The invention relates to a preparation method of isomaltooligosacharide. The preparation method comprises the following steps of 1, adding water into a
starch raw material, adjusting a concentration and a pH value of the
slurry, and adding high temperature-resistant alpha-
amylase into the
slurry to obtain
starch slurry, 2, carrying out liquidation on the
starch slurry to obtain a liquefied liquid, 3, adding
maltotriose generation
enzyme (AMANO AMT1.2L) and
pullulanase (GENENCOR OPTIMAX L-1000) into the liquefied liquid, carrying out thermal-insulation saccharification, and carrying out
enzyme denaturalixation to obtain a saccharified liquid, 4, adding alpha-glucosidetransferase into the saccharified liquid for a reaction to produce a primary trans-
glucoside saccharified liquid, adding alpha-glucosidetransferase into the primary trans-
glucoside saccharified liquid for a reaction, and carrying out
enzyme denaturalixation to obtain a trans-
glucoside saccharified liquid, and 5, carrying out decoloration on the trans-glucoside saccharified liquid, carrying out
filtration, carrying out
ion exchange, carrying out
chromatographic separation, carrying out concentration and carrying out
drying to obtain isomaltooligosacharide. The preparation method greatly improves contents of sugars such as panose, isomaltotriose and
tetrasaccharide having
polymerization degrees greater than or equal to 3 in the
reaction product liquid.