Produção, purificação e formulação da enzima -acetolactato descarboxilase (ALDC) para aplicação em processos industrias de cerveja.

Abstract

The enzyme α-acetolactate decarboxylase (ALDC) converts α-acetolactate, a precursor compound of diacetyl, directly into acetoin, preventing the formation of the off flavor from high concentrations of diacetyl. However, its production cost directly affects its application. For this reason, this study analyzed the impact of different culture media on the productivity and economics of the process obtaining ALDC from the Bacillus subtilis ICA 56, a non-pathogenic microorganism, as well as the characterization, purification and formulation of the enzymatic extract produced, aiming at obtaining a minimum viable product (MVP) for application in the beer industry. Therefore, different carbon and nitrogen sources were evaluated, including the use of agro-industrial byproducts such as sugarcane molasses and corn steep solids (CSS). Pure glucose, used as the standard carbon source, was replaced by six carbon sources: fructose, an equimolar mixture of glucose and fructose, sucrose, lactose, glycerol and sugarcane molasses. As for the nitrogen source, five substitutes of standard source, yeast extract, were evaluated: urea, ammonium sulfate, sodium nitrate, malt powder and corn steep solids (CSS). Cultivation using sugarcane molasses and corn steep solids (CSS) produced an ALDC extract with enzymatic activity of 4.43 ± 0.12 U. mL–1 with a 74 % reduction in enzyme production cost. The ALDC presented an optimal temperature at 40 °C and stable between pH values of 3.6 to 7.0, with higher enzymatic activity at pH 6, 0 with a value of 5.30 ± 0.06 U.mL 1. The enzymatic extract produced was purified using the DEAE-agarose matrix, presenting a specific activity of 18.03 U.mg–1, with a purification factor of 3.22. The liquid formulation study produced a more stable and active enzymatic extract, maintaining 77 % of the ALDC initial activity after 90 days stored at -20 °C. Toxicity tests of the enzymatic extract formulated using Artemia salina showed non-toxicity at a concentration of 1000 mg. mL–1. Thus, this study presented promising and innovative protocols for the production, characterization and formulation of α-acetolactate decarboxylase (ALDC), being low cost, sustainable and low environmental impact alternatives. In addition, this study obtained an MVP with a Technology Readiness Level (TRL) 5.