Recombinant protein detection and its content in total protein, lipids and toxic antinutritional substances in Mexican maize

doi:10.4236/health.2013.510A1002

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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: [email protected]

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

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

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

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.

REFERENCES

[1] James, C. (2004) Executive summary: Global status of

commercialized biotech/GM crops. ISAAA Briefs No. 32.

ISAAA, Ithaca.

[2] Betz, F.S., Hammond, B.G. and Fuchs, R.L. (2000) Sa-

fety and advantages of Bacillus thuringiensis-protected

plants to control insect pests. Regulatory Toxicology and

Pharmacology, 32, 156-173.

http://dx.doi.org/10.1006/rtph.2000.1426

[3] Boisvert, M. and Boisvert, J. (2000) Effects of Bacillus

thuringiensis var. israelensis on target and non target or-

ganisms. A review of laboratory and field experiments.

Biocontrol Science and Technology, 10, 517-561.

http://dx.doi.org/10.1080/095831500750016361

[4] Sevenier, R.M., van der Meer, I., Bino, R. and Koops, A.J.

(2002) Increased production of nutriments by genetically

engineered crops. Journal of the American College of Nu-

trition, 21, 199S-204S.

http://dx.doi.org/10.1080/07315724.2002.10719266

[5] Rubens, O.N. (2009) Calidad de los análisis de riesgo e

inseguridad de los transgénicos para la salud ambiental y

humana. La Revista Peruana de Medicina Experimental y

Salud Pública, 26, 74-82.

[6] Traavik, T and Heinemann, J. (2007) Genetic engineering

and omitted health research: Still no answers to ageing

questions. Third World Network, Penang.

[7] Hails, H. and Kindererer, G. (2003) The GM public de-

bate: Context and communication strategies. Nature Re-

views in Genetics, 4, 819-826.

http://dx.doi.org/10.1038/nrg1182

[8] Rance, F. Allergie alimentaire a l´ arachide et aux fruits a

coque. Allergologie Pedriatic, Paris.

[9] López, M.L.X., Oliart, R. and Valerio, A.G. (2009) An-

tioxidant activity, phenolic compounds and anthocyanins

content of eighteen strains of Mexican maize. Food Sci-

ence and Technology, 42, 1187-1192.

[10] Grisolic, C.K., Olibeira, F. E.C., Ramos, F.R., Lopes,

M.C., Muniz, F.D.H. and Monnerat, R.G. (2009) Acute

toxicity and citotoxicity of Bacillus thuringiensis and Ba-

cillus saphcericus strains in fish and mouse bone narrow.

Ecotoxicology, 18, 22-26.

http://dx.doi.org/10.1007/s10646-008-0252-7

[11] Peterson, G, Cunningham, S., Deutsch, L., Erickso, J.,

Quinla, A. and Raez, L.E. (2000) The risk and benefits of

genetically modified crops: a multidisciplinary perspec-

tive. Conservation Ecology, 4, 13-18.

[12] Chassy, B.M. (2002) Food safety evaluation of crops pro-

duced through biotechnology. Journal of the American

College of Nutrition, 21, 166-173.

http://dx.doi.org/10.1080/07315724.2002.10719261

[13] Einspanier, R., Klotz, A., Kraft, J., Aulrich, K., Poser, R.,

Schwagele, F., Jahreis, G. and Flachowsky, G. (2001) The

fate of forage plant DNA in farm animals: A collaborative

case-study investigating cattle and chicken fed recom-

binant plant material. European Food Research and Te-

chnology, 212, 129-134.

http://dx.doi.org/10.1007/s002170000248

[14] Nowa, K.W. (2007) New perspectives for use of native

and engineered recombinants foods and proteins untreat

of food allergy. Immunology and Allergy Clinics of North

America, 27, 105-127.

http://dx.doi.org/10.1016/j.iac.2006.11.006

[15] Herrera, E.L. and Simpson, J. (1995) Genetically engi-

neered resistance to bacterial and fungal pathogens. World

Journal of Microbiology & Biotechnology, 11, 383-392.

http://dx.doi.org/10.1007/BF00364613

[16] ILSI (1996) Allergy and Immunology Institute and In-

ternational Food Biotechnology Council. Allergenicity of

food produced by genetic modification. CRC Press, Boca

Raton.

[17] ILSI (2004) Comprehensive reviews in food science and

food safety. Food Science & Technology, 3, 44-49.

[18] Wolfenbarger, L.L. and Phifer, P.R. (2000) The ecological

risks and benefits of genetically engineered plants. Sci-

ence, 290, 2088-2093.

http://dx.doi.org/10.1126/science.290.5499.2088

[19] American Association of Cereal Chemists (2000) App-

roved methods of the AACC.

[20] Association of Official Analytical Chemists (1999) Offi-

cial methods of analysis vol II.

[21] Dewanto V., Wu, X. and Liu, R.H. (2002) Processed sweet

corn has higher antioxidant activity. Journal of Agricul-

tural and Food Chemistry, 50, 4959-4964.

http://dx.doi.org/10.1021/jf0255937

[22] Kuiper, H.A., Kleter, G.A., Noteborn, H.P., Kok, E.J.

(2001) Assessment of the food safety issues related to

genetically modified foods. Plant Journal, 27, 503-528.

http://dx.doi.org/10.1046/j.1365-313X.2001.01119.x

[23] Kanny, G., Monret, D., Flabbee, J., Beaudouin, E., Mori-

sset, M. and Thevenin, F. (2001) Population study of food

allergy in France. Journal of Allergy and Clinical Immu-

nology, 108, 133-140.

http://dx.doi.org/10.1067/mai.2001.116427

[24] Cesaretto, L., Vascotto C. and Calligaris, S. (2004) The

importance of redox state in liver damage. Annals of He-

P. B. S. Denise et al. / Health 5 (2013) 9-13

patology, 3, 86-93.

[25] Lipton, C.R., Dautlick, J.X., Grothaus, G.D., Hunst, P.L.,

Magin, K.M., Mihaliak, C.A., Rubio, F.M. and Stave, J.W.

(2000) Guidelines for the validation and use of immu-

noassays for determination of introduced proteins in bio-

technology enhanced crops and derived food ingredients.

Food and Agricultural Immunology, 12, 153-164.

http://dx.doi.org/10.1080/095401000404094

[26] Cockburn, A. (2002) Assuring the safety of genetically

modified foods. The importance of an holistic integrative

approach. Journal of Biotechnology, 98, 79-106.

http://dx.doi.org/10.1016/S0168-1656(02)00088-3

[27] Zolla, L., Rinalducci, S., Antonioli, P. and Righetti, P.G.

(2008) Proteomics as a complementary tool for identi-

fying unintended side effects occurring in transgenic mai-

ze seeds as a result of genetic modifications. Journal of

Proteome Research, 7, 1850-1861.

http://dx.doi.org/10.1021/pr0705082

[28] Ortiz-García, S., Ezcurra, B., Schoel, F, Acevedo, J.

Soberón and Snow, A. (2006) Transgenic maize in Mexico,

BioScience, 56, 709-803.

http://dx.doi.org/10.1641/0006-3568(2006)56[709:TMIM

]2.0.CO;2

[29] Williams, W.P., Gary, C., Buckley, P.M. and Daves, A.A.

(2002) Aflatoxin accumulation in conventional and trans-

genic corn hybrid infested with southern corn borer. J.

Agric. Urban Entomology, 19, 227-236.