Use of electron paramagnetic resonance spectroscopy for characterization of chemical and structural properties of foods and related matrices

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is a widely used tool to unambiguously detect free radicals in food and related matrices. It is a fast, sensitive, and non-destructive technique. The broad analysis range of EPR includes detection and identification of free radicals via direct or indirect methods (e.g., reactive oxygen and reactive nitrogen species), oxidative stability analysis (e.g., lipid oxidation and antioxidant assays), detection and quantification of irradiation-derived radicals (e.g., cellulose derived radicals) in various foods, and structural characterization (e.g., membrane mobility). The first objective is to use EPR spectroscopy as a non-destructive technique to characterize the changes in a bacterial membrane. Bacterial cell characteristics, such as size, morphology, and membrane integrity, are affected by environmental conditions such as thermal treatment. In this objective, the effect of heating on the cell morphology and membrane mobility of E. coli were evaluated by the combined analytical techniques of EPR, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The change in membrane integrity was quantified via the mobility of 16-doxylstearic acid (16-DSA) spin probe, a stable nitroxide that can align with the membrane, using EPR spectroscopy. Two order parameters S1 and S2 defined on x- and y-axes, respectively, decreased with increasing temperature indicating loss of membrane integrity (0.78 and 0.65 at 65 °C for S1 and S2, respectively). The size of E. coli cells increased from 2.3 μm to 3.0 μm with heating up to 50 °C followed by a shrinkage with further heating up to 70 °C. Our findings suggested the analysis of cell size, morphology, and membrane mobility can be used in parallel to provide a deeper understanding of structural changes related to bacterial thermal resistance. Therefore, the combined approach proposed in this study is helpful to characterize survival behavior and inactivation kinetics of microorganisms. The second objective is to use EPR spectroscopy for the detection and quantification of irradiation-derived radicals in cellulose-rich foods. Dried sweet potatoes (SP), which are often irradiated and consumed as human and pet foods, can provide a crystalline cellulose-rich environment to stabilize the irradiation-specific free radicals. SP samples were prepared at two moisture contents (48.3 and 9.7 % by drying at 150 °F for 24 or 48 h) and irradiated at 0, 5, 10, 20, 30, and 50 kGy. The irradiation-derived radicals were analyzed using EPR spectroscopy at X-band. The signal characteristics (intensity and peak shape) were evaluated at different sample locations (skin and flesh), as a function of the sample preparation method (grinding, sieving, and pelletizing). The flesh of irradiated SP showed complex EPR spectra with multiple satellite peaks of cellulose radicals (333.5 and 338.8 mT) and a split peak of dextrose radicals (337.4 mT); while skin spectra were distinctive of cellulose radicals. In this study, the effects of sample composition and preparation method on the formation and analysis of irradiation-specific radicals were detected using EPR spectroscopy. However, the quantification of free radicals in multiline spectra due to complex food matrices is challenging. The third objective of this dissertation is to improve the quantification of free radicals and the performance of EPR analysis by implementing a peak enhancement method. Peak enhancement is an artificial intelligence tool applied for the analysis of complex spectra to improve resolution in various spectroscopy data. The complex EPR spectra were analyzed as a function of irradiation dose by calculating total areas under all peaks (TPA) and areas of irradiation-specific satellite peaks (SPA) using GRAMS software. TPA increased with irradiation dose at a rate of 573.4 AU/dose (R² = 0.98) and 14.7 AU/dose (R² = 0.65) for low- and high-moisture samples, respectively. High-field SPA was shown to be more sensitive to irradiation dose as compared to low-field SPA, however with high variability for both. The resolution of satellite peaks was further improved by peak enhancement procedure: higher linearity (R² of SPA increased from 0.98 to 0.99 for low moisture and 0.77 to 0.94 for high-field of high moisture SP) and lower variability (coefficient of variation of low field SPA of high-moisture SP samples were less than 25% at all doses). The technique proposed in this study can be used to detect and quantify irradiation-specific cellulose satellite peaks and glucose split peak in EPR spectra in both low- and high-moisture plant-based foods rich in sugar and cellulose, such as dried sweet potatoes. The fourth objective of this dissertation is to use EPR spectroscopy for the detection and quantification of irradiation-derived radicals in other matrices such as chicken jerky treats and pig ears. Chicken jerky treats (CJT) and pig ears (PE) are irradiated foods that were commercially analyzed with gas chromatography (GC-MS). These lipid-rich products produce irradiation-specific 2-dodecylcyclobutanone (2-DCB), a radiolysis product of palmitic acid during irradiation. EPR spectroscopy and solid-phase microextraction (SPME)-coupled gas chromatography were used to estimate the irradiation history of these products. In addition, the factors such as IS concentration, matrix properties, and analyte concentrations that are important for the sensitivity of GC-MS analysis were investigated. Two irradiation levels (10 and 50 kGy) and different internal standard (IS) concentrations (8 and 80 ng/g sample for CJT; 8, 80, and 800 ng/g sample for PE) were studied to evaluate the interaction of IS and 2-DCB as a function of their concentrations and matrix properties to improve the precision and accuracy of SPME-coupled GC-MS analysis. IS and 2-DCB were quantified by calculating the area under IS peak (ISA) and the area under 2-DCB peak (DCA), respectively. EPR spectra of non-irradiated PE and CJT exhibited a singlet line. After irradiation, irradiated PE had a signal centered at g = 1.996 ± 0.003 due to isotropic CO₂- radical, while the signal intensity of singlet line in CJT increased. Although the irradiation-specific peak in PE and the increased signal intensity of the central peak in CJT can be used for irradiation detection, they could not be resolved to quantify irradiation doses. For GC-MS analysis, ISA of CJT irradiated at 50 kGy was significantly higher (p<0.01) than that of 10 kGy at IS concentration of 8 ng/g CJT. ISA remained unchanged at high IS concentration. Similar results were obtained for PE samples. The significant increase in IS areas with increasing 2-DCB concentration suggests an interaction and competition phenomena between IS and 2-DCB at low IS concentrations. The results of this study showed that EPR analysis can indicate the irradiation process, it was limited for dose identification in CJT and PE. Choosing the correct IS concentration can solve the problems and improve the accuracy and precision of the GC-MS analysis. This study showed that when used for membrane characterization, EPR analysis can provide information on the structural characterization of biological membranes under external stresses. In addition, it can be used as a non-destructive technique to detect and quantify irradiation-derived radicals in cellulose-rich foods. The peak enhancement method proposed in this study can improve the quantification of irradiation-specific cellulose satellite peaks and glucose split peak in EPR spectra of plant-based foods. In contrast, EPR spectroscopy can serve for the detection of irradiation process in lipid-rich products, however, it needs more studies for dose identification analysis. The EPR methods used in the present work can be used and further be developed to understand thermal inactivation kinetics on microorganisms and establish guidelines for the irradiation detection and the irradiation dose quantification in cellulose-rich plant-based products.