Fate of fumonisin B

1
in the processing of whole maize kernels during dry-milling
Francesca Vanara
*
, Amedeo Reyneri, Massimo Blandino
Turin University, Department of Agronomy, Forest and Land Management, Via Leonardo da Vinci 44, 10095 Grugliasco, TO, Italy
a r t i c l e i n f o
Article history:
Received 28 November 2007
Received in revised form 15 April 2008
Accepted 13 May 2008
Keywords:
Fumonisins
Maize
Meal
Dry-mill
Distribution
a b s t r a c t
The aim of this research was to evaluate how the amount of fumonisins in a kernel is re-distributed over
the different processing products. The study focused on the description of the dry-milling process, with
details on the products and by-products, milling yield, and the granulometric and chemical composition.
Maize kernels and four derived milling fractions from twenty-four lots were sampled from 2002 and
2006.
The main results were: (a) the animal meal and germ had a higher fumonisin content than the unpro-
cessed grain, while human meals were less contaminated; (b) there is an inverse relationship between
the particle size and fumonisin contents in meals; (c) toxin tends to concentrate in the bran and germ,
while the endosperm is only partially contaminated.
2008 Elsevier Ltd. All rights reserved.
1. Introduction
Fumonisins are a crucial problem concerning food products
based on maize grain. In South Europe, and in particular in Italy,
all maize lots are contaminated by these toxins, which are often
present at undesirably high concentrations (Bennett & Klich,
2003; Pietri, Bertuzzi, Pallaroni, & Piva, 2004).
Fumonisins are secondary metabolites produced by fungi of the
Fusarium genus and particularly by F. verticillioides, one of the most
common eld moulds associated with maize and maize-based
foods and feeds (Humpf & Voss, 2004). The contamination occurs
mainly in the eld during the last part of the growing season and
is favoured by warm temperatures and high air humidity: under
such climatic conditions, F. verticillioides is able to infect maize ker-
nels by 100% (Logrieco, Mul, Moretti, & Bottalico, 2002). Post har-
vest and storage conditions, when kernel humidity is lower than
14%, prevent Fusaria activity (Ominski, Marquardt, Sinha, & Abram-
son, 1994); consequently, eld prevention plays an important role,
and crop practices that are able to reduce Fusarium ear rot and
fumonisin content are very relevant to obtain maize kernels of high
quality (Munkvold, 2003; Reyneri et al., 2004). However, crop prac-
tices are unable to exert a satisfactory control of F. verticillioides;
therefore decontamination, with the removal of the most contam-
inated part of the kernel during cleaning and milling processes be-
comes rather crucial, since detoxication of fumonisins with
chemical, biological and physical treatments are not allowed and
are economically unprotable (Castells, Marin, Sanchis, & Ramos,
2005; Scott, 1991; Sinha, 1998). The cleaning process is the rst
way of removing fumonisins concentrated on the pericarp of the
kernel and in the cracked, broken and damaged grains. The simple
step of cleaning maize can reduce fumonisin residues to less than
half that of uncleaned maize (Saunders, Meredith, & Voss, 2001).
The different ways of processing maize, such as dry-milling,
wet-milling, the masa-type process, cooking and extrusion, are
able to improve the nal quality of food products by reducing
mycotoxin concentrations. Moreover, it has been demonstrated
that the levels of mycotoxins in contaminated commodities may
be reduced or re-distributed by food processing procedures which
may involve physical and/or chemical steps (Saunders et al., 2001).
Milling processes by themselves are ways of decontaminating the
maize mycotoxin content (Castells et al., 2005). Dry-milling is a
process that is able to separate grain components by grinding
maize into various particle sizes through the use of roller mills.
Decontamination takes part during the germ separation, a step
usually carried out because the inclusion of its high oil content in
the product reduces shelf-life, due to oxidation and consequent
rancidity. Germs have shown a higher fumonisin content than
the whole kernel (Brera, Debegnach, Grossi, & Miraglia, 2004). An-
other by-product of milling is animal meal (bran), a mixture of the
grain pericarp and a part of the mealy endosperm, used for feeds
(Kent & Evers, 1994). Even bran and animal meal have demon-
strated particularly high fumonisin content (Brera et al., 2004; Bro-
ggi et al., 2002). The main fractions intended for human
consumption are derived from the milling of the horny and mealy
endosperm. The endosperm texture is important because grits and
meals, the products with the highest value, are essentially derived
from the horny endosperm.
0956-7135/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2008.05.014
* Corresponding author.
E-mail address: francesca.vanara@unito.it (F. Vanara).
Food Control 20 (2009) 235238
Contents lists available at ScienceDirect
Food Control
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodcont
Recently, the introduction of European Commission Regulation
(EC) No 1126/2007 (Ofcial Journal of the European Communities,
2007) has stressed the necessity of controlling the fumonisin con-
tent in the chain from the unprocessed grains to the processed
maize-based foods.
Information on the distribution and contamination levels of
fumonisins in food-grade maize and the fate of fumonisins
during the dry-milling of maize has been collected from an
Italian commercial dry-mill. The objectives of this work were:
(1) to determine the distribution of fumonisins in the frac-
tions obtained from dry-milled maize and (2) to establish
the maximum fumonisin content in the whole maize that
could be processed to achieve an acceptable nal fumonisin
contamination in the fractions destined for human
consumption.
2. Material and methods
2.1. Milling process and sampling (commercial dry-milled maize)
From 2002 to 2006, samples of naturally contaminated maize
and its products were collected from a commercial dry-mill used
only for maize processing, in Northern Italy.
The industrial process is based on a dry-milling technology
coupled with a dry degermination. The raw material is cleaned
through a dry stoner, an intensive horizontal scourer, and a vibrat-
ing aspirator. In this step, corn is separated from waste (stones,
earth, and other foreign matter) and from other types of foliage
(cobs, broken kernels, etc.). The foliage is then sent to the ham-
mermill for the production of animal meal. After the cleaning pro-
cess, the corn is sent to the degermination step, which is
composed of three machines: a breaker, a plansichter and gravity
tables. The rst one breaks the kernel into germs, bran, aking
grits and other ne fractions. The plansichter separates all the
products and, at the end, the gravity tables make a nal separation
between the germs and aking grits. The aking grits are pro-
cessed through a rolling and classication system, composed of
rollermills, plansichters, a sieving machine and an aspirator. The
objective of the rening is to reduce the grits to a standard meal
size while producing the minimum quantity of nes. The rening
of the aking grits denes one (two) of these different main prod-
ucts according to the machines adjustments: (a) brewery grit, (b)
pearl and break meal, (c) precooked meal. The last one is derived
from aking grits that are transformed by a steam cooker, a ake
rolling press, and a our dryer. A common by-product of aking
grit rening is maize meal.
Twenty-four lots were analysed, with 164 samples shared out
between the whole maize kernel, germ, animal meal (bran), maize
our and meal.
The sampled products were chosen during milling so that the
sum of the products represented the lot of origin. Samples of each
product listed above were drawn from opening slits of the plant
and the adopted sampling procedure was derived from European
Commission Regulation (EC) No 401/2006 (Ofcial Journal of the
European Communities, 2006). Considering that the plant mills
56 t h
1
of maize kernel, a dynamic sampling procedure was
planned in which each aggregate sample was the result of careful
the blending of 40 incremental samples of 100 g each, collected in
1 h at regular intervals. The fumonisin B
1
content and the chemical
compounds of the raw processed material were derived from the
analysis of whole maize kernel samples collected before the clean-
ing process. The samples were maintained at 18 C until the
mycotoxin analysis was performed, and a sub-sample was imme-
diately employed for granulometric and chemical composition
analysis.
2.2. Granulometric and chemical composition analysis
The particle size, total fat and raw ash contents were detected
using the following methods.
The granulometric analysis was carried on with a meal sifter
(Retsch Analytical Sieve Snakers AS200), an instrument with a
series of riddles placed on a turning platform and an engine that
performs a movement like that of the plansichter in the com-
mercial mill. The analysis was carried out with 300 g of starting
meal and, after 10 min, the quantity of each fraction was
weighted in each riddle in which there was a different range
of particle sizes.
The fat and ash contents were determined following the meth-
ods described in the ISTISAN Report 1996/34 (Baldini et al., 1996a,
b). The fat analyse was carried out with an organic solvent and a
soxhlet extractor, while the ashes were determined as the residue
of complete burning of the sample at 550 C.
2.3. Fumonisin analysis
A 50 g representative sub-sample of the milled material was
analysed for the toxin content.
The fumonisin B1 analysis was carried out using a high perfor-
mance liquid chromatography (HPLC) method. Samples were
extracted by shaking for at least 15 min with 100 ml of water/
methanol 80/20 containing 5 g of NaCl. The supernatant was l-
tered through lter paper (0.45 lm), then 10 ml of solution was
diluted with 40 ml of PBS (8.0 g of NaCl + 1.2 g of Na
2
HPO
4
+ 0.2 g of KCl + 0.2 g of KH
2
PO
4
in 1 l of water). The extract was
cleaned using a FumoniTest

column (Vicam

) by rinsing with
10 ml of PBS. The fumonisin B1 was then eluted using 1.5 ml of
methanol and the eluate was collected in a sample vial. The
methanol eluate was dried and then redissolved in 200 lL meth-
anol: water (50:50). Well-mixed redissolved eluate (25 lL) was
mixed with 225 lL OPA reagent (40 mg of o-phthaldialdehyde
in 1 ml of methanol, and a 0.1 M solution of disodium tetraborate
and 50 ll of 2-mercaptoethanol). Ten microlitres was injected
into an HPLC column (Hypersil C18 column, 100 2.1 mm,
0.2 ml min
1
; uorescence detector, with an excitation wave-
length of 250 nm and an emission wavelength of 440 nm). Toxin
quantication was performed using external standards and peak
height measurements. The analytical method used presented an
average recovery for FB1 of 83% and the detection limit was
10 lg kg
1
.
2.4. Statistical analysis
An analysis of variance was performed for the fumonisin levels
detected in each fraction using the SPSS Inc., Chicago, IL, Version
12.0 programme. Appropriate statistical multiple comparisons
among the means were made using the SNK test. The fumonisin
data were log-transformed before analysis.
3. Results
The chemical composition of the milled fractions is summa-
rized in Table 1. As expected, the germ was rich in oil and ashes
and it was the most different fraction in terms of chemical com-
position. The feed fraction, commercially dened as the animal
meal, is a complex fraction constituted by the pericarp and part
of the mealy endosperm separated from the food products be-
cause of its too ne particle size. It had an ashes and fat mean
content similar to that of the whole kernel. The meal and maize
our had a signicantly lower ash and fat content than the whole
kernel, but the latter had a signicantly higher fat and ash
content.
236 F. Vanara et al. / Food Control 20 (2009) 235238
The 24 lots presented an extreme variability of fumonisin con-
centration, with a range from 273 to 8480 lg kg
1
in the whole
kernel.
The fumonisin B
1
contamination data of each fraction were cal-
culated as the mean of the 24 sampled lots (Table 2). The most con-
taminated fractions were germ and animal meal, with a fumonisin
B
1
content of 3450 and 11,400 lg kg
1
, respectively, in comparison
to 4580 lg kg
1
of the whole kernel. Statistical analyses showed a
signicant difference (P < 0.001) between the animal meal and
whole kernel, while the germ had a similar fumonisin content
compared to whole kernel. The maize our and meal were less
contaminated than the whole kernel, with a mean content of
1690 and 499 lg kg
1
respectively; moreover the meal was signif-
icantly lower (P < 0.001) than all the other analysed fractions.
If fumonisin is expressed as the weighted percentage of the sum
of fumonisins in the fractions, the animal meal fraction contained
the highest fumonisin amount, about 7589% of the recovered
fumonisin, while the food fractions only accounted for about 6
8% of the total fumonisin and the germ for about 69% (Table 2).
The different products for human consumption from the sam-
pled dry-mill and their particle size distribution are summarized
in Table 3. These fractions, described before as meal, include three
different commercial products, obtained from different rening
procedures during the process: (1) brewery grit; (2) pearl and
break meal; (3) precooked meal. The rst one is an ingredient used
for beer production, while pearl and break meal are used for pole-
nta, and are produced during the same process. The coarse grits
must be cooked before grinding in order to obtain a precooked
meal, while heating is not necessary for any of the other products.
The last product for human consumption, the maize our, is an
ingredient that is usually used in the baking and confectionery
industry and is produced during each type of rening.
The main difference between the products is their particle size
composition. Maize our is the only one with more than 50% of
particles under 350 lm, while all the other products have less than
10% under this size. Brewery grit instead, is the only product with
more than 15% above 1000 lm. Pearl and break meal differ because
the rst one has more than 80% of particles above 500 lm, while
break meal has about 80% of particles under 500 lm. Precooked
meal is an intermediate product, with about 90% of particles be-
tween 350 and 840 lm. Taking into consideration European Com-
mission Regulation (EC) No 1126/2007, the maize our is the only
milling fraction with a particle size completely <500 lm.
The analytical results of this study showed that all the food
products had a lower mean fumonisin B
1
content than the whole
kernel (Table 4). The maize our had a higher fumonisin content
(P < 0.001) than all the other meals, while there were no signicant
differences within this last group. The most important difference
between the maize our and meal/grits is the granulometric com-
position; the smallest fractions, derived fromthe mealy endosperm
that surrounds the germ, are found in maize our. All the other
products are the result of grinding and sieving, at different levels,
especially of the horny endosperm; therefore no signicant differ-
ence in fumonisin content was observed.
4. Discussion
The data underline the interaction between the chemical com-
position, particle size and fumonisin content.
Fumonisins are not uniformly distributed in maize kernels and
the high content in animal meal is mainly due to the presence of
the pericarp, the rst part of the kernel colonized by fungi because
of its peripheral location and also the part to which kernel dusts
adhere. The presence, in this fraction, of part of the endosperm lo-
cated near the germ (mealy endosperm) probably contributes to its
high fat and fumonisin content.
In a study conducted with a collection of samples derived from
both commercial dry-milled maize and from experimental dry-
milled maize, Katta, Cagampang, Jackson, and Bullerman (1997) re-
ported the same results, with high fumonisin concentrations in
germ, bran and nes. They suggested that higher fumonisin con-
centrations, together with higher Fusarium counts in germ, bran
and nes may be due to localization of the fungus in the tip cap
and germ areas just beneath the pericarp.
The increase infumonisinconcentrationwiththe decreasingpar-
ticle size of meal is another relevant aspect that these results have
pointedout. The fumonisincontent inthe humanfoodproducts ana-
lysed in this study increased as the particle size decreased. Katta et
al. (1997) reported the same results and formed the hypothesis that
this may be due to contamination of the meal with the germ. Our
data also suggest the hypothesis that the horny and mealy endo-
Table 1
Chemical composition of the dry-milled fractions
Fraction Samples
(N.)
Ashes Fat
Mean
(%)
Min
(%)
Max
(%)
Mean
(%)
Min
(%)
Max
(%)
Whole kernel 7 1.66c 1.51 1.80 4.11d 3.40 4.80
Germ 7 4.84d 4.00 6.35 17.4e 15.8 19.0
Animal meal (bran) 7 1.51c 0.90 1.80 3.23c 3.00 3.80
Maize our 7 0.72b 0.60 0.90 1.97b 1.62 2.20
Meal 7 0.50a 0.40 0.62 1.19a 0.99 1.60
Post-hoc SNK test. Different letters mean statistically signicant differences.
Table 2
Distribution of fumonisin B
1
in the whole kernel and in its dry-milled fractions
Fraction Kernel weight Fumonisin B
1
Min
(%)
Max
(%)
Mean
(lg kg
1
)
Min
(lg kg
1
)
Max
(lg kg
1
)
Min (%
of total)
Max (%
of total)
P < 0.001
Whole kernel 4580c 273 8480
Germ 8 12 3450bc 175 9920 6.0 9
Animal meal
(bran)
30 36 11,400d 1310 26,800 74.7 89.6
Maize our 3 5 1690b 120 4420 1.1 1.9
Meal 49 54 499a 85 1340 5.3 5.9
Post-hoc SNK test. Different letters mean statistically signicant differences.
Table 3
Distribution of the particle sizes of the food products
Fraction Particle size percentage (lm)
<350 350500 500840 8401000 >1000
Maize our 56 44 0 0 0
Break meal 9 69 22 0 0
Pearl meal 1 15 67 14 3
Precooked meal 2 35 58 5 0
Brewery grits 3 78 19
Table 4
Distribution of fumonisins B
1
in the food products
Fraction Fumonisin B
1
Mean (lg kg
1
) Min (lg kg
1
) Max (lg kg
1
)
P < 0.001
Maize our 1690b 120 4430
Break meal 724ab 96 1230
Pearl meal 491a 85 845
Precooked meal 437a 108 1340
Brewery grits 454a 305 537
Post-hoc SNK test. Different letters mean statistically signicant differences.
F. Vanara et al. / Food Control 20 (2009) 235238 237
spermare contaminatedto different extent, witha higher fumonisin
content in the latter, from which the ne particles are derived.
Broggi et al. (2002), in a study in a commercial dry-mill in
Argentina, found a three times higher fumonisin contamination
level in germ and bran than in whole corn. In this Italian study,
the germ had the same fumonisin content as the whole kernel,
while the bran had a three times higher content. However, consid-
ering the reduction coefcients from the whole kernel, the values
were approximately 3 and 9 for maize our and meal, respectively.
Similar coefcient values of 4 and 11 were reported in the Argen-
tinean study.
Dry-millers are concerned about these reduction coefcients be-
cause they are used to dene the maximum acceptable content of
fumonisins in the whole kernel in order to respect the maximumle-
vel in the products for the direct human consumption and to assure
consumers health. Considering European legislation regarding
fumonisins (sum of FB
1
and FB
2
), different maximum levels are set
for smaller and larger milling fractions than 500 lm, for maize-
based food for direct consumption and for baby foods; these levels
reect the contamination level of the different fractions and range
between 1400 and 2000 lg kg
1
for milling fractions and between
200 and 1000 lg kg
1
for maize-based food for direct consumption.
As a consequence, in a mill with a similar coefcient of reduction as
the one analysed in this study, it is necessary to mill raw kernels
with an approximately lower fumonisin B
1
and B
2
content than
6000 lg kg
1
. This value is derived from the reduction coefcient
of maize our (three times), which is the most restrictive coefcient
used to assure 2000 lg kg
1
in the smallest fraction.
In conclusion, the research has dened a fumonisin re-distribu-
tion in milled products in this commercial mill and has found ker-
nel components with clearly different toxin contents. This work
agrees with other studies on fumonisin distribution in milled frac-
tions. Bran is the fraction with the highest fumonisin content; as a
consequence, the wholemeal formed by the endosperm and bran
fractions has a higher risk of contamination than common meal.
The germ is also a part of the kernel which has an important con-
tamination. For this reason, this study conrms that there is a dif-
ference between fractions obtained from degermed corn kernels
and those from non-degermed or partially-degermed corn (US
Food, 2007). Finally, the contamination of food products depends
on two main aspects: rst of all, the whole kernel content in
fumonisins, and second, the kernel component that is used for
the nal products.
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