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Cereal starch digestibility by porcine pancreatic α-amylase isoforms in vitro: Effect of non-starch polysaccharides

Anitha, Gopal B. (2008) Cereal starch digestibility by porcine pancreatic α-amylase isoforms in vitro: Effect of non-starch polysaccharides. PhD thesis, University of Mysore.

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Abstract

α-Amylases (EC 3.2.1.1) are a family of endo-amylases which catalyze the hydrolysis of α-D-(1,4) glycosidic linkages in starch and glycogen releasing malto-oligosaccharides. The amylose contents of ragi, rice, wheat and maize starches were 26.6, 25.1, 26.4 and 27.3% respectively. The gelatinization temperatures and the enthalpy of gelatinization of ragi, rice, wheat and maize starches were 64.9, 60.7, 62.2 & 66.5°C and 11.9, 8.9, 10.4 and 13.4 J/g respectively. X-ray crystallography revealed that all the starches showed typical A-type diffraction pattern. Pancreatic alpha amylase (PPA) was separated into two activity peaks i.e PPA-I and PPA-II on DEAE-cellulose chromatography. The molecular weights of PPA-I and PPA-II were found to be ~55 kDa, hence isoforms. The MS fragmentation pattern of the sugars released from starches by pancreatic α-amylase treatment indicated preponderantly maltose, maltotriose and maltotetraose. The pH and temperature optima of PPA, PPA-I and PPA-II were 6.9 and 45°C respectively. PPA was found to be stable in the pH range of 7.0-8.0 whereas PPA-I and PPA-II were stable in the pH range of 6.0-8.0. PPA, PPA-I and II were stable up to 45°C. Km and Vmax calculated from the Lineweaver Burk double reciprocal plot showed that maize starch was the least digestible among all the starches as indicated by its high Km value. Ca2+, Ba2+, Co2+ and Mg2+ were found to have both activating and stabilizing effect whereas Al3+ and Hg2+ completely inactivated PPA and its isoforms. Citric acid and oxalic acid inhibited PPA and its isoforms in concentration dependent manner. The viscosity of water soluble non-starch polysaccharides (WSNSP) increased with increase in concentration (0.2 to 1.0%) and decreased with increase in temperature (20 to 70°C). Maize WSNSP reduced the activity of PPA and its isoforms to a greater extent compared to the WSNSP of ragi, wheat and rice. The glycoprotein nature of PPA and its isoforms were ascertained by colorimetric and periodic acid Schiff’s (PAS) staining methods. The completeness of deglycosylation was observed after 24 h of PNGase F treatment. ESI-MS of the released oligosaccharide of these amylases showed the molecular fragmentation at 365 and 709 m/z. The deglycosylated enzymes were slightly less thermo-stable than the glycosylated forms. The excitation of the glycosylated enzymes were observed at 280 nm and that of deglycosylated samples were 286 nm indicating a red shift. The differences between the glycosylated and deglycosylated PPA and its isoforms with respect to the spectral characteristics in the far-UV region did not show any significant differences. The thesis ends with summary and conclusions along with a list of references.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Endo-amylases; Hydrolysis; Ragi; Rice; Wheat; Maize
Subjects: 600 Technology > 08 Food technology > 21 Cereals
600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions: Dept. of Biochemistry
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 12 Mar 2010 09:49
Last Modified: 18 Nov 2016 08:18
URI: http://ir.cftri.com/id/eprint/9380

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