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                            Development of Fourier Transform Mid-Infrared Spectroscopy as a Metabolomic Technique for Characterizing the Protective Properties of Grain Sorghum Against Oxidation
	
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University of Nebraska - Lincoln University of Nebraska - Lincoln

[email protected] of Nebraska - Lincoln [email protected] of Nebraska - Lincoln

Dissertations, Theses, & Student Research in
Food Science and Technology Food Science and Technology Department

Spring 5-2010

Development of Fourier Transform Mid-Infrared Spectroscopy as Development of Fourier Transform Mid-Infrared Spectroscopy as

a Metabolomic Technique for Characterizing the Protective a Metabolomic Technique for Characterizing the Protective

Properties of Grain Sorghum Against Oxidation Properties of Grain Sorghum Against Oxidation

Emily Diane Sitorius
University of Nebraska at Lincoln, [email protected]

Follow this and additional works at: https://digitalcommons.unl.edu/foodscidiss

Part of the Food Science Commons

Sitorius, Emily Diane, "Development of Fourier Transform Mid-Infrared Spectroscopy as a Metabolomic
Technique for Characterizing the Protective Properties of Grain Sorghum Against Oxidation" (2010).
Dissertations, Theses, & Student Research in Food Science and Technology. 3.
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in polar based extraction, which in turn can enhance or compromise the overall

antioxidant capacity (Adom and Liu, 2002; Awika et al, 2003).

Specific Aim 2: FT-mIR Metabolomic Method for Monitoring Oxidative Stress in

Caco-2 Cells

FT-Method Development

Solvent selection: The literature has demonstrated the importance of consistent

and precise experimental protocol, i.e. incubation time, medium, temperature, harvest,

and sample preparation, to obtain reproducible FT-mIR data from biological systems

(Melin et al, 1999; Diem et al, 1999; Holman et al, 2000). In a cell cycle study of human

lung fibroblasts, Holman et al (2000) showed that high quality cell monolayers were

needed to produce optimal FT-mIR data. To ensure that homogeneous biofilms were

consistently prepared, the treated cells were washed two times with PBS, pipetted onto

gold-coated glass slide pieces, and spread out with a pipette tip. The researchers were

also careful to apply a consistent cell density to the slides, as well as to control

temperature and humidity. Another group, Diem et al (1999), noted that large spectral

differences in the DNA bands were dependant on cell growth cycle. As a result, the

DMEM culture medium was removed from the cell culture immediately after the

treatment period was completed to stop cell growth and inhibit metabolism (ATCC,

2009).

Accordingly, an appropriate solvent for our study was selected based upon three

criteria: that the solvent could not induce cell death, influence the spectra, or support

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post-harvest metabolism. Based on results presented by Diem et al (1999), the DMEM

growth medium was removed from the cell culture as soon as the treatment period was

completed. Because phosphate buffered saline (PBS) can be adjusted to a physiological

pH and had been successfully used for developing the Caco-2 oxidative model (Wijeratne

and Cuppett, 2005; 2006), it was the logical cell suspension solvent for the next step, i.e.,

biofilm preparation. To ensure that PBS did not influence the spectra, 20 μL of Caco-2

cells suspended in 1x PBS was applied to 15 windows of a bio-wheel and dried under

vacuum. At this concentration, the salt in the PBS crystallized resulting in non-uniform

films and noisy spectra (data not shown). PBS concentrations ranging from 0x-1x PBS

were thus evaluated. Homogenous biofilms were produced with PBS concentrations less

than 1x resulting in FT-mIR spectra with high signal to noise ratios. Using a light

microscope at 10x power, it was further determined that cell death was not induced at any

of these concentrations. Therefore, a concentration of 0.1x PBS solutions was used

throughout the studies to prepare the biofilms. No nutrients were added to the PBS and

the cell suspension was kept at 4 °C until applied to the wheel to inhibit post-harvest

metabolism as much as possible. Once dried, the biofilm was examined using a light

microscope to confirm that cells dried in a monolayer.

Optimizing Optical Density (OD): Previous work within our group completed

with bacteria (Plantz, personal communication, 2008) has shown that high and consistent

signal to noise ratios were only obtained when the spectral peak absorbance of the amide

I peak (1695-1610 cm-1, Figure 6, window 2) was between 0.60 and 0.90. To

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United States Department of Agriculture. Oxygen radical absorbance capacity (ORAC)
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Wijeratne, S. S. K. and Cuppett, S. L. Soy Isoflavones Protect the Intestine from Lipid
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