Novel process to obtain agave sap (aguamiel) by directed enzymatic hydrolysis of agave juice fructans

Abstract Kluyveromyces marxianus and Saccharomyces paradoxus, which are the native microorganisms found in aguamiel, were used successfully for exo- and endo-fructanase synthesis to hydrolyze agave fructans important in agave sap production. We studied culture media for enzyme synthesis, as well as the physicochemical properties and enzyme activity, and we optimized the bioprocess parameters and the enzyme extract mixture to maximize agave fructan hydrolysis. The results showed that the best culture medium for fructanase synthesis contained 14.0, 20.0, and 1.0 (g-L−1) of sucrose, peptone, and K2HPO4, respectively. The best enzyme-to-substrate ratio for agave juice containing 90 g-L−1 of fructans was 10:1. Under constant operating conditions of 45.25 °C, 65.47 rpm, and pH 4.28, a product with 93.62 g-L−1of total reducing sugars (TRS) and fructooligosaccharides (FOS) with a polymerization degree (PD) from 3 to 10 hexose units was obtained using a mixture of 98.4% K. marxianus and 1.6% S. paradoxus enzyme extracts. The resulting agave sap preserves the natural flavor and tastes closest to natural agave sap (aguamiel); hence, the results show, for the first time, a resolution of the technical challenge for stable agave sap production and confirm the importance of using native strains to evolve from spontaneous to directed fermentation.