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Three main in vitro approaches can be distinguished for obtaining amylose (AM): enzymatic synthesis, AM leaching, and AM complexation following starch dispersion. The first uses α-d-glucose-1-phosphate (G1P), a glucosyl primer with a degree of polymerization (DP) of at least 4 and phosphorylase (EC 2.4.1.1), commonly from potatoes. Such approach provides AM chains with low polydispersity, the average DP of which can be manipulated by varying the reaction time and the ratio between G1P, primer, and enzyme dose. AM leaching is the result of heating a starch suspension above the gelatinization temperature. This approach allows isolating AM on large scale. The AM DP, yield, and purity depend on the heating rate, leaching temperature, shear forces and botanical origin. High leaching temperatures (80-85°C) result in mostly pure AM of DP >1000. At higher temperatures, lower purity AM is obtained due to amylopectin leaching. Annealing as pretreatment and ultracentrifugation or repetitive organic solvent-based precipitations after leaching are strategies, which improve the purity of AM extracts. When AM is separated by complex formation, complete dispersion of starch is followed by bringing AM into contact with, e.g., n-butanol or thymol. The resultant complex is separated from amylopectin as a precipitate. Complete starch dispersion without degradation is critical for obtaining AM of high purity. Finally, higher DP AM can be converted enzymatically into AM fractions of lower DP.