An analytical method for measuring α‐amylase activity in starch‐containing foods

Abstract

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.

Critical review on conventional spectroscopic α‐amylase activity detection methods: merits, demerits, and future prospects

“…The quality of starch‐containing foods is significantly influenced by the action of naturally present α ‐amylase in foods, which can hydrolyse starch and eventually result in viscosity loss. Starch is mainly used as a thickening agent in many food preparations, such as sauces, soups, and canned stews and curries . The presence of even a small amount of α ‐amylase can hydrolyse the starch and result in significant viscosity loss and eventually degrade the quality of the final product .…”

“…Starch is mainly used as a thickening agent in many food preparations, such as sauces, soups, and canned stews and curries . The presence of even a small amount of α ‐amylase can hydrolyse the starch and result in significant viscosity loss and eventually degrade the quality of the final product . In egg processing, α ‐amylase activity is measured to ensure the efficiency of the pasteurization process .…”

“…Industrially, α ‐amylase monitoring is critical in maintaining the quality of final products, and clinically it is mainly used to identify some disease conditions associated with the pancreas and salivary glands. Naturally present α ‐amylases in foodstuffs, such as honey, raw fruits and vegetables, cereal grains, and fermented foods, degrade the quality of some processed food items . Both high and low levels of α ‐amylase are reported to have a huge impact on the final quality of bread, thus making it essential to measure the activity of the enzyme to maintain the final quality of food products .…”

“…In the literature, several papers describing different kinds of analytical techniques to determine α ‐amylase activity can be found . For example, techniques such as spectrometry, fluorometry, amperometry, electrophoresis, isoelectric focusing, chromatography, and immunological methods have been adapted to detect α ‐amylase activity. In addition, several methods have emerged that use different substrates, including starch, amylose, amylopectin, and some chemically modified derivatives of polymers and maltooligosaccharides of varying chain length linked to a chromophore, such as 4‐nitrophenyl or 2‐chloro‐4‐nitrophenyl .…”

Effect of Starch on the Inactivation of Amylase in Starch-Containing Foods

“…The α-amylase activity in model foods was measured by modifying our previous method (Koyama et al, 2012). The principle of this method is based on monitoring the fluorescent substrate digested by α-amylase, combined with steps to avoid interference caused by starch, dye and impurities in food samples.…”