Procedure for amylase digestions To facilitate the release of lipid interacting with the starch fraction, a thorough digestion

of the residues obtained from the C-m 2:1extractions described above was performed. An 0.86 mg/ml alfa-amilase (2050 units/mg) solution was prepared in 100mM 2-(Nmorpholino)- ethanesulfonic acid (MES) buffer, pH 5.9 6.0. Calcium chloride was added to the solution at 1mM as an activator. Residues from the original C-M 2:1 extractions were placed in 125 mL Erlenmeyer flasks to wich 20 mL of the foregoing enzyme solution was added. Samples were digested by incubation at 60C with constant shaking at 250 Rpm for 2 hours. Following this digestion the flasks contents were quantitatively transferred into preweighted 50mL polyethylene centrifuge tubes. The insoluble residues were separated by centrifugation at 24.000 x g for 10 minutes. Supernatants were poured off and the pellets obtained were washed twice with approximately 30 mL of distilled water. All three supernatant fractions were combined and their total weight was recorded. Pellets were quantitatively transferred to preweighed bottles and freeze-dried, after which their weights were determined. The quantitative transfer of pellets from their freeze-drying bottles to Erlenmeyer flasks for extraction was accomplished with 50mL of C- M 2:1 finally, C-M 2:1 extraction was carried out exactly as on the original cornmeal samples. Lipid Available to chloroform-methanol (2:1) extractions before and after digestion with alfaamylase Table 2 shows the results obtained from initial C-M 2:1 extraction and those following amylase digestion of unextruded cornmeal along with the extruded samples displaying the least and most interaction (by hexane extraction ). Even with efficient solvent system such as C-M 2:1, unextruded cornmeal shows at least 50% more lipid available to extraction than extruded samples. Furthermore, the most-interaction sample still shows less lipid extractable (3mg/mg) than the least-interaction sample (5mg/g), in agreement with the hexane extraction data. The ratio of nonpolar lipid to polar lipid in the extracts stays relatively constantly for all three samples, suggesting that as a whole, one fraction is not bound preferentially over the other. It can be seen from Table 2 that the amylase digestion was effective in releasing the great majority of the bound lipid, with about 13mg of total lipid extracted from both the unextruded standard and the extruded samples. More lipid was rendered available to extraction after amylase digestion even in the unextruded sample. This can be explained as follows. In the unextruded cornmeal te starch is in granules embedded in a continuous protein matrix (glutein), which also contains the protein bodies (zein). Therefore, there may he some regions into which solvent does not penetrate simply because the rather rigid structure of cornmeal prevents it. After amylase digestion of the unextruded sample, the starch granules are degraded to certain extent, thus opening in the cornmeal matrix channels that allow solvent to enter and extract previously unextractable lipid. It is apparent that the extrudate defined as displaying the most interaction by the hexane extractions is also the sample that showed the largest release of both nonpolar and polar lipid after digestion with amylase. Thus the interactions favored by high temperature low moisture conditions during extrusion appear to be largely overcome by disruption of the starch structure followed by C-M 2:1 extraction. These data suggest that lipid-carbohydrate or three-way lipidcarbohydrate-protein interactions may be responsible for the lipid binding observed. Before the

mechanisms of these samples should be examined, to permit the identification of interacting lipids. Nonpolar Lipid Analysis of extruded cornmeal extracts Table 3 shows the quantitative results of nonpolar lipid class analysis of initial C-M 2:1 extraction and those following amylase digestion. It can be seen that in all cases, the bulk of the lipid extracted was triglyceride. Availability of triglyceride decreased on the average by 40% in the least-interaction sample and 51% in the most-interaction sample. Fatty acid availability showed a still greater decrease during extrusion, dropping 48% in the least-interaction sample and 88% in the most-interaction sample. Diglyceride availability in the least-interaction extrudate dropped 53%, while the most interaction sample showed a 62% decrease. These data have allowed us to identify classes of nonpolar lipid involved in interactions during extrusion. The structures of these lipid classes can now be used to help determine the molecular interactions in which they can participate and thus the mechanisms of interaction. There are basically two types of mechanism by which the lipid can interact with the cornmeal system. The first is a physical entrapment or encapsulation mechanism, and the second is actual molecular-level interactions. If the lipids are simply entrapped within the cornmeal matrix formed during extrusion, the percentages of decreases in all lipid species would be expected to be about the same. The spaces in which lipids are entrapped would not be the size of the singlelipid molecules, and therefore the small differences in size between triglyceride, diglyceride and fatty acid should have not effect on the entrapment mechanism. On the other hand, if molecularlevel interactions were occurring, each lipid class would interact by a slightly different mechanism as a result of the types of bond each is capable of forming. The data show that the percentage decrease in extractable lipid is different for each lipid class, supporting the theory that molecular-level interactions are involved. As discussed previously, C-M 2:1 should have a potent ability to disrupt hydrophobic bonds. Triglyceride, in its unoxidized state, will interact predominantly by hydrophobic bonding. Therefore, triglyceride seems to interact by entrapment mechanism, since although the solvent system should be strong enough to disrupt hydrophobic interactions, triglyceride remains bound. In addition to forming hydrophobic bonds, fatty acid and diglyceride have the ability to participate in hydrogen bonding. Although methanol will have some ability to disrupt hydrogen bonds, there may be molecules more strongly hydrogen bound, which methanol cannot disrupt. Furthemore, the fatty acid molecules have the ability to form inclusion complexes and may be interacting with amylase by this mechanism. Very little additional triglyceride can be extracted from unextruded cornmeal after digestion with amylase. The negligibleadditional amount that those become available can be explained by the slightly more open structure of the amylase-digested unextruded cornmeal thus facilitating greater solvent penetration