Free Radical Generation during Extrusion - American Chemical Society

Chapter 3

Free Radical Generation during Extrusion: A Critical Contributor to Texturization

Downloaded by CORNELL UNIV on September 22, 2016 | http://pubs.acs.org Publication Date: March 4, 2002 | doi: 10.1021/bk-2002-0807.ch003

Κ. M. Schaich

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520

EPR has detected free radicals in extruded cornmeal, wheat flour, potato granules, and plantain flour. The g-values in all cases are in the range g=2.0055-2.0060, indicating nitrogen -centered radicals, most likely on proteins. Extensive EPR and chemical analyses have shown that the radicals are integrally involved in the chemical changes leading to texturization in these materials. High heat causes peptide scission, which yields N• radicals that are the major component of EPR signals. Under conditions of high shear, S-S bonds are also broken, and sulfur radicals then are evident in the EPR spectra. Oxidizing lipids transfer radicals to protein side chains, and this is reflected in spectral lineshapes. Addition of free radical scavengers during extrusion dramatically diminishes and alters both EPR spectra and extrudate textures. Chemical and microscopic evidence supports EPR data and shows that free radical-mediated protein-protein and protein -starchcrosslinking are key determinants of extrudate physical structure.

© 2002 American Chemical Society Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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Introduction Despite extensive industrial use of extrusion, the molecular mechanisms of extrusion texturization remain poorly understood. In the Center for Advanced Food Technology at Rutgers University, we have been interested in determining specific reactions responsible for texturization during extrusion so that we can learn how to control these reactions to generate desired textures. During early work in this project, adventitious discovery of EPR (Electronic Paramagnetic Resonance) signals in cornmeal extrudates prompted exploratory investigations of free radical formation during extrusion. The EPR signals in cornmeal were broad singlets nearly identical to signals from lysozyme and other proteins irradiated or reacted with oxidizing lipids, and g-values were 2.0045-2.0059, indicating nitrogen-centered radicals (Figure 1A). Furthermore, signal intensities and lineshapes correlated with the degree of crosslinking in the extrudates (Figure IB), suggesting a functional role for the radicals.

EPR Signals from Extruded Cornmeal at Room Temperature

Figure 1. A. EPR spectrum of extruded cornmeal; spectrum of lysozyme reacted with oxidizing lipids included for comparison. Adapted from (1). Copyright American Assoc. of Cereal Chemists, 1999. B. EPR spectra from cornmeal extruded at 195 °C and different moistures correlated with degree of protein crosslinking in the extrudates. Relative signal intensities: cornmeal, 9; least crosslinking (30% moisture), 42; most crosslinking (20% moisture), 122.

Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

37 This discovery led us to study free radical production systematically in extruded wheat flour to determine whether free radical production was related to extrusion conditions and chemical changes in wheat proteins, and to distinguish the relative contributions of heat, shear, and oxidizing lipids in formation of protein radicals.


Materials and Methods Extrusion was conducted on a Werner and Pfleiderer ZSK30 twin screw extruder (Werner and Pfleiderer, Ramsey, NJ) equipped with two co-rotating self-wiping screws. Commercial hard red wheat flours (Bay State Milling Co., Minneapolis, MN) were extruded under puffing conditions according to conditions described by Schaich (1), Rebello (2), Koh et al (3), and Wang ( 4). EPR analyses were conducted on a Varian E-12 EPR spectrometer interfaced to and controlled by a MassComp 5500 minicomputer and equipped with an X-band (9.5 GHz) microwave bridge, 100 kHz modulation, variable rate signal averaging, and Hewlett Packard 505Cfrequencycounter (1,5). Typical instrumental setting were 5-10 mW power, 5-10 G modulation amplitude, 10,000 gain, 0.03 sec time constant.

Results and Discussion EPR signals from wheat flour extrudates were a mixture of nitrogen and sulfur centered radicals, with evidence of alkoxyl or peroxyl radicals as well (Figure 2). They varied quantitatively and qualitatively with extrusion conditions. The dominant signal in all extrudates was from nitrogen-centered radicals with broadened shoulders consistent with oxyl radicals. Weaker downfield peaks (left of the central line) from sulfur radicals appeared in some extrudates (1). EPR signal intensities were not random; they varied with extrusion conditions and showed strong correlations with high temperature and low moisture (1). This indicates that the free radicals were not experimental artifacts but were integrally related to the extrusion process. Furthermore, EPR signals also correlated with SH/SS and HPLC changes and with protein loss (2). Most importantly, extrudates with high EPR signals also showed the most dramatic alterations in protein mol wt distributions. HPLC peaks from gliadins, some low mol wt glutenins, and albumins and globulins were lost, and polymeric and low mol wtfractionsincreased (2). These observations supported a critical role for free radicals in the chemical mechanisms of protein changes during extrusion. To distinguish the relative contributions of heat, shear force, and lipid oxidation in production offreeradicals during extrusion, rawflourwas heated in


38 an oven at 180 °C for varying periods, cooled, and analyzed by EPR at 77 K. EPR spectra were narrow singlets (g=2.0056) that increased with heating time (Figure 3). These are highly localized nitrogen radicals that arise from peptide scission. No sulfur radicals were evident even with long heating times and with addition of moisture. Either heat does not induce disulfide bond scission or the sulfur radicals were too short-lived for detection. The very weak nitrogen signals detected in starch derived from traces of nitrogenous contaminants. Starch radicals were never detected. At the present time, it is not known whether starch radicals are not formed or, more likely, they anneal too rapidly for detection (1).


N« or NO*

20 gauss RSS*

Figure 2. Low temperature EPR spectrafromextruded wheatflour,g-values of major peaks and associated radical species are noted. Number sequences at right ofspectra — % protein, moisture, temperature, SME. G37-G46: extrusion run code numbers. Adaptedfrom(1) with permission. Copyright American Assoc of Cereal Chemists, 1999.



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Figure 3. Heat production of nitrogen radicals. A. Room temp. EPR spectra fromflourand Hylon 7 amylose, 180 °C for 10 min. Relative intensities noted at left B. Nitrogen radical production as a function of heating time at 180 °C. Adaptedfrom(1) with permission . Copyright American Assoc. of Cereal Chemists, 1999.

Figure 4. EPR signals of wheatflourextruded at 185 °C die temperature, 16% moisture, 225 g/min massflowrate, and 500 rpm screw speed, then ground for analysis by various methods. Sulfur peaks in EPR signals show that shear stress (grinding) produces sulfur radicals via scission of S-S bonds.



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Disulfide bonds are broken by shear force, however, as was evident in grinding experiments (Figure 4). Grinding is known to produce free radicals, so sample preparation for EPR analysis normally uses gentle grinding by hand in a mortar and pestle. EPR signals of extrudates ground in this manner are qualitatively identical to EPR signals from intact strands of extrudates (data not shown). The signal remained largely unchanged when extrudates were ground in a Wiley mill to 40 mesh (e.g. for chemical analysis), but at 60 mesh sulfur radicals increased. Samples ground to 60 mesh in a centrifugal mill showed marked shifts in the spectra, with diminished nitrogen radicals (center line), dominant sulfur radicals (shoulder from sulfur oxyl radicals and downfield peaks from thiyl and disulfide radicals), and much higher signal intensity. Chemical analyses of SH/SS contents showed an order of magnitude increase in available sulfhydryls These results show that shear force breaks disulfide bonds and, further, provide a note of caution that materials in which SH/SS or other protein analyses are to be conducted must be handled gently to avoid artifacts and misleading results. Raw flour and starch reacted with oxidizing methyl linoleate to determine the ability of lipids to induce radicals in flour proteins gave EPR signals that were broad singlets with significant wing structure but no sulfur radicals (Figure 5), consistent with previous evidence that oxidizing lipids can only react with surface residues on proteins and generally cannot break disulfide bonds (6).

Figure 5. Free radicals induced in wheatfloursand starch by oxidizing methyl linoleate, MLox (10:1 weight proportions). Adapted from (1) with permission, Copyright American Assoc. of Cereal Chemists, 1999.



41 The EPR lineshape is typical of composite spectra in which signals from many different but closely related radicals overlap. In this case, signals probably result from radicals on multiple side chains, particularly tryptophan, histidine, arginine, and lysine which are accessible to the lipids and have easily abstractable nitrogens (6,7). The weak radical signals in starch again were nitrogen-centered, not carbon, arisingfromnitrogenous contaminants rather than the starch itself. From these results, we propose that in EPR signals from wheat flour the dominant center line is from nitrogen-centered radicals produced during heatinduced peptide scission, down-field peaks and shoulders are from sulfur radicals induced by shear scission of disulfide bonds, and the line broadening and wings are from protein side chain nitrogen or oxyl radicals transferred from oxidizing lipids. Integrating results to this point, we proposed an initial Working Hypothesis to explain protein changes and texturization extrusion. Under pressure, proteins denature; they become increasingly susceptible tofragmentationby heat and shear, and more amino acid side chains also become accessible to reaction with lipids. Fragmentation or reaction with lipids forms protein radicals, some of which recombine to generate a crosslinked network. Extensive crosslinking of large proteins yields an insoluble coarse network that provides the structural framework. Solubility, microscopic, chemical, and textural analyses suggest that starch acts primarily as the filler in the dispersed phase (fine network), along with some soluble proteins, protein-starch complexes, and protein fragments (Schaich, unpublished data; Wang, 2000). The overall texture in a given product is determined by the balance between the twofractions,as shown in Scheme 1.

Native proteins

—

Denature

Free Radical Generation during Extrusion - American Chemical Society

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