Vol.5, No.10A, 9-13 (2013) Health
http://dx.doi.org/10.4236/health.2013.510A1002
Recombinant protein detection and its content in
total protein, lipids and toxic antinutritional
substances in Mexican maize
Peña Betancourt Silvia Denise1*, Posadas Manzano Eduardo2,
Valladares Carranza Benjamín3
1Laboratorio de Toxicología, Departamento de Producción Agrícola y Animal, UAM-Xochimilco, México, D.F., Mexico;
*Corresponding Author:
2Departamento de Rumiantes, Facultad de Medicina Veterinaria y Zootecnia UNAM, México, D.F., Mexico
3Centro de Investigación y Estudios avanzados en salud animal, FMVZ-UAEM, México
Received 18 July 2013; revised 19 August 2013; accepted 15 September 2013
Copyright © 2013 Peña Betancourt Silvia Denise et al. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
The engineering genetic technology has devel-
oped Bt maize events which contain recombi-
nant protein that will be safe for the consumer.
The aflatoxins are contaminants present in
maize capable of producing cancer and de-
creasing the immune response in human, addi-
tionally contained polyphenols compounds con-
sidered non nutritive. The objective of this study
was to identify the presence of recombinant
protein in hybrid and local varieties of corn and
evaluate the content of aflatoxins and tannins.
25 samples of white grain maize for human con-
sumption were collected, 12 were for hybrid
maize and 13 local varieties, from the states of
Hidalgo, Mexico and Morelos. Samples were ana-
lyzed for Cry1Ab/Cry1Ac, using lateral flow strip
method, crude protein and lipids by standard
methods. Aflatoxins were assessed by comer-
cial Elisa kit and tannins by spectroscopy me-
thod. The data were grouped in a completely
random model and an analysis of variance was
performed. The results indicated that 44.5% of
hybrid corn was positive by Bt-Cry1Ab/1Ac pro-
teins, containing 9.02% ± 2.5 lipids and 11.33% ±
2.2 crude protein, 189 ± 0.92 mg/g of tannins and
6.36 ± 3.3 µg·g−1 aflatoxins. The local maize
samples (55.5%) were negative to Bt-Cry1Ab/
Cry1Ac, which protein content was of 8.68% ±
0.90, 6.14% lipids ± 2.3, 273 ± 0.40 mg/100g tan-
nin and 7.15 ± 3.3 µg·g−1 of aflatoxins. In con-
clusion, we observed an improvement of nutri-
ent composition in hybrid maize with Bt proteins,
and decrease in tannins content comparing with
some local varieties without Bt proteins. The
effectiveness of Bt maize expressing the Cry1Ab/
Cry1Ac in reducing aflatoxin contamination was
not observed, therefore, additive affects of afla-
toxins contamination in maize Bt-Cry need to be
further investigated in cancer disease develop-
ment.
Keywords: Transgenic Maize; Mycotoxins; Safety;
Toxicants; Anti-Nutritional Protein
1. INTRODUCTION
Recombinant DNA technology has been running for over
30 years in various agricultural crops worldwide [1].
Corn is a staple in the food and feed because of its high
energy content and low cost. The genetically modified
maize events have a higher protection against insects and
the herbicide glyphosate, due to the inclusion of genes
from bacterias such as Bacillus thuringiensis, Esche-
richia coli and some species of Agrobacterium. The cry
genes express proteins with toxic action on mainly Le-
pidoptera insects [2,3]. The events of Bt-176, MON-810
and Bt-11, contain one or more genes (Cry1Ab, CP4 and
esps) expressing Cry1A proteins, Cry1Ac, Cry2Ab. Bt
events have many advantages over isogenic counterpart
(non Bt maize), such as an increase in lysine amino acid,
enhanced ability to prevent fungal pathogens [4], and
their toxins [5], between others, however, not enough
evidence is currently available in Mexico to support or
refuse the safety of proteins Cry present in Bt maize [6,7].

P. B. S. Denise et al. / Health 5 (2013) 9-13
10
The introduction of Bt corn seed in Mexico has gener-
ated a considerable debate in public due to ignorance of
its implications for human health, principally in the de-
velop of allergy diseases [8,9]. In the literature it exists
information about the toxicity principally made with la-
boratory animals in which they have been probed a very
low acute oral toxicity [10,11], on the other hand sub-
stantial equivalence studies have failed to show differ-
ences from isogenic corn (non Bt maize), other works
have shown resistant to fungal contamination of genus
Fusarium verticillioides and Aspergillus flavus, both
found in the mycoflora present in maize produced under
high temperatures and humidity, climatic conditions in
states of Mexico, such as Hidalgo, Mexico and Morelos
[12]. The aflatoxins produced by Aspergillus flavus, fil-
amentous fungi, causes a decrease in immune function
and tumor formation in the liver. Fusarium verticillioides
synthesize fumonisins, and can lead to esophageal cancer
[13]. The antinutritional substances which comprise tan-
nins, a group of polyphenols, present in small corn con-
centrations, are known for its role as natural protective
agents against pathogenic microorganisms during plant
growth in the field, and by the capacity to precipitate
proteins and starch which causes a decrease in the crop
nutritional value [14]; the gallic acid is involved in a
lymphocytes Th1 inhibition, then interleukine IL-1b se-
cretion, and cyclooxygenase-2 gene expression. Also it is
important to remember that epigenetic modifications in
DNA play a role in tumorigenesis [15], like to emphasize
the studies in which rats were fed with Bt maize (MON810)
for 30 and 90 days. It was observed a decrease in T and
B cells specific for CD4 and CD8 sub-populations in the
treated rats, which shows a decrease in lymphocytes Th1
and interleukine response [16].
The protein products of the expression of a transgene
can be determined using immunological methods, like
strip test (strips-reactive), which may have a nitrocellu-
lose support for a specific antibody conjugated to a target
protein staining reagent. The result is qualitative (posi-
tive or negative) and the detection limit is between 0.5
and 2% for corn events MON810 and Bt11 which are
protected from insects by the cry1Ab gene from Bacillus
Thuringiensis.
The aim of this study was to identify the presence of
Bt-Cry1Ab protein/Cry1Ac in 25 commercial corn sam-
ples from the center of Mexico and to compare among
nine pairs about the protein content, lipid, aflatoxins and
tannins.
2. MATERIAL AND METHODS
2.1. Materials
Grain Collect
There were two visits INIFAP Experimental Fields in
the states of Hidalgo and Morelos. The State of Mexico
samples were provided by the FES-Cuautitlán, UNAM.
A total of 25 accessions of white grain maize, identified
commercially as P30G40, P30V46, P30T26, panther,
leopard, bear, bengala, puma 1167, puma 1085, H-155,
H-159, H-515, H-519, H-516, DK-2027, DK-2060,
cronos, aspros, V5370, toluqueño, bola, chalco, puebla,
tlaxcala, añejo and chapingo. All samples were milled
into whole grain flour passed through 250 µm screen
mesh, prior their analyses.
2.2. Methods
2.2.1. Physical Characterization
It was determined moisture content of each sample
grain by duplicate according to standard method [17,18] .
2.2.2. Chemical Analysis
The content was determined by the crude protein
MacroKjeldahl method and the fat by the Goldfish
method 7044 [19], performing every duplicate analysis.
The identification of Cry1Ab-1Ac was assay by lateral
flow strip method (ImmunoStrip test) of Agdia laborato-
ries, USA.
2.2.3. Aflatoxins Analysis
Aflatoxin analysis was performed by enzyme immu-
noassay ELISA (Aflatoxin Ridascreen kit from R-Bio-
pharm AG Germany) to cereals. The assay detection
limit is 2 mg·kg. It was prepared by 5 g ground corn
flour finely and the extraction was performed with 10 mL
of the methanol: water (50:50 v/v), according to the
manufacturer’s procedure, the absorbance was read at
650 nm on a plate reader and the data was passed to EXE
RIDAWIN Software multiplying by the dilution factor
generating results in µg·g−1.
2.2.4. Poliphenols Compounds Analysis
The extraction of free phenolic compounds was per-
formed according to Dewanto et al., (2002): 5 g of
ground whole grain was blended with aqueous methanol
(80:20, v/v), for 15 minutes, using achilled waring
blender. The absorbance readings at 725 nm were taken
in spectrophotometer UV-visible light, for quantification.
Total phenolic content was expressed as mg tannic acid/
100 g, based on a standard curve of 0 - 500 µg de tannic
acid/mL.
2.2.5. Bt-Cry1Ab/1Ac Protein Detection
Corn kernels were ground into a fine powder and the
extraction was performed with double-distilled water,
placing it in a beaker and placed the strip immune
(ImmunoStrip Bt-Cry1Ab/1Ac Test, Agdia Laboratory,
USA).

P. B. S. Denise et al. / Health 5 (2013) 9-13 11
2.2.6. Statistical Analysis
It was made an analysis of variance to determine dif-
ferences between means in a completely randomized de-
sign.
3. RESULTS
From the total of the samples collected, only eighteen
could be evaluated due to the loss of the material and
insufficient quantity for four chemical analysis. The
44.5% of the samples were positive for Bt-Cry1Ab/1Ac
protein. From twelve maize hybrids analyzed, all were
white grain by human consumption, most of them
(33.33%) from Hidalgo State, (22.2%) from Mexico
State, and (11.11%) Morelos, in all of them were de-
tected qualitatively Bt-Cry1Ab/Cry1Ac proteins, and
none of local varieties studied. The results showed that
crude protein content in corn hybrids positive to Bt-
Cry1Ab/Cry1Ac was significantly higher that the com-
posite in corn non Bt-Cry proteins (11.33% versus
8.68%); as well as humidity (12.27 % and 10.75%). The
mean lipid content was significantly (p ≤ 0.05) higher
(9.02% compared to 6.14%) (Table 1). Aflatoxins were
identified in all samples analyzed in an average level of
6.75 µg·g−1, a range to 2.0 - 13.0 µg·g−1; in maize hybrids
positive to Cry1Ab/Cry1Ac proteins showed an average
content of 6.36 ± 3.3 µg·g−1 compared to 7.15 ± 3.3
µg·g−1 non Bt-Cry protein, with any significant differ-
ence. The ranges of total phenolic levels observed among
the 18 Mexican maize studied were 140 - 360 mg/100 g
of grain flour. Among the group of maize with Cry pro-
teins was 130 - 300 mg/100 g and a median to 189 ± 0.92
mg/100 g, and for the group of maize without Cry pro-
teins was 200 - 360 mg/100 g, with a median of 273 ±
0.40 mg/100 g, highlighting the hybrids 30T26, H-515,
H-519 and Cronos by contented the greatest phenolic
levels (250 - 270 mg/100 g), however their content was
slightly lower than levels founded in local varieties (300
- 360 mg/100 g) (Table 2).
4. DISCUSSION
The protein 1Ab/1Ac which was observed with im-
munological strips was quickly and effective, which
agrees with the views expressed previously [20,21]. This
finding, should alert epidemiologists as the food allergy
due to importance in health, like, in Mexico, the allergy
diseases increased in the last 40 years by effects of pollu-
tion, in others countries has been reported a prevalence
of food allergy in 3.52%, with the 9% due to allergens in
vegetables, more than eggs or milk [22]. Moreover this
study emphasizes the increase risk of food allergy in
well-developed countries; it has been mentioned that
food allergies are increasing due to physico-chemical
modifications of food proteins and the development of
Table 1. Chemical analysis and identification of proteins
Cry1Ab/1Ac in 18 commercial corn samples from the states of
Hidalgo, Morelos and Mexico.
No. of
samples
Moisture
(%)
Lipids
(%)
Proteins
(%)
Local and hybrid
Corn negative hybrid
Cry protein 1Ab/1Ac
10 10.75 ± 0.58 6.14 ± 2.38.68 ± 0.86
Hybrid Corn positive
hybrid Cry protein
1Ab/1Ac
8 12.27 ± 0.89 9.02 ± 2.511.33 ± 2.19
Table 2. Aflatoxins and tannins content in different corn strains
and varieties.
Maize varieties name Aflatoxin B1
(µg·g−1)
Tannins
(mg/100 g)
Local and hybrid Corn negative
hybrid Cry protein 1Ab/1Ac 7.15 ± 3.3 273 ± 0.40
Hybrid Corn positive hybrid Cry
protein 1Ab/1Ac 6.36 ± 3.3 189 ± 0.92
new food processing agro-technologies, considering the
emergence of new allergens [23]. Furthermore the results
suggest that Bt maize hybrids have been commercially
introduced to the country, which is because of importa-
tions and the need to increase the human grain consump-
tion, it helps lower the risk of cardiovascular disease,
type II diabetes and gastrointestinal cancer [24].
In general, the content view of humidity in all maize
samples was appropriate because the law allows up to
15% for marketing. The higher content of protein and
lipids in maize with recombinant protein (Bt-Cry1Ab/
1Ac), suggests a benefit due to hybridization and trans-
genesis, which is similar to the previously published,
however it is necessary to consider that increased lipids
in maize can promote the formation of reactive species
(ROS), and subsequent liver damage, including cancer in
humans [25].
The aflatoxin levels observed in all samples contained
less than 20 µg AFB1 kg, so they are within allowed by
Mexican legislation in corn for human consumption, but
it is important to consider their genotoxic and carcino-
genic effect and their chronic exposition, it can lead to
DNA epigenetic modification, then in cancer disease, by
the ingestion of low levels aflatoxins [26]. Additionally
we did not found a significant reduction of aflatoxins
accumulation as has been mentioned earlier [27,28],
these differences may be caused by the influence of cli-
matic conditions in the places of maize source and by
different forms of production. The ranges of tannins lev-
els founding in the 18 Mexican maize were lower than
those reported previously [29], nevertheless confirms
that most of the phenolic compounds present in bound
form, not to mention the environmental effects on me-

P. B. S. Denise et al. / Health 5 (2013) 9-13
12
tabolism vegetal.
5. CONCLUSIONS
The result of this research indicates differences among
hybrid and local maize commercialized in three states of
Mexico about the presence of recombinant protein (Bt-
Cry1Ab/Cry1Ac).
The effectiveness of Bt maize expressing the Cry1Ab/
Cry1Ac in reducing aflatoxin contamination was not ob-
served under climatic conditions of three states of Mex-
ico, therefore, the additive effects of aflatoxins contami-
nation in maize Bt-Cry need to be further investigated in
cancer disease development.
We observed an improvement of nutrient composition
in hybrid maize with Bt proteins, and a decrease in tan-
nins content in comparison with some local varieties.
The immunological test strips for the identification of
proteins Bt-Cry1Ab/Cry1Ac are efficient and fast.
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