4. RESULTADOS 50.
4.2 ANÁLISIS DE RESULTADOS OBJETIVO 2 59.
ANDREIA BENTO DA SILVA1*,MARIA FRANCESCA GRECO2,ANA LIMPO3,CLÁUDIO ALMEIDA4,ELSA MECHA1,ANA TERESA SERRA1,ADELE
PAPETTI2,MARIA CARLOTA VAZ PATTO1,MARIA DO ROSÁRIO BRONZE1,3,4 1 Instituto de Tecnologia Química e Biológica, Oeiras, Portugal,
2 Dipartamento di Scienze del Farmaco, Università di Pavia, Pavia, Italia, 3 Faculdade de Farmácia da Universidade de Lisboa, Lisboa, Portugal, 4 Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal Key words: maize, broa, polyphenol content, antioxidant capacity
Summary: Maize is one of the most consumed cereals worldwide and, in Portugal, it can be used to produce “broa”, a traditional maize bread. In order to develop a prediction model for determination of total phenolic content and antioxidant capacity in maize flour and broas, Fourier transform infrared spectroscopy (FTIR) attenuated total reflectance (ATR) analyses were performed. Results show that the proposed method may be used for a rapid screening of total phenolic content in maize flour.
Background
In Portugal, maize bread is consumed mainly in the form of a traditional bread named broa (Vaz Patto et al. 2007). Phenolic compounds, mainly hydroxycinnamic acids (ferulic and p-coumaric acids) represent important maize constituents (Lopez-Martinez et al., 2011) which exhibit antioxidant capacity (Ramos-Escudero et al., 2012).
Total phenolic content of samples can be determined using Folin-Ciocalteu colorimetric method (based on oxidation of phenolic compounds by Folin- Ciocalteu reagent) and by HPLC (high performance liquid chromatography) analyses, considering chromatogram total areas at 280nm.
The antioxidant capacity can be determined by ORAC (Oxygen Radical Absorbance Capacity) assay. In this method, the antioxidant capacity of samples is evaluated by the inhibition of fluorescein (FL) oxidation by peroxyl radicals (ROO·) originated by AAPH – 2’,2’-Azobis (2-amidinopropano) dihydrochloride (Ou et al., 2001).
FTIR-ATR, prediction models for total phenolic content and antioxidant capacity, in maize flours and maize breads, were developed, using Partial Least Squares (PLS). Infrared spectrometry is an excellent tool for screening foods, since it is a rapid and simple method (Lu and Rasco, 2012).
Main chapter Introduction
Eleven maize varieties cropped by farmers in the central region of Portugal were selected based on a diversity criteria, and they were used to produce broa, a Portuguese traditional maize bread, produced with more than 50% of maize flour, and 20 to 50% of rye and wheat flours. A commercial flour was also studied for comparison. The same recipe was used in all breads prepared. The total phenolic content and antioxidant capacity of the extracts prepared from the 12 flours and breads were determined, and FTIR-ATR analyses were also performed.
Material & Methods
Total phenolic content of both maize flours and maize breads were determined using Folin-Ciocalteu method and HPLC analyses. Concerning the Folin- Ciocalteu assay, the absorbance of the resulting blue colour originated by Na2CO3 was measured at 725nm using a Beckman_DU®–70
Spectrophotometer. For the HPLC analyses, the total phenolic content was determined by measuring the total peak areas detected in the chromatograms at 280nm, using a HPLC Thermo Finnigan (model Surveyor) equipped with a RP-18 (5µm) 250×4–Lichrocart® column and a RP-18
(5µm) pre-column.
Antioxidant capacity by ORAC assay was also performed. The fluorescence emitted by the reduced form of FL was measured and read per minute at 515 nm after excitation at 493 nm (fluorescence spectrophotometer with thermostatic bath, model Cary Eclipse, Varian Ltd, Surrey, UK) during 30 minutes.
The 12 maize flours and 12 maize breads were analysed using a Thermo Scientific FTIR Spectrometer (San Jose, USA), Class 1 Laser Product Nicolet 6100, which include an accessory with a ZnSe ATR crystal. All the data were imported to Unscrambler X software. PLS regression was used to analyse data from FTIR spectra, in order to predict total phenolic content and antioxidant capacity. It was considered the spectral region from 1700.935 to 649.9039 cm-1 to avoid water interference.
Results
Maize breads showed higher total phenolic content and antioxidant capacity than maize flours. Using Folin method, the total phenolic content of maize flours ranged from 66.33 to 101.74mg GAE (gallic acid equivalents)/100g dw (dry weight). The total phenolic content of maize breads ranged from 152.16 to 225.08mg GAE/100g dw. The antioxidant capacity of maize flours ranged from 931.46 to 2358.59µmol TEAC (trolox equivalents antioxidant
capacity)/100g dw and maize breads from 1674.02 to 3776.19 µmol TEAC/100g dw. Traditional maize breads showed more antioxidant capacity than the commercial variety.
As expected, it was possible to strongly correlate the total phenolic content determined by HPLC and Folin method (r2 = 0.925). Antioxidant capacity was
moderately correlated with phenolic content (r2=0.584 and r2=0.677 for Folin method and HPLC areas at 280nm, respectively).
It was also possible to correlate the total phenolic content of maize flour obtained by Folin-Ciocalteu method and FTIR data, using the PLS calibration model. However, it wasn’t possible to correlate FTIR data with ORAC assay.
Discussion
FTIR-ATR analyses of maize flour may be used for a rapid screening of total phenolic content in maize flour, since it was possible to obtain a good correlation between FTIR data and Folin-Ciocalteu method. However, FTIR-ATR analysis may not be useful to predict antioxidant capacity determined by ORAC assay. Since ORAC and Folin methods aren’t strongly correlated, the antioxidant compounds present in samples may not interact with peroxyl radicals, but instead with other oxidant reagents. These are preliminary results and a higher amount of data must be used in order to validate this model. References
Lopez-Martinez L.X., Parkin K.L., Garcia H.S., 2011. Phase II-inducing, polyphenols content and antioxidant capacity of corn (Zea mays L.) from phenotypes of white, blue, red and purple colors processed into masa and tortillas. Plant foods for human nutrition. 66(1) pp. 41-7. Available at: <http://link.springer.com/article/10.1007%2Fs11130-011-0210-z> [Accessed 9 May 2014].
Lu X., Rasco B., 2012. Determination of Antioxidant Content and Antioxidant Activity in Foods using Infrared Spectroscopy and Chemometrics: A Review. Critical Reviews in Food Science and Nutrition. 52(10), pp. 853-875. Available at: <http://www.tandfonline.com/doi/abs/10.1080/10408398.2010.511322#.U20aiShIGP8> [Accessed 9 May 2014].
Ou B., Hampsch-Woodill M., Prior R.L., 2001. Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J. Agric. Food Chem. 49 (10), pp 4619–4462. Available at <http://pubs.acs.org/doi/abs/10.1021/jf010586o> [Accessed 9 May 2014].
Ramos-Escudero F., Muñoz A.M., Alvarado-Ortíz C., Alvarado Á., Yáñez J.A., 2012. Purple corn (Zea mays L.) phenolic compounds profile and its assessment as an agent against oxidative stress in isolated mouse organs. Journal of medicinal food. 15(2), pp. 206-15. Available at: <http://online.liebertpub.com/doi/abs/10.1089/jmf.2010.0342> [Accessed 9 May 2014].
Vaz Patto M., Moreira P., Carvalho V., Pego S., 2007. Collecting maize (Zea mays L. convar. mays) with potential technological ability for bread making in Portugal. Genetic Resources and Crop Evolution. 54(7), pp. 1555-63. Available at: <http://link.springer.com/article/10.1007%2Fs10722-006-9168-3> [Accessed 9 May 2014].
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Session C Poster C3