Effect of pepsin hydrolysis on amylase activity and lack of amylase inhibitory activity of sweet potato protein

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Title: Effect of pepsin hydrolysis on amylase activity and lack of amylase inhibitory activity of sweet potato protein
Author: Patterson, Katherine Worth
Advisors: Dr. Van-Den Truong, Committee Member
Dr. Sarah Ash, Committee Member
Dr. Jonathan C. Allen, Committee Chair
Abstract: Caiapo, an extract of Japanese white-skinned sweet potato, has been shown to possess antidiabetic properties. Caiapo treatment has been associated with a decrease in fasting plasma glucose, an increase in insulin sensitivity, and a decrease in HbA1c. A 22 kDa acidic glycoprotein has been isolated and deemed the active component of Caiapo. Beauregard sweet potatoes, one of the most common commercial cultivars in the United States, have been shown to elicit a low glycemic response and may possess antidiabetic properties similar to Caiapo. Protein band patterns of Caiapo and protein isolated from Beauregard sweet potato were found to be almost identical. Inhibition of pancreatic amylase by sweet potato protein was proposed as a possible mechanism for the low glycemic response to sweet potatoes because the addition of Caiapo to white potato decreased the glycemic response while the addition of Caiapo to glucose did not decrease the glycemic response. The first objective of this study was to determine the most efficient method for extracting protein from Beauregard sweet potato. Protein in solution after homogenization with buffers of varying concentrations of sodium phosphate (0.05 M and 0.025 M) and sodium chloride (0.3 M, 0.2 M, and 0.1 M) was compared to homogenization with distilled water by BCA assay. The concentration of protein in solution was similar for all buffers. Protein in solution after precipitation of protein with calcium chloride and resuspension was also compared by BCA assay. The highest concentration of protein in solution was achieved with the use of the 0.05 M sodium phosphate and 0.1 M sodium chloride buffer, however, none of the protein concentrations were statistically different. The second objective of this study was to determine the effect of digestive conditions on sweet potato protein. In order for a protein to inhibit pancreatic amylase in the small intestine the active portion of the protein must not be destroyed by the acidic conditions of the stomach or pepsin digestion. Sweet potato protein was subjected to pepsin digestion under acidic condition and then run on a gel next to protein not subjected to digestive conditions. Proteins resistant to pepsin digestion were found in Beauregard sweet potato flesh, peel, and leaf. The third objective of this study was to quantify the native amylase activity of Beauregard sweet potato protein. Protein extracted from the flesh exhibited higher amylase activity (32.7 ± 5.93 units/mg protein) than protein extracted from the peel (13.0 ± 1.12 units/mg protein). Digestion with pepsin eliminated amylase activity in the flesh (0.013 ± 0.812 units/mg protein) and peel extracts (0.609 ± 0.795 units/mg protein). No amylase activity was found in the leaf extract (-1.40 ± 0.469 units/mg protein) or the leaf extract treated with pepsin (-0.625 ± 0.179 units/mg protein). The fourth objective of this study was to determine if proteinaceous amylase inhibitors are present in Beauregard sweet potato. The amylase activity of pancreatic amylase and sweet potato protein were determined separately and then in combination by measuring the degradation of starch over time with a dinitrosalicylic acid (DNS) reagent. DNS reacts with sugar reducing ends to produce a compound that absorbs at 540 nm. The amylase inhibitory activity was calculated by subtracting the sum of the individually determined pancreatic and sweet potato amylase activities from the amylase activity of the combination determined experimentally. Amylase inhibitors were not apparent in any of the fractions.
Date: 2008-12-02
Degree: MS
Discipline: Nutrition
URI: http://www.lib.ncsu.edu/resolver/1840.16/2557

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