with 2 ml of a 70:30 mixture of acetonitrile/water. The ®nal extract
was collected in Eppendorf tubes and kept at ÿ208C until use.
Two hundred microliters of the ®nal extract were derivatized with 50 ml of a solution of ophthaldialdehyde (OPA) (40 mg of OPA dissolved in 1 ml of methanol and diluted in 5 ml of
0.1 M sodium tetraborate containing 50 ml of mercaptoethanol). The product of this reaction
was analyzed by a reverse-phase isocratic HPLC system consisting of a Shimadzu SCL-6B
pump, an RF55 ¯uorescent detector (Shimadzu; excitation and emission wavelength of 355 and
440 nm, respectively) and a 150 4.6 mm C18 column (5ODS-20, Phenomenex). The eluent
was methanol/sodium acetate buer (77:23), pH 3.6. Calibration of the apparatus was
undertaken using stock solutions of standard fumonisins (Sigma) at the concentrations 0.0125,
0.025 and 0.05 mg/ml for FB1 and 0.005, 0.01 and 0.02 mg/ml for FB2. A recovery experiment
was carried out in quadruplicate with toxin levels that varied from 4±24 ng of FB1 and 8±56
ng of FB2 per gram of ground maize. The recoveries of FB1 and FB2 were 88% and 94%,
respectively. The detection limit was 50 ng/g for both FB1 and FB2.
2.5. Climatic data
During storage, the climatic conditions relating to temperature (8C) and relative humidity
(%) were recorded as monthly averages.
2.6. Statistical analysis
The experimental design used for each hybrid was totally randomized, with 13 storage time
periods analyzed as orthogonal polynomials to obtain the regression equations. The dependent
variables (CFU/g of Aspergillus spp, Fusarium spp, Penicillium spp and fumonisins B1, and B2)
were transformed into loge (x + 1) (Steel and Torrie, 1960). Stepwise multiple regression and
studentized residuals were used to obtain the regression models.
Table 1
Absolute and relative (%) frequencies of fungi recovered from 195 samples of maize grains (hybrids Br 201, C 125
and Cx 322) collected over a one-year period in Brazila
Aspergillus
Fusarium
Penicillium
Trichosporon
Cladosporium
N. S. F.b
a
b
Br 201
C 125
Cx 322
22.5
55.0
50.5
16.0
29.0
2.5
20.0
60.0
47.0
13.5
20.0
3.0
13.0
56.0
33.5
7.5
31.5
4.0
(34.6)
(84.6)
(77.7)
(24.6)
(44.6)
(3.8)
For each hybrid, 65 samples were analyzed.
Non-sporulated fungi.
(30.8)
(92.3)
(72.3)
(20.8)
(30.8)
(4.6)
(20.0)
(86.1)
(51.5)
(11.5)
(48.5)
(6.1)
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
79
3. Results and discussion
The fungi identi®ed in the 195 samples of maize analyzed (hybrids Br 201, C 125 and Cx
322) showed that Fusarium spp were the most frequent molds, followed by Penicillium spp,
Aspergillus spp, Trichosporon spp, Cladosporium spp, and non-sporulated fungi (Table 1).
Another 8 genera of fungi were isolated at very low frequencies: Epicoccum spp, Mucor spp,
Rhizopus spp, Neurospora spp, Rhizoctonia spp, Curvularia spp, Trichoderma spp, and
Nigrospora spp. Fusarium moniliforme was the predominant Fusarium species and, within the
genus Aspergillus, A. alutaceus Berkeley and Curtis (A. ochraceus ) had the greatest frequency
of recovery. Penicillium isolates were not identi®ed to species.
The majority of the fungal isolates were recovered at aw values of 0.7±0.79 (Table 2). The
speci®c frequencies of the total species identi®ed were: F. moniliforme 59.2%, F. proliferatum
36.1%, F. anthophilum A. Braun 4.6%, A. alutaceus 10.8%, A. chrysellus Kwon and Fennell
3.8%, A. tamani Kita 3.8%, A. niger Van Tieghem 3.1%, A. ornatus Raper, Fennell and
Tresner 2.3%, A. cremeus Kwon and Fennell 2.3%, A. nidulans Eidam 1.5%, A. candidus Link
1.5% and A. ¯avus Link 0.8% for hybrid Br 201; F. moniliforme 55.4%, F. proliferatum 42.3%,
F. anthophilum 9.2%, A. alutaceus 10.8%, A. tamarii 5.4%, A. chrysellus 4.6%, A. cremeus
3.1%, A. niger 2.3%, A. ¯avus 2.3%, A. glaucus Link 2.3%, and A. versicolor Tiraboschi 0.8%
for hybrid C 125; and F. moniliforme 69.2%, F. proliferatum 38.5%, F. anthophilum 15.4%, F.
clamydosporum Wollenw 0.8%, F. nygamai Burgess 0.8%, A. alutaceus 8.5%, A. ornatus 3.1 %,
A. ¯avus 1.5%, A. chrysellus 0.8%, A. cremeus 0.8%, A. glaucus 0.8%, A. niger 0.8% and A.
raperi Stolk 0.8%. for hybrid Cx 322. The greater prevalence of Fusarium spp found in the
present investigation agrees with the data reported by other authors (Pozzi et al., 1995; Lillehoj
and Zuber, 1988; Asevedo et al., 1994; Castro et al., 1995) who also describe this genus as that
Table 2
Absolute and relative (%) frequencies of the genera Fusarium, Aspergillus and Penicillium in 195 samples of maize
grains (hybrids Br 201, C 125 and Cx 322) and range of water activity (aw) values recorded over a one-year period
in Brazil. For each hybrid, 65 samples were analyzed; AF=Absolute frequency; RF=Relative frequency; nd=not
detected at dilution 10ÿ3
Hybrids
Br 201a
C 125b
Cx 322a
a
Range of aw
Samples in each
aw group
0.90±0.80
0.79±0.70
0.69±0.67
0.90±0.80
0.79±0.70
0.69±0.66
0.90±0.80
0.79±0.70
0.69±0.67
3
7
3
3
7
3
1
11
1
Aspergillus
AF (RF%)
Fusarium
AF (RF%)
Penicillium
AF (RF%)
9(6.9)
26(20.0)
10(7.7)
nd a A
23 (17.7) b A
17 (13.0) b A
nd
23(17.7)
3(2.3)
30(23.0)
52(40.0)
28(21.5)
20(15.4) a B
66(50.8) b B
34(26.2) b B
10(7.7)
93(71.5)
9(6.9)
27(20.8)
51 (39.2)
23(17.7)
18 (13.8) a B
50 (38.5) b B
26 (20.0) b B
8(6.2)
56(43.0)
3(2.3)
(P > 0.05).
Frequencies followed by dierent lower case letters denote dierences in the rows. Frequencies followed by dierent capital letters denote dierences in the columns. Analysis among hybrids by w-square.
b
80
Table 3
Fumonisin levels and number of colony forming units (CFU/g) of the fungi isolated from 195 freshly harvested and stored maize grains (hybrids
Br 201, C 125 and Cx 322) collected over a one-year period in Brazila
Fumonisin levels
(mg/g)b
Br 201
c
FH
S1e
S2
S3
S4
S5
S6
S7
S8
S9
Cx 322
Genera
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
ndd
594.6
6.9
1
1096
276
280
680
1480
60
2616
866
90
5340
1430
187
872
2096
50
460
1790
60
360
600
20
36
163
10
15
nd
460.3
18.7
nd
62.5
104
3
1216
329
72
1523
444
55
286
196
21
2790
121
70
550
620
140
290
120
14
22
34
14
24
nd
669.8
14.9
1
1415
41
nd
868
8
2
677
102
31
170
75
30
2940
260
50
490
50
120
160
100
3
36
13
2
89
Br 201
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
24.0
15.0
13.1
9.8
12.1
7.2
17.1
11.7
17.2
8.4
7.5
6.5
4.5
7.4
9.4
8.0
7.4
5.3
8.0
7.2
11.0
7.1
9.1
6.0
14.1
9.8
6.8
10.1
3.3
7.9
13.0
7.3
26.0
9.2
18.0
8.7
3.6
6.5
4.1
7.5
6.0
7.2
11.0
7.1
10.1
7.5
11.2
6.8
3.0
7.3
3.6
5.8
3.6
7.7
10.5
8.3
11.6
9.0
6.6
5.3
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
Samples
C 125
Table 3 (continued )
Fumonisin levels
(mg/g)b
Br 201
Samples
S11
S12
Meanf/SDg
a
Genera
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
Aspergillus
Fusarium
Penicillium
6
2
11
2
6
11
54
5
107
94
59.3/ 2 81.3
938.3/ 2 1442.3
681.8/ 2 734.5
34
9
18
10
2
69
13
5
148
45
31.1/ 2 40.2
573.7/ 2 785.1
160.6/ 2 183.4
For each hybrid, 65 samples were analyzed.
Mean of 5 samples.
c
Freshly harvested.
d
Not detected at dilution 10ÿ3.
e
Storage month.
f
Mean of 65 samples.
g
Standard deviation.
b
Cx 322
11
3
25
11
1
37
2
10
82
37
19.4/ 232.7
589.1/ 2789.6
55.7/ 267.6
Br 201
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
8.9
6.9
15.4
8.0
10.3
7.4
15.3
7.5
5.2
5.3
3.7
6.6
4.4
4.2
4.1
5.7
5.9
6.3
10.5/2 5.9
8.1/ 2 2.5
10.5/2 6.1
7.8/2 1.4
8.0/ 2 3.9
6.8/2 0.9
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
S10
C 125
81
82
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
most frequently associated with Brazilian maize grains. The predominance of F. moniliforme
had already been observed in Brazil, as well as the occurrence of F. proliferatum as the second
most frequent species in maize (Pozzi et al., 1995). However, our results show higher
frequencies of these fungi in the maize hybrids studied.
The three most important fungal genera in terms of toxigenicity produced the greatest
numbers of colony forming units (CFU/g) in hybrid Br 201, varying from 11 104 to
5340 104 with Fusarium spp, from 0 to 280 104 with Aspergillus spp and from 2 104 to
2096 104 with Penicillium spp. The values recorded for these fungal genera in the other two
hybrids were 18 104 to 2790 104 Fusarium spp, 0 to 140 104 Aspergillus spp and 10 104
to 620 104 Penicillium spp for hybrid C 125 and 25 104 to 2940 104 Fusarium spp, 0 to
120 104 Aspergillus spp and 2 104 to 260 104 Penicillium spp for hybrid Cx 322 (Table
3). These numbers exceed the tolerance limits set by the International Commission on
Microbiological Speci®cation for Foods (Elliott, 1980), which are of the order of 102 to 104
CFU/g.
The genus Aspergillus was absent from the myco¯ora of the 15 samples of freshly harvested
grains for the three hybrids studied (Table 3). In the remaining samples analyzed, species of
Aspergillus were found in grains with moisture content of 10.2±16.4%, the greater number of
recoveries occurring between 12.4±16.4% and at water activities of 0.7±0.79 (Tables 2 and 4).
The linear correlation analysis showed a signi®cant positive correlation between the
Table 4
Moisture content (MC) and water activity (aw) values of 195 samples of maize grains (hybrids Br 201, C 125 and Cx
322) listed with the climatic data recorded over a one-year period in Brazil
Mean of 5 samples per month
Samples
FHa
S1b
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
Mean
SIDc
a
Br 201 MC % ± aw
C 125 MC % ± aw
Cx 322 MC % ± aw
Relative humidity
(%)
Mean temperature
(8C)
17.0±0.91
16.4±0.82
16.2±0.81
16.2±0.74
14.2±0.75
12.3±0.68
12.0±0.69
12.6±0.73
11.7±0.70
11.8±0.71
12.6±0.68
10.3±0.72
10.8±0.71
13.3±0.74
2 2.2ÿ 2 0.06
16.8±0.87
16.8±0.82
16.6±0.79
16.4±0.74
14.0±0.80
12.6±0.70
12.0±0.66
12.4±0.74
11.8±0.67
11.9±0.71
12.6±0.69
10.2±0.72
10.9±0.71
13.4±0.74
2 2.2ÿ2 0.06
16.0±0.83
15.8±0.78
15.6±0.78
15.6±0.74
14.2±0.76
12.8±0.74
12.2±0.67
12.0±0.75
12.6±0.71
12.7±0.72
12.7±0.72
10.2±0.74
11.1±0.72
13.3±0.74
2 1.8ÿ 2 0.03
86.9
82.0
82.3
80.6
82.3
83.2
83.0
83.7
85.5
92.7
93.0
92.9
91.6
86.1
2 4.5
20.2
19.8
18.8
19.4
20.0
21.4
22.7
22.2
24.4
24.1
23.5
24.3
23.3
21.8
2 1.9
Freshly harvested.
Storage month.
c
Standard deviation.
b
Average per month
83
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
populations of Aspergillus spp and those of Penicillium spp (P < 0.05; 0.34) and signi®cant
negative correlations between Aspergillus spp and both relative humidity (P < 0.05; ÿ0.35) and
aw (P < 0.05; ÿ0.34). Signi®cant negative correlations were also found between the genus
Penicillium and both mean temperature (P < 0.01; ÿ0.41) and relative humidity (P < 0.01;
ÿ0.49). The regression analysis of number of CFU/g versus storage time showed a statistically
signi®cant correlation in the three hybrids analyzed for the genera Aspergillus and Penicillium
(P < 0.01).
The stepwise multiple regression analysis for number of CFU/g, storage time, aw, moisture
content, mean temperature and relative humidity revealed the most important variables in each
model: for the genus Aspergillus they were storage time in Br 201 (P < 0.05) and storage time,
relative humidity, mean temperature and mean temperature2 in C 125 (P < 0.05); for the genus
Penicillium they were time of storage and aw in Br 201 (P < 0.01) and mean temperature
(P < 0.05) in Cx 322.
The highest numbers of CFU/g of the genus Fusarium were recovered during the ®rst ®ve
storage months (Table 3) at mean temperature values of 18.8±21.48C, grain moisture contents
of 12.6±16.8%, relative humidity of 80.6±83.2% (Table 4). Fusarium spp showed signi®cant
negative correlations with mean temperature (P < 0.01; ÿ0.67) and relative humidity
(P < 0.01; ÿ0.57) and positive correlations with grain moisture content (P < 0.01; 0.52). These
results corroborate the ®nding that this mold, despite being able to survive on desiccated
grains, thrives in more humid conditions. The regression analysis of number of CFU/g versus
storage time showed a statistically signi®cant correlation (P < 0.01) for Fusarium spp. The
stepwise multiple regression analysis for number of CFU/g, storage time, aw, moisture content,
mean temperature, and relative humidity revealed the most important variables for each model
of the genus Fusarium: mean temperature and mean temperature2 for Br 201 (P < 0.01); aw
and relative humidity for C 125 (P < 0.05); storage time for Cx 322 (P < 0.01).
The lowest frequencies of occurrence were observed in hybrid Br 201 for Fusarium spp and
in hybrid Cx 322 for Aspergillus spp and Penicillium spp (Table 1). Hybrids Br 201 and CX
322 did not show statistically signi®cant dierences of Aspergillus, Fusarium and Penicillium
contamination at the aw values detected. However, in hybrid C 125, the higher numbers of
CFU/g for Aspergillus spp were statistically signi®cant when compared to contamination levels
for the other two genera (P < 0.05) at aw values of 0.90±0.80 (Table 2).
Tables 5 and 6 show the occurrence of fumonisins in grains of the Brazilian maize hybrids
analyzed. Out of a total of 195 samples (65 belonging to each of the three hybrids), 176
Table 5
Fumonisin levels in 195 samples of freshly harvested and stored maize grains (hybrids Br 201, C 125 and Cx 322)
from Brazil
BR 201
Positive samples
Mean concentration
Concentration range (mg/g)
C 125
Cx 322
FB1
FB2
FB1
FB2
FB1
FB2
62(93.38%)
10.54
0.90±46.39
64(98.46%)
8.17
2.51±29.16
56(86.15%)
10.57
0.98±49.31
63(96.92%)
7.80
3.1±20.65
58 (89.2%)
8.05
0.87±37.14
63(96.92%)
6.83
1.96±11.74
84
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
Table 6
Distribution of FB1 and FB2 levels in 195 samples of freshly harvested and stored maize grains (hybrids BR 201, C
125 and Cx 322) in Brazil
BR 201
C 125
CX 322
Total
% of samples
Fumonisins levels (mg/g)
FB1
FB2
FB1
FB2
FB1
FB2
FB1
FB2
FB1
FB2
< 1.0
1.0±3.0
3.1±5.0
5.1±8.0
8.1±10.0
10.1±13.0
13.1±15.0
15.1±18.0
18.1±20.0
20.1±23.0
23.1±25.0
25.1±28.0
28.1±30.0
30.1±50.0
nda
4
20
15
3
5
2
nd
3
4
1
1
nd
5
nd
2
nd
43
5
8
2
nd
1
2
nd
nd
1
nd
1
6
12
7
5
6
2
2
3
4
1
nd
2
5
nd
nd
3
33
16
7
3
1
nd
nd
nd
nd
nd
nd
1
6
14
16
3
5
3
2
2
3
1
nd
nd
1
nd
2
1
45
10
4
1
nd
nd
nd
nd
nd
nd
nd
2
16
46
38
11
16
7
4
8
11
3
1
2
11
±
1.0
8.2
23.6
19.5
5.6
8.2
3.6
2.0
4.1
5.6
1.5
0.5
1.0
5.6
±
2
2
62
15.9
9.7
3.1
0.5
0.5
1.0
±
±
0.5
±
a
4
4
121
31
19
6
1
1
2
±
±
1
±
nd=not detected.
(90.2%) were contaminated with FB1 at concentrations that ranged from 0.87±49.31 mg/g and
190 (97.4%) were contaminated with FB2 at concentrations that ranged from 1.96±29.16 mg/g.
Studies on fumonisin contaminants in maize and maize-based products from several countries
(Shephard et al., 1996; Bullerman, 1996) revealed levels of contamination lower than those
presently reported. A similar ®nding was observed when comparing our results with data on
FB1 and FB2 contamination in maize hybrids or inbred lines grown in countries from three
continents. Of these, Argentina showed the highest concentration of fumonisins (37 mg/g) in 51
samples analyzed (Visconti, 1996). In Brazil, Hirooka et al. (1996) reported maximum levels of
18.5 mg/g for FB1 and 19.3 mg/g for FB2 in 48 samples of maize grains.
With respect to possible dierences in fumonisin contamination between the maize hybrids
studied, their pro®les are compared in Tables 5 and 6. The lowest contamination occurred in
hybrid CX 322. This applied to both FB1 and FB2 mean levels (8.05 and 6.83 mg/g,
respectively) and range of concentrations (0.87±37.14 mg/g for FB1 and 1.96±11.74 mg/g for
FB2). The highest FB1 levels were found in 140 day-storage samples of the C 125 hybrid
(49.31 mg/g) and in freshly harvested samples of the BR 201 and CX 322 hybrids (46.59 and
37.14 mg/g, respectively). A more uniform distribution of mean concentrations of FB2 as
compared to FB1 occurred in all the hybrids.
After 140 days of storage, an overall decrease in the number of CFU/g for Fusarium spp
together with a tendency towards decreased FB1 mean concentrations was observed in the
three hybrids analyzed, although FB1 levels showed some variability. The diversi®ed myco¯ora
of these hybrids, demonstrated by the recovery of 13 distinct fungal genera, presupposes a
competition for nutrients. According to Horn and Wicklow (1983), each fungus carries a factor
that determines its growth and ability to compete with other fungal species. In this respect, it
R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87
85
should be mentioned that studies carried out by Marin et al. (1998) indicated that fungal
interactions could act as an additional factor in the control of Fusarium spp contaminants in
maize and lead to decreased contamination by this genus, mainly at lower aw. On the other
hand, sample size should also be considered as an in¯uencing factor when trying to account for
the decrease and variability of fumonisin levels recorded during our investigation (Whitaker et
al., 1998).
The statistical analysis showed a negative correlation between fumonisins B1 and relative
humidity (P < 0.001; ÿ0.68769). Negative correlations were also found between the fumonisin
B2 and relative humidity (P < 0.001; ÿ0.64384) and mean temperature (P < 0.001; ÿ0.48662).
FB2 correlated positively with grain moisture content (P < 0.001; 0.56913).
To conclude, the signi®cant levels of contamination with Fusarium spp reported herein, both
in terms of prevalence and number of CFU/g, and the detection of fumonisins in freshly
harvested and stored maize grains highlights the relevance of studies on the occurrence of
fusariotoxins in maize and its by-products in the tropics.
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