DEGRADATION OF STARCH SPHERULITES BY α-AMYLASE
Open Access
- Author:
- Suwanayuen, Nuttanit
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 23, 2009
- Committee Members:
- Gregory Ray Ziegler, Thesis Advisor/Co-Advisor
Gregory Ray Ziegler, Thesis Advisor/Co-Advisor - Keywords:
- degradation
starch spherulites
starch
α-amylase - Abstract:
- The goals of this study were to investigate the structure of starch spherulites using α-amylase and to understand the effect of degree of crystallinity on starch resistance. Common corn amylose of two different chain lengths, 600 or 120 degree of polymerization (DP), were heated to 180°C and rapidly cooled to 10°C in a pressure vessel to form amylose spherulites. In order to determine enzyme susceptibility of amylose spherulites, the spherulites formed from 600 and 120 DP amylose were hydrolyzed by pancreatic α-amylase and amyloglucosidase at 37°C for 16 hours according to the official AOAC 2002.02/AACC 32-40 method, and the spherulite residues were analyzed for resistant starch (RS) content. Amylose spherulites before and after enzyme digestion were characterized using optical microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and differential scanning calorimetry (DSC) . A two-stage digestion experiment was performed to analyze for RS content of enzyme-hydrolyzed residues. Before hydrolysis, starch spherulites made from both 600 and 120 DP amylose exhibited B-type crystalline pattern containing about 24% and 34 % crystallinity, respectively. Most spherulites had an average size of about 10 µm, round shape, rough texture, no pores and exhibited a characteristic “Maltese” cross extinction pattern between cross polarizers. A small number of spherulites had a smaller diameter (<5µm), exhibited weak to no “Maltese” cross pattern, and perhaps contained non-spherulitic materials on the surfaces. After hydrolysis, spherulites were found to be partially digested by enzymes and had enzyme resistant starch contents of approximately 37% (600 DP) and 62% (120 DP). Despite the substantial hydrolysis, as determined by RS assay, microscopy showed no extensive erosion of the treated spherulites. The spherulite residues appeared mostly intact and revealed no significant differences from untreated spherulites, unlike obvious changes normally observed in digested starch granules. In general, digested spherulites had smoother surfaces and formed larger aggregates than fresh spherulites. It was likely that the size of spherulite residues also decreased; however, this was difficult to quantify from the images. The results led to speculation that the enzyme digested a large portion of spherulites from the inside-out. Internal structure of amylose spherulites did not show radially oriented crystalline growth as expected of an ordinary spherulitic material. There were no cavities or holes present inside of digested spherulites revealing evidence of endo-corrosion pattern as expected from amylolysis. A blocklet structure was present, which ranged from 20-60 nm and 50-90 nm for 600 DP and 120 DP spherulites, respectively. The AFM phase images of spherulite residues showed an increase in the crystalline phase evidenced by larger blocklet size. For all spherulite residues, DSC profiles showed an increasing trend of melting temperature and melting enthalpy of treated spherulites after hydrolysis, suggesting an increase in molecular order and crystallinity. The two-stage digestion experiment was performed to investigate the formation of enzyme-resistant material after enzyme hydrolysis, higher RS content of treated spherulites after the second round of 16 hours hydrolysis was observed. Micrographs and DSC profiles of the first and second digestions showed similar finding as of typical spherulite residues. The results obtained in the experiments led to three possible explanations. First, enzymes preferentially digested a spherulite population which were not fully developed (i.e. exhibited partial or no “Maltese” cross or some non-spherulitic material that might be presented), and thus, this digested population largely contributed to the amount of RS contents measured by RS assay. Second, the outer surface layer of amylose spherulites (i.e. dangling/ hair like structure or rough surface) was preferentially hydrolyzed by α-amylase, hence, leaving the intact morphology and smooth surface observed on spherulites residues. Third, α-amylase partially hydrolyzed amylose spherulites creating defect crystallites and shorter amylose chains which were re-crystallized or orderly rearranged during the hydrolysis, thereby, inhibiting further enzyme digestion. As evident from accretion of spherulitic crystals and higher thermostability of digested spherulites, it is likely that additional resistant material was formed during hydrolysis. This was confirmed by the observation that there were no significant differences in spherulites morphology before and after enzyme hydrolysis but RS contents and crystallinity of amylose spherulites increased.