An analytical method for measuring α-amylase activity in starch-containing foods.
The quality of starch-containing foods may be significantly impaired by contamination with very small amounts of α-amylase, which can enzymatically hydrolyze the starch and cause viscosity loss. Thus, for quality control, it is necessary to have an analytical method that can measure low amylase activity. We developed a sensitive analytical method for measuring the activity of α-amylase (from Bacillus subtilis) in starch-containing foods. The method consists of six steps: (1) crude extraction of α-amylase by centrifugation and filtration; (2) α-amylase purification by desalting and anion-exchange chromatography; (3) reaction of the purified amylase with boron-dipyrromethene (BODIPY)-labeled substrate, which releases a fluorescent fragment upon digestion of the substrate, thus avoiding interference from starch derivatives in the sample; (4) stopping the reaction with acetonitrile; (5) reversed-phase solid-phase extraction of the fluorescent substrate to remove contaminating dye and impurities; and (6) separation and measurement of BODIPY fluorescence by HPLC. The proposed method could quantify α-amylase activities as low as 10 mU/mL, which is enough to reduce the viscosity of starch-containing foods.
Somatech Center, House Foods Corporation, 1-4 Takanodai, Yotsukaido-shi, Chiba, 284-0033, Japan. k-koyama@housefoods.co.jp
Kazuo
K
Koyama
Takashi
T
Hirao
Akira
A
Toriba
Kazuichi
K
Hayakawa
D002851:Chromatography, High Pressure Liquid; D005504:Food Analysis / Q000379:methods*; D052616:Solid Phase Extraction; D013050:Spectrometry, Fluorescence; D013213:Starch / Q000032:analysis / Q000378:metabolism*; D014783:Viscosity; D000516:alpha-Amylases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D013213:Starch; D000516:alpha-Amylases