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The rate and extent of starch hydrolysis in the digestive tract impacts blood glucose levels, which may influence an individual's susceptibility to diabetes and obesity. Strategies for decreasing starch digestibility are therefore useful for developing healthier foods. β-amylase is an exo -hydrolase that specifically cleaves α-1,4 glycosidic linkages of gelatinized starches. In this study, starch granules were disrupted by extrusion under different feed moisture conditions, and then subjected to β-amylolysis. The degree of starch gelatinization increased with increasing feed moisture content during extrusion, leading to faster β-amylolysis. The hydrolysis of in vitro starch digestion study was reduced for extruded samples treated with β-amylase, which was attributed to an increase in resistant starch (RS) after β-amylase treatment. Indeed, X-ray diffraction (XRD) indicated that the crystalline structure in the extruded starch was either partially or fully lost after β-amylase treatment. Similarly, Fourier transform infrared (FTIR) analysis indicated there was a higher level of amorphous regions in the starch after β-amylase treatment. Overall, our results suggest that enzymatic treatment of extruded starch with β-amylolysis reduces the ratio of crystalline-to-amorphous regions, which increases the level of resistant starch, thereby slowing down digestion. These results have important implications for the development of healthier starch-based foods. [ABSTRACT FROM AUTHOR]